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• RAF-isotype switching: from B to C through PDE
  - Nat Struct Mol Biol 18(5):517-518 (2011)
  Nature Structural & Molecular Biology | News and Views RAF-isotype switching: from B to C through PDE * Eric Lau1 * Ze'ev Ronai1 * Affiliations * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:517–518Year published:(2011)DOI:doi:10.1038/nsmb.2063Published online04 May 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. A new study reveals that rewiring of MAPK signaling in cells expressing mutant RAS includes ERK-mediated BRAF inactivation and the concomitant activation of CRAF, partly through amplification by the phosphodiesterase 4 isoform. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Eric Lau and Ze'ev Ronai are in the Signal Transduction Program at the Sanford Burnham Medical Research Institute, La Jolla, California, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ze'ev Ronai Author Details * Eric Lau Search for this author in: * NPG journals * PubMed * Google Scholar * Ze'ev Ronai Contact Ze'ev Ronai Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• Research highlights
  - Nat Struct Mol Biol 18(5):519 (2011)
  Nature Structural & Molecular Biology | Research Highlights Research highlights Journal name:Nature Structural & Molecular BiologyVolume: 18,Page:519Year published:(2011)DOI:doi:10.1038/nsmb0511-519Published online04 May 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Neuronal switch Type IIa receptor protein tyrosine phosphatases (RPTPs), such as RPTPσ, LAR and RPTPδ, are cell-surface receptors with important roles in neuronal development, function and repair. Their ectodomains interact with heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs), which typically have opposing effects on cell function. How these effects are mediated at the molecular level has until now been unknown. The CSPG neurocan was previously shown to reduce outgrowth of dorsal root ganglion neurons, and this inhibitory effect is decreased in RPTPσ−/− (Ptprs−/−) neurons. Now Aricescu, Flanagan, Jones and colleagues show that the HSPG glypican-2 strongly promotes outgrowth of the same neuronal population, also in an RPTPσ-dependent manner. The glycosaminoglycan (GAG) chains of neurocan and glypican-2 are involved in these opposing activities, through a common receptor, RPTPσ. Crystallographic analysis of a shared GAG-binding site in RPTPσ r! eveals a V-shaped arrangement of immunoglobulin domains 1 and 2 (Ig1 and Ig2), which is stabilized by conserved interactions. Residues of RPTPσ known to mediate GAG binding lie on loops between Ig1 β-strands C–D and E–F, forming an extended positively charged surface. The crystal structure of human LAR Ig1-2 in complex with a synthetic heparin mimic reveals a conformational plasticity of the C–D loop. The modified topology of the GAG-binding site maintains an overall positive charge, suggesting that the combination of basic side chains used by the GAG-binding site may vary to accommodate chemically diverse GAGs. Heparan sulfate and heparin analogs were shown to induce RPTPσ clustering, whereas excess chondroitin sulfate inhibited heparan sulfate–induced RPTPσ clustering, suggesting that the HSPG:CSPG ratio and its effect on receptor clustering may influence neuronal function. Indeed, exogenous addition of HSPG or CSPG shifts the HSPG:CSPG ratio, thereby switchin! g the cellular response. The authors propose a model in which ! high levels of HSPG promote clustering of RPTPσ molecules, causing an uneven distribution of phosphatase activity on the cell surface and the formation of microdomains with high phosphotyrosine levels that support neuronal extension. Conversely, increasing the CSPG:HSPG ratio shifts the balance away from growth-promoting RPTPσ clusters and results in stalled axon growth. (Science doi:10.1126/science.1200840, published online 31 Mar 2011)AH A tale of parasitic tails Production of functional mRNAs in the mitochondria of trypanosomes involves several steps after transcription of the kinetoplastid DNA. The initial polycistronic precursor mRNAs are cleaved to yield pre-mRNAs and rRNAs. Most pre-mRNAs require editing (either insertion or deletion of U residues) to generate usable transcripts. Before editing, the pre-mRNAs possess a short (20–40 nt) polyadenylated tail, but the fully edited version contains a much longer extension (200–300 nt) composed of both A and U nucleotides. The mitochondrial poly(A) polymerase KPAP1 adds the initial short tail, and it was speculated that the post-editing formation of the A/U tail involved KPAP1 and the TUTase RET1. It was not clear how these enzymes collaborated to make the A/U tail or what its physiological role was. Aphasizheva and colleagues have now investigated A/U addition by looking for factors that interact with KPAP1. This led to the isolation of two pentatricopeptide repeat (PPR) proteins! , KPAF1 and KPAF2, that form a heterodimer; a small amount of RET1 was also found to bind KPAF1. In the absence of KPAF1, the short poly(A) tail is formed, but the long A/U tail is not, and this compromises parasite viability. A/U tail formation is able to be reconstituted in vitro when KPAFs are added to KPAP1 and RET1. KPAFs change the activity of both enzymes: in its presence, RET1 UMP addition is limited to ~18 nt, whereas the addition of poly(A) by KPAP1 is stimulated. The long A/U tail promotes the association of fully edited mRNAs with translating mitochondrial ribosomal complexes, specifically through an interaction with the small ribosome subunit. Functionally, loss of the A/U tails due to knockdown of KPAF1 results in inhibition of mitochondrial translation. Therefore, KPAF1–2 heterodimer-dependent extension of the short poly(A) tail into a full A/U heteropolymer constitutes a rate-limiting, possibly regulatory step that additionally discriminates which mRNAs ar! e fully edited and can form productive translation complexes. ! (Mol. Cell42, 106–117, 2011) AKE View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• Constructing and decoding unconventional ubiquitin chains
  - Nat Struct Mol Biol 18(5):520-528 (2011)
  Nature Structural & Molecular Biology | Review Constructing and decoding unconventional ubiquitin chains * Christian Behrends1 * J Wade Harper2 * Affiliations * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:520–528Year published:(2011)DOI:doi:10.1038/nsmb.2066Published online04 May 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg One of the most notable discoveries in the ubiquitin system during the past decade is the extensive use of diverse chain linkages to control signaling networks. Although the utility of Lys48- and Lys63-linked chains in protein turnover and molecular assembly, respectively, are well known, we are only beginning to understand how unconventional chain linkages are formed on target proteins and how such linkages are decoded by specific binding proteins. In this review, we summarize recent efforts to elucidate the machinery and mechanisms controlling assembly of Lys11-linked and linear (or Met1-linked) ubiquitin chains, and describe current models for how these chain types function in immune signaling and cell-cycle control. View full text Figures at a glance * Figure 1: Schematic overview of polyubiquitin chains and linkage-specific assembly. () Possible ubiquitin modifications including monoubiquitination (Mono Ub), multi-monoubiquitination (Multimono Ub) and polyubiquitination (Poly Ub). () Conformation of linkage-specific polyubiquitin (Ub4 in diagram) chains. Lys11-, Lys48-, Lys63- and Met1-linked polyubiquitin chains are shown as examples (Lys11 model, PDB 2XEW38; Lys48, PDB 2O6V64; Lys63, PDB 2JF565; Met1, PDB 2W9N65). Lys11- and Lys48-linked chains adopt compact structures, but Lys63-linked and linear structures are elongated5. () Ubiquitination cascade of E2-E3 pairs. Ubiquitin-charged E2 engages substrate-bound E3 for ubiquitin transfer. () Mechanism of linkage-specific ubiquitin chain assembly. Residues in the E2 UBE2S and the substrate-bound ubiquitin (acceptor) contribute to the spatial orientation of thioester-bound ubiquitin (donor), which is crucial for formation of Lys11-linked chains. Glu34 facilitates deprotonation of Lys11 and subsequent linkage-specific isopeptide bond formation. () Structure ! of UBE2S (magenta) showing both the donor (cyan) and acceptor (green) ubiquitin (PDB 2OB4)30. The closeup of the active site shows the positions of Cys95 in yellow, Lys11 in blue and Glu34 in red. The dotted line represents the thioester bond between the donor ubiquitin and Cys95 of UBE2S. * Figure 2: Linear chain formation and mechanism of RBR E3s. () Model of LUBAC formation and ubiquitin binding by HOIP, HOIL-1 and Sharpin; aa, amino acid. () Cysteine and lysine reactivity of ubiquitin-charged E2s. UBE2D and UBE2L3 differentially undergo ubiquitin discharge in the present of free cysteine or lysine, respectively. Thioester bonds are represented by the thick black curly lines. () Scheme of ubiquitin transfer reactions. RING, HECT and RBR E3 ubiquitin ligases use distinct mechanisms for substrate ubiquitination. Straight arrows, transthiolation; curved arrows, isopeptide bond formation. * Figure 3: Chain linkage specificity in cellular signaling. () Schematic model of TNFα-mediated NF-κB activation. TNFα-induced trimerization of TNF receptor 1 (TNF-R1) at the cell membrane results in recruitment of TRADD, RIP1 and TRAF2, which in turn provides a binding and activation platform for the E3 RING ligases cIAP1 and cIAP2 (1). cIAPs polyubiquitinate components of the TNF-R1 complex, including RIP1, TRADD, TNF-R1 and the cIAPs themselves, through Lys63 and possibly other chain linkages, including Lys11, which promotes recruitment of TAK1–TAB1–TAB2, and LUBAC complexes through their respective ubiquitin-binding domains (2). LUBAC linearly polyubiquitinates NEMO in IKK complexes (3), which in turn yields further recruitment of IKK complexes by means of linear chain binding by NEMO's UBAN domain. Binding of NEMO to linear ubiquitin chains has been hypothesized to cause a conformational change that favors autophosphorylation of IKKα and IKKβ subunits, leading to kinase activation (4). Upon activation, IKK phosphorylate! s the inhibitor of κB (IκB) (5), which retains NF-κB composed of p65 and p50 in an inactive state in the cytoplasm. Phosphorylated IκB is specifically recognized by the SCFβTrCP E3 RING ligase complex as a substrate for Lys48-linked polyubiquitination (6). Subsequent proteasomal degradation of IκB releases the p65–p50 dimer, which then translocates into the nucleus and induces expression of target genes (7). Bent arrow, phosphorylation. () Structural basis for recognition of Lys63-linked chains by the NZF domain of TAB2. NZF domain, magenta; Lys63 diubiquitin (di-Ub), green. () Structure of the UBAN domain of NEMO bound to two linear diubiquitin molecules (left, one diubiquitin in yellow and the other in green) or Lys63 diubiquitin (right). NEMO is a dimeric complex of the UBAN domain made up of coiled coils (magenta and cyan). Author information * Abstract * Author information Affiliations * Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt, Germany. * Christian Behrends * Departments of Pathology and Cell Biology, Harvard Medical School, Boston, Massachusetts, USA. * J Wade Harper Competing financial interests J.W.H. is a consultant for Millennium Pharmaceuticals. Corresponding author Correspondence to: * J Wade Harper Author Details * Christian Behrends Search for this author in: * NPG journals * PubMed * Google Scholar * J Wade Harper Contact J Wade Harper Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• Structural basis for CRISPR RNA-guided DNA recognition by Cascade
  - Nat Struct Mol Biol 18(5):529-536 (2011)
  Nature Structural & Molecular Biology | Article Structural basis for CRISPR RNA-guided DNA recognition by Cascade * Matthijs M Jore1, 11 * Magnus Lundgren1, 10, 11 * Esther van Duijn2, 3, 11 * Jelle B Bultema4, 11 * Edze R Westra1 * Sakharam P Waghmare5 * Blake Wiedenheft6, 7, 8 * Ümit Pul9 * Reinhild Wurm9 * Rolf Wagner9 * Marieke R Beijer1 * Arjan Barendregt2, 3 * Kaihong Zhou6, 7, 8 * Ambrosius P L Snijders5, 10 * Mark J Dickman5 * Jennifer A Doudna6, 7, 8 * Egbert J Boekema4 * Albert J R Heck2, 3 * John van der Oost1 * Stan J J Brouns1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:529–536Year published:(2011)DOI:doi:10.1038/nsmb.2019Received06 May 2010Accepted24 January 2011Published online03 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unu! sual seahorse shape that undergoes conformational changes when it binds target DNA. View full text Figures at a glance * Figure 1: Core complexes of Cascade retain crRNA. () Schematic diagram of the CRISPR-Cas locus in E. coli K12 containing cas3 (ygcB), casA (cse1, ygcL), casB (cse2, ygcK), casC (cse4, ygcJ), casD (cas5e, ygcI), casE (cse3, ygcH), cas1 (ygbT) and cas2 (ygbF)9, 10. () Coomassie blue–stained SDS-polyacrylamide gel showing StrepTactin-purified Cascade, CasBCDE and CasCDE. Protein marker sizes in kDa. Asterisk, Strep-tagged subunits. () Ethidium bromide–stained denaturing PAA-gel showing nucleic acids isolated from purified Cascade (sub)complexes. RNA marker sizes in nucleotides. () RNase A or DNase I treatment of Cascade-bound nucleic acids. () Size exclusion elution profiles of CasCDE, CasBCDE and Cascade before and after DNase I treatment. * Figure 2: Architecture of crRNA. () Ion-pair reversed-phase HPLC purification of mature R44 crRNA at 75 °C. () Multiple-charged ESI-MS spectrum of the purified mature crRNA. () Enhanced view of the −18 charged species before (top) and after (bottom) acid treatment indicating hydrolysis of the 2′,3′-cyclic phosphate. () Diagram of mature crRNA derived from the R44 CRISPR. * Figure 3: Target recognition by Cascade. (,) Effect of the type of crRNA bound. Cascade was loaded with either targeting crRNA (R44 CRISPR, Supplementary Fig. 2) or nontargeting crRNA (K12 CRISPR). The binding of these two types of Cascade complex to one type of probe is shown. DNA probes are 86-nucleotide ssDNA or dsDNA sequences containing the R44 protospacer (32 nucleotides) flanked by 27 nucleotides on either end. (–) Effect of uniform crRNA-loaded (sub)complexes (R44 CRISPR) on the binding of single- or double-stranded target and nontarget DNA. Nontarget DNA probes contain a scrambled R44 protospacer sequence. (,) Effect of uniform crRNA-loaded Cascade (R44 CRISPR) on the binding of target and nontarget ssRNA and dsRNA. (–) Labeled probe concentration 1 nM; DNA competitor concentration 2,500, 500, 50, 5 and 0.5 ng μl−1 (highest concentration not used for CasCDE); protein concentration 200–300 nM except in , where the Cascade concentrations were 200, 100, 50, 25 and 12.5 nM. * Figure 4: R-loop formation by Cascade. () Competition assay between R44-crRNA–loaded Cascade and CasBCDE for R44 ssDNA target. Total protein concentration was 500 nM in each reaction, and the Cascade:CasBCDE ratio was 1:0, 100:1, 10:1, 1:1, 1:10, 1:100 and 0:1. DNA competitor concentration was 1 μg μl−1. (,) Effect of labeling the complementary or noncomplementary strand of a target dsDNA with 27-bp flanks () or without protospacer-flanking sequences (). Cascade concentrations were 1,500, 300, 60 and 12.5 nM. (,) Mapping of ssDNA regions in the Cascade–target DNA complex using nuclease P1 and KMnO4. Sensitive regions are indicated by dashed lines and the protospacer by a solid line according to the G+A sequencing lanes of each strand. Cascade loaded with K12-derived crRNA was used as a control. () Exonuclease III mapping of accessible dsDNA regions upstream and downstream of the Cascade–DNA complex. The borders of the Cascade-protected regions are indicated by arrows. () Detection of the R-loop in a tar! get plasmid. Agarose gel indicating the mobility of the different plasmid forms (SC, supercoiled; L, linear; OC, open circular) and the mobility shifts caused by R44-Cascade and R44-crRNA binding. () Schematic diagram of the R-loop formed in crRNA-guided dsDNA recognition by Cascade. Regions sensitive to nuclease P1 and KMnO4 are indicated by hash and asterisk signs, respectively. * Figure 5: Subunit composition of Cascade. () Native nano-ESI mass spectrum of Cascade. Two charge state distributions are present at high m/z values, corresponding to complexes of 405 kDa (purple) and 349 kDa (pink). The charge state distribution in red corresponds to the CasB dimer. () Cascade (sub)complexes analyzed by native mass spectrometry. The subcomplexes were formed in solution after adding 5% 2-propanol to the buffer solution containing Cascade. * Figure 6: EM structure of Cascade. (–,) Three Cascade projections showing an elongated, seahorse-shaped particle with dimensions 20 × 10 nm. (–) Six Cascade projections bound to target ssDNA. The arrow indicates an anticipated rotation along the vertical axis. The six regularly arranged CasC subunits are indicated by asterisks. (–) Difference map () of Cascade () and Cascade with target ssDNA bound () indicating morphological changes upon DNA binding in mainly the head and back areas. (–) Difference map () of Cascade () and CasBCDE () with target ssDNAs bound, showing the location of the CasA subunit. (–) Difference map () of CasBCDE () and CasCDE (), showing the location of the CasB subunits. () Enlargement of defining the seahorse-like morphological features of Cascade. A typical indentation that contributes to the head feature of the seahorse is indicated with a hash sign. The number of particle projections to create the average shown is depicted on the bottom-right corner of each image. The § ! sign indicates that the depicted particle is a mirrored view of the original to match the predominant particle orientation in . Raw electron micrographs and an overview of the particle analysis method are given in Supplementary Figures 7 and 8, respectively. Scale bar, 10 nm. () Structural model of Cascade. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Matthijs M Jore, * Magnus Lundgren, * Esther van Duijn & * Jelle B Bultema Affiliations * Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Wageningen, The Netherlands. * Matthijs M Jore, * Magnus Lundgren, * Edze R Westra, * Marieke R Beijer, * John van der Oost & * Stan J J Brouns * Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and the Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands. * Esther van Duijn, * Arjan Barendregt & * Albert J R Heck * The Netherlands Proteomics Center, Utrecht, The Netherlands. * Esther van Duijn, * Arjan Barendregt & * Albert J R Heck * Department of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands. * Jelle B Bultema & * Egbert J Boekema * ChELSI Institute, Department of Chemical and Process Engineering, University of Sheffield, Sheffield, UK. * Sakharam P Waghmare, * Ambrosius P L Snijders & * Mark J Dickman * Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA. * Blake Wiedenheft, * Kaihong Zhou & * Jennifer A Doudna * Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California. * Blake Wiedenheft, * Kaihong Zhou & * Jennifer A Doudna * Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. * Blake Wiedenheft, * Kaihong Zhou & * Jennifer A Doudna * Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany. * Ümit Pul, * Reinhild Wurm & * Rolf Wagner * Present addresses: Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden (M.L.); MRC Clinical Sciences Centre, Imperial College London, London, UK (A.P.L.S.). * Magnus Lundgren & * Ambrosius P L Snijders Contributions M.M.J., M.L., E.R.W., M.R.B., J.J.B. and J.v.d.O. purified Cascade and performed binding assays and plaque assays. E.v.D., A.B. and A.J.R.H. conducted protein MS experiments. J.B.B. and E.J.B. performed EM. S.P.W., A.P.L.S. and M.J.D. conducted RNA MS experiments. B.W., K.Z. and J.A.D. performed SAXS. Ü.P., R. Wurm and R. Wagner did the footprint analyses. All the authors analyzed the data, and S.J.J.B., M.M.J. and J.v.d.O. wrote the manuscript with input from all authors. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * John van der Oost Author Details * Matthijs M Jore Search for this author in: * NPG journals * PubMed * Google Scholar * Magnus Lundgren Search for this author in: * NPG journals * PubMed * Google Scholar * Esther van Duijn Search for this author in: * NPG journals * PubMed * Google Scholar * Jelle B Bultema Search for this author in: * NPG journals * PubMed * Google Scholar * Edze R Westra Search for this author in: * NPG journals * PubMed * Google Scholar * Sakharam P Waghmare Search for this author in: * NPG journals * PubMed * Google Scholar * Blake Wiedenheft Search for this author in: * NPG journals * PubMed * Google Scholar * Ümit Pul Search for this author in: * NPG journals * PubMed * Google Scholar * Reinhild Wurm Search for this author in: * NPG journals * PubMed * Google Scholar * Rolf Wagner Search for this author in: * NPG journals * PubMed * Google Scholar * Marieke R Beijer Search for this author in: * NPG journals * PubMed * Google Scholar * Arjan Barendregt Search for this author in: * NPG journals * PubMed * Google Scholar * Kaihong Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Ambrosius P L Snijders Search for this author in: * NPG journals * PubMed * Google Scholar * Mark J Dickman Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer A Doudna Search for this author in: * NPG journals * PubMed * Google Scholar * Egbert J Boekema Search for this author in: * NPG journals * PubMed * Google Scholar * Albert J R Heck Search for this author in: * NPG journals * PubMed * Google Scholar * John van der Oost Contact John van der Oost Search for this author in: * NPG journals * PubMed * Google Scholar * Stan J J Brouns Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figures 1–10, Supplementary Tables 1–3 and Supplementary Methods Additional data Entities in this article * Uncharacterized protein ygbT ygbT Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Uncharacterized protein ygcI casD Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Uncharacterized protein ygcK casB Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Uncharacterized protein ygcJ casC Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Uncharacterized protein ygcB ygcB Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Uncharacterized protein ygcH casE Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Uncharacterized protein ygcL casA Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Uncharacterized protein ygbF ygbF Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene
• The client protein p53 adopts a molten globule–like state in the presence of Hsp90
  - Nat Struct Mol Biol 18(5):537-541 (2011)
  Nature Structural & Molecular Biology | Article The client protein p53 adopts a molten globule–like state in the presence of Hsp90 * Sung Jean Park1, 2 * Brendan N Borin1 * Maria A Martinez-Yamout1 * H Jane Dyson1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:537–541Year published:(2011)DOI:doi:10.1038/nsmb.2045Received27 September 2010Accepted25 January 2011Published online03 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg It is not currently known in what state (folded, unfolded or alternatively folded) client proteins interact with the chaperone Hsp90. We show that one client, the p53 DNA-binding domain, undergoes a structural change in the presence of Hsp90 to adopt a molten globule–like state. Addition of one- and two-domain constructs of Hsp90, as well as the full-length three-domain protein, to isotopically labeled p53 led to reduction in NMR signal intensity throughout p53, particularly in its central β-sheet. This reduction seems to be associated with a change of structure of p53 without formation of a distinct complex with Hsp90. Fluorescence and hydrogen-exchange measurements support a loosening of the structure of p53 in the presence of Hsp90 and its domains. We propose that Hsp90 interacts with p53 by multiple transient interactions, forming a dynamic heterogeneous manifold of conformational states that resembles a molten globule. View full text Figures at a glance * Figure 1: Schematic of domains of Hsp90 and p53. () Domains of Hsp90 with position of charged linker and C-terminal tetratricopeptide-binding sequence. () Domains of p53. AD, N-terminal activation domain; PRD, proline-rich domain; DBD, DNA-binding domain; TD, tetramerization domain; BD, C-terminal regulatory domain. () 1H-15N HSQC spectrum of 15N-labeled p53 alone (black) and with addition of unlabeled Hsp90 NM at indicated concentration ratios. All spectra are plotted at the same contour level. Two examples of cross-peaks that shift as NM is added are circled. The p53 sample used in these experiments contained two mutations (Y346F and T253I)13. * Figure 2: 1H-15N spectra of 15N-labeled p53 with added Hsp90. (–) Each panel shows the same portion of the 1H-15N HSQC spectrum of 15N-labeled p53 alone (black, left cross-peaks) and with addition of unlabeled Hsp90 domains at molar ratios represented by green (middle) and red (right) cross-peaks. The three spectra in each panel are plotted at the same contour level, and the levels in the various panels are adjusted to give comparable intensity for the black spectrum in each case. The three spectra are offset for clarity. Rectangles indicate Hsp90 construct, colored as in Figure 1a. Concentration of proteins: p53, 148 μM; Hsp90 N, 0, 163 and 311 μM (); p53, 163 μM; Hsp90 M, 0, 180 and 342 μM (); p53, 130 μM; Hsp90 NM, 0, 46 and 96 μM (). p53, 156 μM; Hsp90 MC, 0, 55 and 115 μM (); p53, 100 μM; Hsp90Δ, 0, 35 and 74 μM (). * Figure 3: p53 resonance attenuation by Hsp90 proteins. (–) For each amino acid residue in p5394–312, the concentration ratio (conc1/2) between p53 and added Hsp90 titrant for which the intensity (measured as cross-peak volume) of a given cross-peak is halved compared with that of the free protein is plotted for all residues for which cross-peaks could be resolved in the 1H-15N HSQC spectrum. Cross-peaks that show no attenuation are represented by an arbitrary value of 5. Horizontal lines show the positions of average conc1/2 values used to estimate Kd values. () Ribbon diagram of residues 94–297 of a single structure from the family of NMR structures of the human p53 DBD13, with backbone colored to show attenuation of resonances by the addition of Hsp90 M. Red spheres show the position of the amide N of residues where conc1/2 < (mean – s.d.); orange, residues where conc1/2 < mean. Figure prepared using MolMol28. * Figure 4: Schematic of model for NMR titration results. () Cross-peaks are observed in the 1H-15N HSQC spectrum of free p5394–312 for both surface and core residues (blue dots). Upon addition of Hsp90 or its constituent one- and two-domain constructs, the core resonances disappear (green dots), but many of the flexible surface resonances remain. The complex exchanges with any remaining free p53 in the solution. Therefore, at submaximal concentration ratios of Hsp90 titrant, the cross-peaks of the core residues are visible but lower in intensity, because they correspond to resonances of the remaining free protein. () When the buffer is exchanged for D2O, the surface amide protons of free p53 are exchanged for D, leading to disappearance of the corresponding cross-peaks (red dots). Core amides that are hydrogen bonded and sequestered from bulk solvent are exchanged more slowly, and their cross-peaks therefore appear in the HSQC spectrum in D2O. () If p53 resembles a molten globule in the presence of Hsp90, buffer exchange of p53 ! to which Hsp90 has been added should lead to a faster H/D exchange of the core amides than is observed for the free protein because the loosened structure of p53 promotes faster exchange of the core amide H for D while the p53 is bound to Hsp90. Because p53 is exchanging on and off the Hsp90, the core amide cross-peaks in the HSQC spectrum (observed as for , for the remaining free p53) should disappear more rapidly after D2O exchange. The addition of a small amount of Hsp90 acts as a catalyst to speed up H/D exchange in the core of p53. * Figure 5: ANS fluorescence spectra and H/D exchange. () Fluorescence spectrum of 11.3 μM ANS in 25 mM phosphate buffer, pH 7.1 (blue dotted line) or pH 6.0 (black dotted line), with addition of 5 μM p53 DBD (pH 7.1, blue line; pH 6.0, pink line), with addition of 5 μM Hsp90Δ (pH 7.1, green line; pH 6.0, orange line) and with further addition of 5 μM p53 DBD to form a 1:1 complex (pH 7.1, dark blue; pH 6.0, red). Arrows, extent of blue shift and increase of emission maximum at each pH after addition of p53 to Hsp90Δ. () Plot of normalized peak volume difference (calculated according to equation shown) between cross-peak intensities of amides of p53 that are persistent (slowly exchanging) in D2O. Inset, selected sets of cross-peaks from four 1H-15N HSQC spectra overlain and then offset for clarity, for p53 (123 μM) in H2O buffer (black), in D2O buffer (orange), in the presence of a 1:1 concentration ratio of Hsp90 M (green) and in the presence of a 1:1 concentration ratio of Hsp90 M and subsequently exchanged into D2O buf! fer (pink). All spectra were acquired under otherwise identical conditions, for an identical length of time and are plotted at the same contour level. The complete plot is in Supplementary Figure 4. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA. * Sung Jean Park, * Brendan N Borin, * Maria A Martinez-Yamout & * H Jane Dyson * Present address: College of Pharmacy, Gachon University School of Medicine and Science, Incheon, Korea. * Sung Jean Park Contributions S.J.P. and H.J.D. designed experiments; S.J.P. carried out NMR and fluorescence experiments; B.N.B. carried out H/D exchange experiments; S.J.P., M.A.M.-Y. and H.J.D. analyzed data; S.J.P., M.A.M.-Y. and H.J.D. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * H Jane Dyson Author Details * Sung Jean Park Search for this author in: * NPG journals * PubMed * Google Scholar * Brendan N Borin Search for this author in: * NPG journals * PubMed * Google Scholar * Maria A Martinez-Yamout Search for this author in: * NPG journals * PubMed * Google Scholar * H Jane Dyson Contact H Jane Dyson Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (596 K) Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Discussion Additional data Entities in this article * Cellular tumor antigen p53 TP53 Homo sapiens * View in UniProt * View in Entrez Gene * Heat shock protein HSP 90-alpha HSP90AA1 Homo sapiens * View in UniProt * View in Entrez Gene * Cyclin-dependent kinase 4 CDK4 Homo sapiens * View in UniProt * View in Entrez Gene * Heat shock protein HSP 90-beta HSP90AB1 Homo sapiens * View in UniProt * View in Entrez Gene * Hsp90 co-chaperone Cdc37 CDC37 Homo sapiens * View in UniProt * View in Entrez Gene * Prostaglandin E synthase 3 PTGES3 Homo sapiens * View in UniProt * View in Entrez Gene * ATP-dependent molecular chaperone HSP82 HSP82 Saccharomyces cerevisiae * View in UniProt * View in Entrez Gene * Chaperone protein htpG htpG Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene
• Munc13 mediates the transition from the closed syntaxin–Munc18 complex to the SNARE complex
  - Nat Struct Mol Biol 18(5):542-549 (2011)
  Nature Structural & Molecular Biology | Article Munc13 mediates the transition from the closed syntaxin–Munc18 complex to the SNARE complex * Cong Ma1, 2, 3 * Wei Li1, 2, 3 * Yibin Xu1, 2 * Josep Rizo1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:542–549Year published:(2011)DOI:doi:10.1038/nsmb.2047Received15 October 2010Accepted08 February 2011Published online17 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg During the priming step that leaves synaptic vesicles ready for neurotransmitter release, the SNARE syntaxin-1 transitions from a closed conformation that binds Munc18-1 tightly to an open conformation within the highly stable SNARE complex. Control of this conformational transition is important for brain function, but the underlying mechanism is unknown. NMR and fluorescence experiments now show that the Munc13-1 MUN domain, which plays a central role in vesicle priming, markedly accelerates the transition from the syntaxin-1–Munc18-1 complex to the SNARE complex. This activity depends on weak interactions of the MUN domain with the syntaxin-1 SNARE motif, and probably with Munc18-1. Together with available physiological data, these results provide a defined molecular basis for synaptic vesicle priming, and they illustrate how weak protein-protein interactions can play crucial biological roles by promoting transitions between high-affinity macromolecular assemblies. View full text Figures at a glance * Figure 1: Weak interactions can dramatically accelerate transitions between tight complexes. () Domain diagrams of rat syntaxin-1A and Munc13-1. Residue numbers indicate selected domain boundaries. The position of the loop sequence removed in MUN* (residues 1408–1452) is also indicated. CaMb, calmodulin binding region. () Diagrams of the closed syntaxin-1–Munc18-1 complex (Munc18-1 in purple; syntaxin-1 regions colored as in ) and the SNARE complex. Only the SNARE motifs are shown for synaptobrevin (red) and SNAP-25 (green). () Free-energy diagram illustrating how the energy barrier for the transition from the closed syntaxin-1–Munc18-1 complex to the SNARE complex (with Munc18-1 bound to it) could be decreased through weak interactions with the MUN domain, which might lead to the formation of MUN–SNARE complex–Munc18-1 assemblies. * Figure 2: Formation of MUN*–SNARE complex–Munc18-1 macromolecular assemblies. () Superposition of expansions of 1H-13C HMQC spectra of 12 μM [2H-13CH3]-MUN* before and after addition of 20 μM Munc18-1 (M18) and 20 μM SNARE complex (SC). The inset was plotted at lower contour levels to show that binding did not induce large cross-peak shifts. () Average decrease in the intensities of well-resolved cross-peaks from 1H-13C HMQC spectra of [2H-13CH3]-MUN* upon addition of 20 μM Munc18-1, 20 μM SNARE complex, or both. () Superposition of expansions of 1H-13C HMQC spectra of 15 μM [2H-13CH3]-SNARE complex before or after addition of 20 μM Munc18-1 and 30 μM MUN*. The [2H-13CH3]-SNARE complex was formed with [2H-13CH3-Ile]-syntaxin-12–253, [2H-13CH3-Leu, Val]-synaptobrevin29–93, [2H]-SNAP-2511–82 and [2H]-SNAP-25141–203. () Average decrease in the intensities of well-resolved cross-peaks from 1H-13C HMQC spectra of 15 μM [2H-13CH3]-SNARE complex upon addition of 20 μM Munc18-1, 30 μM MUN*, or both. Color-coded bars represent data obtained ! for cross-peaks from the syntaxin-1 Habc domain, from the syntaxin-1 and synaptobrevin SNARE motifs (SNARE motif), or both (all). In and the error bars denote the standard error calculated from two consecutive spectra acquired on the same samples. () 1D 1H-15N HSQC spectra of 20 μM [15N]MUN* before and after addition of 40 μM Munc18-1 and SNARE complex. () Plots of normalized integrals of the amide region of 1D 1H-15N HSQC spectra of 20 μM 15N-labeled MUN* upon addition of different concentrations of Munc18-1, syntaxin-12–253–Munc18-1 complex (Syx–M18)), SNARE complex and Munc18-1 plus SNARE complex. Curves show the fits obtained with a standard one-to-one protein-ligand binding model. * Figure 3: MUN* binds to the syntaxin-1 SNARE motif. () Plots of normalized integrals of the amide region of 1D 1H-15N HSQC spectra of 20 μM [15N]SNARE complex containing syntaxin-12–253 (SC with Syx Habc) or syntaxin-1191–253 (SC without Syx Habc) upon addition of different concentrations of MUN*. () 2D 1H-15N HSQC spectra of 20 μM [15N]syntaxin-1 N-terminal region (residues 2–180) before or after addition of 30 μM MUN*. () 2D 1H-15N HSQC spectra of 20 μM [15N]syntaxin-1 SNARE motif (residues 191–253) before or after addition of 30 μM MUN*. Cross-peaks that are strongly broadened by MUN* binding are labeled with their corresponding residue number. The diagram above indicates in blue the location of the MUN*-binding region (showing strong broadening) within the syntaxin-1 SNARE motif, including selected residue numbers above. () Plots of normalized integrals of the amide region of 1D 1H-15N HSQC spectra of 10 μM wild-type (WT) or R210E mutant (R210E) [15N]syntaxin-1 SNARE motif upon addition of different concentr! ations of MUN*. In and , curves show the fits obtained with a standard one-to-one protein-ligand binding model. () Ribbon diagram of the structure of the syntaxin-1–Munc18-1 complex39. Munc18-1 is in purple and syntaxin-1 in orange (Habc domain), gray (linker) and yellow (SNARE motif), except that residues that show strong broadening upon binding of MUN* to the isolated syntaxin-1 SNARE motif (see panel ) are in blue. Arg210 is represented by pink hard spheres. The ribbon diagram was generated with PyMOL (DeLano Scientific). () 2D 1H-15N HSQC spectra of 20 μM [15N]syntaxin-1-R210E SNARE motif before or after addition of 30 μM MUN*. * Figure 4: The MUN domain accelerates the transition from the syntaxin-1–Munc18-1 complex to the SNARE complex. () Proposed model whereby the MUN domain (pink) promotes the transition from the syntaxin-1–Munc18-1 complex to the SNARE complex through its weak interactions with the syntaxin-1 SNARE motif (color-coding as in Fig. 1b). () Superposition of expansions of 1H-13C HMQC spectra of 15 μM [2H-13CH3-lle] syntaxin-12–253 bound to Munc18-1 or incorporated into SNARE complex formed with [2H-13CH3-Leu, Val] synaptobrevin(29–93), [2H]SNAP-2511–82 and [2H]SNAP-25141–203. The cross-peak assignments that are available are indicated with the residue number; H3 identifies cross-peaks that are known to belong to the SNARE motif but do not have residue-specific assignment; * indicates tentative assignments (Supplementary Methods). (,) 1H-13C HMQC spectra of samples of 15 μM [2H-13CH3-lle] syntaxin-12–253 bound to Munc18-1 acquired immediately after addition of 15 μM [2H-13CH3-Leu, Val] synaptobrevin29–93, 25 μM [2H]SNAP-2511–82 and 25 μM [2H]SNAP-25141–203, in the absen! ce () or presence of 30 μM MUN* (). (,) The progress of the transition from the syntaxin-1–Munc18-1 complex to the SNARE complex as a function of time was monitored through the intensity of the disappearing Ile203 cross-peak of the syntaxin-1–Munc18-1 complex and the intensity of the appearing Ile203 cross-peak of the SNARE complex in consecutive 1H-13C HMQC spectra (2.3 h each) obtained after mixing samples as in (,), in the absence of () or in the presence of () 30 μM MUN*. The vertical axis represents the fraction of reaction completed based on normalized cross-peak intensities (see Supplementary Methods). () Progress of the transition from the syntaxin-1–Munc18-1 complex to the SNARE complex in the presence of 30 μM MUN* monitored as in but acquiring 10 min 1H-13C HMQC spectra. In –, the curves show the fits of the data to an exponential rise to a maximum. * Figure 5: MUN* activity depends on interactions with the syntaxin-1 SNARE motif and is bypassed by the syntaxin-1 LE mutation. (–) SNARE complex assembly reactions monitored by the time-dependent development of FRET between a BODIPY fluorescence donor on residue 61 of synaptobrevin29–93 and a rhodamine fluorescence acceptor on residue 249 of syntaxin-12–253. Reactions were started by adding 2 μM labeled synaptobrevin, and 10 μM SNAP-2511–82 and SNAP-25141–203 to 10 μM WT syntaxin-12–253 or the R210E or LE syntaxin-12–253 mutants alone or bound to Munc18-1 (+ M18) in the absence or in the presence of 30 μM MUN* (+ MUN*), as indicated. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Cong Ma & * Wei Li Affiliations * Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA. * Cong Ma, * Wei Li, * Yibin Xu & * Josep Rizo * Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, USA. * Cong Ma, * Wei Li, * Yibin Xu & * Josep Rizo Contributions C.M. did the kinetic studies of SNARE complex formation. C.M., W.L. and J.R. conducted the NMR experiments. Y.X. did initial biochemical studies of the MUN domain. J.R. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Josep Rizo Author Details * Cong Ma Search for this author in: * NPG journals * PubMed * Google Scholar * Wei Li Search for this author in: * NPG journals * PubMed * Google Scholar * Yibin Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Josep Rizo Contact Josep Rizo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–9, Supplementary Notes 1 and 2, and Supplementary Methods Additional data Entities in this article * Protein unc-13 homolog B Unc13b Mus musculus * View in UniProt * View in Entrez Gene * Protein unc-13 homolog A Unc13a Mus musculus * View in UniProt * View in Entrez Gene * Regulating synaptic membrane exocytosis protein 2 Rims2 Mus musculus * View in UniProt * View in Entrez Gene * Regulating synaptic membrane exocytosis protein 1 Rims1 Mus musculus * View in UniProt * View in Entrez Gene * Synaptosomal-associated protein 25 SNAP25 Homo sapiens * View in UniProt * View in Entrez Gene * Protein unc-13 homolog A Unc13a Rattus norvegicus * View in UniProt * View in Entrez Gene * Rab-3-interacting molecule unc-10 unc-10 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Phorbol ester/diacylglycerol-binding protein unc-13 unc-13 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Sec1-like protein Loligo pealeii * View in UniProt * Ras-related protein Rab-3A Rab3a Rattus norvegicus * View in UniProt * View in Entrez Gene * Synaptotagmin-1 Syt1 Rattus norvegicus * View in UniProt * View in Entrez Gene * Syntaxin-binding protein 1 Stxbp1 Rattus norvegicus * View in UniProt * View in Entrez Gene * Syntaxin-1A Stx1a Rattus norvegicus * View in UniProt * View in Entrez Gene * Vesicle-associated membrane protein 2 Vamp2 Rattus norvegicus * View in UniProt * View in Entrez Gene
• Tuning protein autoinhibition by domain destabilization
  - Nat Struct Mol Biol 18(5):550-555 (2011)
  Nature Structural & Molecular Biology | Article Tuning protein autoinhibition by domain destabilization * Jae-Hyun Cho1 * Vasant Muralidharan2, 4 * Miquel Vila-Perello2 * Daniel P Raleigh3 * Tom W Muir2 * Arthur G Palmer III1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:550–555Year published:(2011)DOI:doi:10.1038/nsmb.2039Received29 September 2010Accepted15 February 2011Published online01 May 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Activation of many multidomain signaling proteins requires rearrangement of autoinhibitory interdomain interactions that occlude activator binding sites. In one model for activation, the major inactive conformation exists in equilibrium with activated-like conformations that can be stabilized by ligand binding or post-translational modifications. We established the molecular basis for this model for the archetypal signaling adaptor protein Crk-II by measuring the thermodynamics and kinetics of the equilibrium between autoinhibited and activated-like states. We used fluorescence and NMR spectroscopies together with segmental isotopic labeling by means of expressed protein ligation. The results demonstrate that intramolecular domain-domain interactions both stabilize the autoinhibited state and induce the activated-like conformation. A combination of favorable interdomain interactions and unfavorable intradomain structural changes fine-tunes the population of the activated-lik! e conformation and allows facile response to activators. This mechanism suggests a general strategy for optimization of autoinhibitory interactions of multidomain proteins. View full text Figures at a glance * Figure 1: Scheme for the autoinhibition and activation of Crk-II. () Solution structure of Crk-II (SH2, blue; nSH3, orange; cSH3, green) (PDB 2EYZ19). Active-site residues of each domain are shown in stick-and-ball format. () Expanded view of the interface between the active site of the nSH3 and SH2 domains in Crk-II. Active-site residues are shown in red on the surface representation of the nSH3 domain. Interface residues of the SH2 domain are shown in stick format. () Schematic representation of a three-state equilibrium of ligand binding to the nSH3 domain in Crk-II (SH2, blue; nSH3, orange; cSH3, green; ligand, red). KO is the equilibrium constant between the closed and open state, and Kint is the intrinsic binding affinity of the ligand to the nSH3 domain in the open state. C and O represent the closed and open state of Crk-II, respectively. L represents the ligand that binds to the nSH3 domain. () Change in the chemical shift of Trp169 (top) and Ala172 (bottom) as a function of C3G-ligand concentration. The ratios of protein to ligan! d are 1:0 (red), 1:0.22 (green), 1:0.63 (cyan), 1:0.79 (black) and 1:1.19 (blue). * Figure 2: NMR analysis of the cSH3 domain within full-length Crk-II. () Schematic diagram outlining preparation of segmentally labeled Crk-II (residues 208–304 are labeled with 15N). COSR represents the C-terminal α-thioester derivative. () Overlay of the 1H-15N HSQC spectrum of uniformly 15N-labeled Crk-II (black) and segmentally labeled Crk-II (red). () NMR-detected H/D-exchange free energy of unfolding of the cSH3 domain in isolation (black) and Crk-II (gray). The error bars represent s.d. of three repeated experiments. () Residues detected in the H/D-exchange experiment are shown as spheres on the structure of the cSH3 domain. * Figure 3: Structural and dynamic analysis of the cSH3 domain within Crk-II. () Chemical-shift differences (Δδ) of the cSH3 domain in Crk-II (black) and C3G ligand–bound Crk-II (green), relative to the isolated cSH3 domain. The inset shows the locations of the residues whose resonances changed markedly between isolated cSH3 and Crk-II (top 25% in the deviation plot). () Chemical-shift differences of the cSH3 domain in C3G ligand–bound Crk-II compared to those within Crk-II. The difference for the 15Nɛ1 resonance of Trp275 is shown as a bar. () Comparison of average R2 rates of the cSH3 domain in isolation (molecular mass = 8,569 Da), in Crk-II, and in Crk-II and C3G (~33,830 Da). The dashed line represents the molecular mass–dependent R2 rates calculated using isotropic rotational correlation times from Stokes' law. Error bars represent the s.d. of average R2. () {1H}-15N heteronuclear NOE measurements for the cSH3 domain in isolation, residues 232–304 (closed circles), and within Crk-II, residues 208–304 (open circles). The residues cor! responding to the linker region, residues 208–236, are shaded in the plot. Error bars represent the propagated uncertainties of two repeated experiments. The background noise of the spectrum was used to estimate the uncertainty. * Figure 4: Conformation of Trp275 modulates the stability of the cSH3 domain. () Comparison of the position of the indole ring of Trp275 in the structures of isolated cSH3 domain (green, PDB 2GGR32) and full-length Crk-II (silver, PDB 2EYZ19). The 7-position of the tryptophan residue is highlighted in red. () Steady-state fluorescence quenching experiments for free indole (closed circles) and 7-azaindole (open circles). () Steady-state fluorescence quenching experiments for isolated cSH3-WT (closed circles) and isolated cSH3-7AW (open circles). The solid lines represent the best-fit model using the Stern-Volmer relationship (see Online Methods). () Schematic representation of the effects of the conformational change of Trp275 on the equilibrium between the open and closed states of Crk-II (red 'W' represents Trp275). Reduced stability of the cSH3 upon interdomain interactions reduces the activation barrier between the open and closed states of Crk-II. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA. * Jae-Hyun Cho & * Arthur G Palmer III * The Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, New York, USA. * Vasant Muralidharan, * Miquel Vila-Perello & * Tom W Muir * Department of Chemistry and Graduate Program in Biochemistry and Structural Biology, State University of New York at Stony Brook, Stony Brook, New York, USA. * Daniel P Raleigh * Present address: Howard Hughes Medical Institute, Washington University School of Medicine, Departments of Molecular Microbiology and Medicine, St. Louis, Missouri, USA. * Vasant Muralidharan Contributions J.-H.C. designed and conducted all experiments, analyzed the data and helped write the paper. V.M. and M.V.-P. helped to synthesize the ligands and prepare the segmentally labeled protein. D.P.R., T.W.M. and A.G.P. designed the experiments, analyzed the data and helped write the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Tom W Muir or * Arthur G Palmer III Author Details * Jae-Hyun Cho Search for this author in: * NPG journals * PubMed * Google Scholar * Vasant Muralidharan Search for this author in: * NPG journals * PubMed * Google Scholar * Miquel Vila-Perello Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel P Raleigh Search for this author in: * NPG journals * PubMed * Google Scholar * Tom W Muir Contact Tom W Muir Search for this author in: * NPG journals * PubMed * Google Scholar * Arthur G Palmer III Contact Arthur G Palmer III Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–7 and Supplementary Methods Additional data Entities in this article * Adapter molecule crk Crk Mus musculus * View in UniProt * View in Entrez Gene * Adapter molecule crk CRK Gallus gallus * View in UniProt * View in Entrez Gene * Adapter molecule crk CRK Homo sapiens * View in UniProt * View in Entrez Gene
• DNA binding alters coactivator interaction surfaces of the intact VDR–RXR complex
  - Nat Struct Mol Biol 18(5):556-563 (2011)
  Nature Structural & Molecular Biology | Article DNA binding alters coactivator interaction surfaces of the intact VDR–RXR complex * Jun Zhang1 * Michael J Chalmers1, 2 * Keith R Stayrook3 * Lorri L Burris3 * Yongjun Wang1 * Scott A Busby1 * Bruce D Pascal1, 2 * Ruben D Garcia-Ordonez1 * John B Bruning4 * Monica A Istrate1 * Douglas J Kojetin1 * Jeffrey A Dodge3 * Thomas P Burris1 * Patrick R Griffin1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:556–563Year published:(2011)DOI:doi:10.1038/nsmb.2046Received22 September 2010Accepted28 January 2011Published online10 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The vitamin D receptor (VDR) functions as an obligate heterodimer in complex with the retinoid X receptor (RXR). These nuclear receptors are multidomain proteins, and it is unclear how various domains interact with one another within the nuclear receptor heterodimer. Here, we show that binding of intact heterodimer to DNA alters the receptor dynamics in regions remote from the DNA-binding domains (DBDs), including the coactivator binding surfaces of both co-receptors, and that the sequence of the DNA response element can determine these dynamics. Furthermore, agonist binding to the heterodimer results in changes in the stability of the VDR DBD, indicating that the ligand itself may play a role in DNA recognition. These data suggest a mechanism by which nuclear receptors show promoter specificity and have differential effects on various target genes, providing insight into the function of selective nuclear receptor modulators. View full text Figures at a glance * Figure 1: The interactions along the dimer interface of the RXR–VDR heterodimer. () The addition of RXR induced conformational changes in VDR LBD. The average differential HDX of VDR LBD versus VDR LBD–RXR (Supplementary Table 1b, column (i–1)) mapped onto the VDR LBD–RXR complex docking model. () The addition of VDR-induced conformational changes in RXR. The average differential HDX of RXR versus RXR–VDR (Supplementary Table 1c, column (i–2)) mapped onto a RXR–VDR heterodimer docking model. The uniform color legends indicating the differential HDX between the two states are referred to throughout the entire manuscript and they are shown in Supplementary Table 1. Gray, no change in HDX between compared conditions; green to blue, slower rates of HDX between compared conditions; white, areas not covered in the analysis. * Figure 2: Ligand-induced domain-domain interactions within the RXR–VDR heterodimer complex. (,) Differential HDX data mapped onto the RXR–VDR docking model demonstrate that 1,25D3 (columns (ii) in Supplementary Table 1b,c) () and 9-cis-RA (columns (iii) in Supplementary Table 1b,c) () induced conformational changes of the RXR–VDR heterodimer complex, as shown by comparing the deuterium incorporation of both receptors in the absence or presence of ligands. () 1,25D3-induced conformational changes of the RXR–VDR heterodimer complex bound by 9-cis-RA (columns (iv) in Supplementary Table 1b,c). () 9-cis-RA induced conformational changes of the RXR–VDR heterodimer complex when it is bound to 1,25D3 (columns (v) in Supplementary Table 1b,c). Gray, no change in HDX between compared conditions; light to dark blue, slower rates of HDX between compared conditions; yellow to orange, faster rates of HDX between compared conditions; purple, 9-cis-RA; cyan, 1,25D3 ligand. * Figure 3: The interactions between RXR and VDR when they are bound to DNA. Differential HDX data mapped onto the RXR–VDR docking model of DBD and LBD domains when it is bound to different ligands and DNA response elements. () The interactions between RXR and VDR on VDRE DR3 in the absence of ligands (columns (vi) in Supplementary Table 1b,c). () The interactions between RXR and VDR on VDRE DR3 in the presence of 1,25D3 only (columns (vii) in Supplementary Table 1b,c). () The interactions between RXR and VDR on VDRE DR3 in the presence of 1,25D3 and 9-cis-RA (columns (viii) in Supplementary Table 1b,c). () The interactions between RXR and VDR on Cyp24 VDRE in the presence of 1,25D3 and 9-cis-RA (columns (ix) in Supplementary Table 1b,c). Gray, no change in HDX between compared conditions; green to blue, slower rates of HDX between compared conditions; yellow, faster rates of HDX between compared conditions; purple, 9-cis-RA ligand; cyan, 1,25D3 ligand. * Figure 4: Ligand dependency of SRC1 RID binding to the RXR–VDR heterodimer complex. Differential HDX data mapped onto the RXR–VDR docking model of LBD domains in the presence of various ligands and SRC1 RID. () In the presence of both 1,25D3 and 9-cis-RA (columns (x) in Supplementary Table 1b,c). () In the presence of 1,25D3 only (columns (xi) in Supplementary Table 1b,c). () In the presence of 9-cis-RA only (columns (xii) in Supplementary Table 1b,c). () In the absence of both ligands (columns (xiii) in Supplementary Table 1b,c). (,) Comparison of differential HDX dynamics of the peptides (residues 411–419 and 412–419) from VDR helix 12 () and the peptides (residues 271–279 and 433–438) from RXR H3 and H10–H11 () induced by SRC1 RID binding. Solid lines represent the deuterium incorporation of the peptides from the heterodimer bound to both 1,25D3 and 9-cis-RA in the presence or absence of SRC1 RID, and the dotted lines represent the deuterium incorporation of the peptides from the heterodimer bound to either 1,25D3 () or 9-cis-RA () only, in t! he presence or absence of SRC1 RID. The values in parentheses represent the charge state of the peptide ions. Data were the mean ± s.d. of triplicate individual measurements. Gray, no change in HDX between compared conditions; green to blue, slower rates of HDX between compared conditions; purple, 9-cis-RA ligand; cyan, 1,25D3 ligand. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA. * Jun Zhang, * Michael J Chalmers, * Yongjun Wang, * Scott A Busby, * Bruce D Pascal, * Ruben D Garcia-Ordonez, * Monica A Istrate, * Douglas J Kojetin, * Thomas P Burris & * Patrick R Griffin * The Scripps Research Molecular Screening Center, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA. * Michael J Chalmers, * Bruce D Pascal & * Patrick R Griffin * Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, USA. * Keith R Stayrook, * Lorri L Burris & * Jeffrey A Dodge * Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA. * John B Bruning Contributions J.Z., M.J.C., K.R.S., J.A.D. and P.R.G. conceived of the project and designed the research; J.Z., L.L.B., Y.W., S.A.B., B.D.P., R.D.G.-O., J.B.B., M.A.I. and D.J.K. conducted the research; J.Z., M.J.C., K.R.S., L.L.B., Y.W., S.A.B., B.D.P., R.D.G.-O., J.B.B., M.A.I., D.J.K., T.P.B., J.A.D. and P.R.G. analyzed the data; and J.Z., K.R.S., T.P.B., S.A.B. and P.R.G. wrote the paper, with contributions from all authors. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Patrick R Griffin Author Details * Jun Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Michael J Chalmers Search for this author in: * NPG journals * PubMed * Google Scholar * Keith R Stayrook Search for this author in: * NPG journals * PubMed * Google Scholar * Lorri L Burris Search for this author in: * NPG journals * PubMed * Google Scholar * Yongjun Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Scott A Busby Search for this author in: * NPG journals * PubMed * Google Scholar * Bruce D Pascal Search for this author in: * NPG journals * PubMed * Google Scholar * Ruben D Garcia-Ordonez Search for this author in: * NPG journals * PubMed * Google Scholar * John B Bruning Search for this author in: * NPG journals * PubMed * Google Scholar * Monica A Istrate Search for this author in: * NPG journals * PubMed * Google Scholar * Douglas J Kojetin Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey A Dodge Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas P Burris Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick R Griffin Contact Patrick R Griffin Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–9 and Supplementary Table 1 Additional data Entities in this article * Vitamin D3 receptor VDR Homo sapiens * View in UniProt * View in Entrez Gene * 1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial CYP24A1 Homo sapiens * View in UniProt * View in Entrez Gene * Retinoic acid receptor RXR-alpha RXRA Homo sapiens * View in UniProt * View in Entrez Gene * Peroxisome proliferator-activated receptor gamma PPARG Homo sapiens * View in UniProt * View in Entrez Gene * Nuclear receptor coactivator 1 NCOA1 Homo sapiens * View in UniProt * View in Entrez Gene * Estrogen receptor ESR1 Homo sapiens * View in UniProt * View in Entrez Gene
• Common architecture of nuclear receptor heterodimers on DNA direct repeat elements with different spacings
  - Nat Struct Mol Biol 18(5):564-570 (2011)
  Nature Structural & Molecular Biology | Article Common architecture of nuclear receptor heterodimers on DNA direct repeat elements with different spacings * Natacha Rochel1, 2, 3, 4 * Fabrice Ciesielski1, 2, 3, 4 * Julien Godet4, 5, 6 * Edelmiro Moman1, 2, 3, 4 * Manfred Roessle7 * Carole Peluso-Iltis1, 2, 3, 4 * Martine Moulin8 * Michael Haertlein8 * Phil Callow8 * Yves Mély4, 5, 6 * Dmitri I Svergun7 * Dino Moras1, 2, 3, 4 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:564–570Year published:(2011)DOI:doi:10.1038/nsmb.2054Received05 November 2010Accepted27 January 2011Published online10 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nuclear hormone receptors (NHRs) control numerous physiological processes through the regulation of gene expression. The present study provides a structural basis for understanding the role of DNA in the spatial organization of NHR heterodimers in complexes with coactivators such as Med1 and SRC-1. We have used SAXS, SANS and FRET to determine the solution structures of three heterodimer NHR complexes (RXR–RAR, PPAR–RXR and RXR–VDR) coupled with the NHR interacting domains of coactivators bound to their cognate direct repeat elements. The structures show an extended asymmetric shape and point to the important role played by the hinge domains in establishing and maintaining the integrity of the structures. The results reveal two additional features: the conserved position of the ligand-binding domains at the 5′ ends of the target DNAs and the binding of only one coactivator molecule per heterodimer, to RXR's partner. View full text Figures at a glance * Figure 1: Solution structure of the RXR–RAR–DR5 and RXR–VDR–DR3 complexes. () Most typical ab initio envelope of RXRαΔAB–RARαΔABF–DR5 generated by DAMMIN shows two domains separated by a narrower region. () Rigid-body refined quasiatomic model (RXR in cyan, RAR in dark blue and DR5 in red) in the ab initio envelope. () The model giving the best agreement to the experimental data (χR = 1.15) of RXRαΔAB–RARαΔABF–DR5 is shown in solvated surface representation. The RAR and RXR hinges for which no atomic structures are available are represented as dashed lines. DR5 is shown as red spheres. The coactivator peptide bound to RAR is shown in purple. () Ab initio envelope of RXRαΔAB–VDR–DR3 generated by DAMMIN reveals two domains connected by a narrower region. () Quasiatomic model refined by rigid-body in the ab initio envelope (RXR in cyan, VDR in orange and DR3 in red). The crystal structure of the VDR DBD includes the VDR hinge. Other hinges were modeled by pseudo-atoms. () Refined model of RXRαΔAB–VDR–DR3 showing the best a! greement (χR = 1.05) to the experimental data. The coactivator peptide bound to VDR is shown in pink. The N-terminal residue of VDR is shown in purple. The darkest areas are shadows. * Figure 2: Solution structures of RAR–RXR and PPAR–RXR complexes to DR1. () Most typical ab initio envelope of RARαΔAB–RXRαΔAB–DR1 generated by DAMMIN shows two domains separated by a region larger than in the envelope of RXRαΔAB–RARαΔABF–DR5. () Refined SAXS model of RARΔAB–RXRΔAB–DR1. The reversed polarity of the DBDs on the DNA and the relative position of the LBD heterodimers were determined using FRET measurements (Supplementary Table 2). (c) Refined model of PPARγΔAB–RXRαΔAB–PPRE DR1. () Model of the PPARγ–RXR–PPRE DR1 as seen in the crystal structure (PDB 3DZY)4. () The PPAR–RXR–DR1 structure in solution differs from that in the crystal. Beginning of the experimental SAXS curve of PPARγΔAB–RXRαΔAB–PPRE (pink dots), with the corresponding fits of the crystal structure model (blue) and of the refined SAXS model (yellow). * Figure 3: Validation of the models. (,) Comparison of the experimental SANS curve of dRXRαΔAB–RARDAB–DR5 in 95% (v/v) D2O (purple dots) with the corresponding fit (, blue line) for the refined SAXS model with masked RXR. (,) Relative positions of the LBDs versus the DBDs analyzed by FRET. The refined SAXS models of RXR–RAR–DR5 () and RAR–RXR–DR1 () agree with the measured distances between the fluorescent probes as indicated on the models. * Figure 4: One molecule of Med1 domain binds to RAR in the RXR–RAR–DR5 complex. () Sedimentation velocity analysis for RXR–RAR (blue) and its 1:1 complexes with DR5 (red) and Med1 (green) by Lamm equation fits using the Sedfit program (http://www.analyticalultracentrifugation.com/). The c(s) distribution plots show one sedimentation species for each complex, with the sedimentation coefficient s020,w and the corresponding molecular mass values (in parentheses) of 4.2 (80 kDa), 4.9 (94 kDa) and 6.4 (120 kDa). () Comparison of the experimental SAXS curve of RXRαΔAB–RARαΔAB–DR5 (blue) and RXRαΔAB(K284A)–RARαΔAB–DR5–Med1 in solution (green) with the corresponding fits for the refined models (black and red lines, χR = 1.08 and 1.46 respectively). A model with two bound Med1 molecules yielded poorer fit (χR = 2.01) (data not shown). () Electron pair distribution function (P (r)) computed from the SAXS experimental data for RXRΔAB–RARΔAB–DR5–Med1. () Refined model of RXRΔAB–RARΔAB–DR5–Med1 with two possible orientations of ! the elongated flexible Med1 attached to RAR that fit the data. The Med1 generated by DAMMIN is shown as gray spheres (same color code as in previous figures). * Figure 5: Functional implication of the conserved relative positions of the RXR's partner and the bound coactivator. () Role of the hinges. Three HREs with different polarities (DR1 versus DR3 and DR5) lead to the same orientation of the LBD heterodimer. () Deletion mutants in the hinges of VDR and RXR are in agreement with the solution structure of the RXR–VDR–DR3 complex. The entire hinge of VDR is required for full transcriptional activation. The region 114–120 (purple line) is a sequence-independent spacer required for transcription but not for binding to vitamin D response element (VDRE). In contrast, mutation of residues 108–114 (black cylinder) to alanine leads to a loss of transcription. The role of the sequence-independent spacer is to establish the correct geometry between the DBD and LBD. For RXR, deletion of up to 14 residues (blue dashed lines) in the hinge area corresponding to the sequence-independent spacer in VDR results in near wild-type transcriptional activity. () The retinoic acid response element (RARE) of the RARβ2 promoter is located on the dyad axis of the! nucleosome37. Left, three-dimensional structure of a mononucleosome (PDB 2NZD)38 with the location of the RARE of the RARBβ gene (black). The TATA box is shown in yellow. Middle, RXR–RAR is able to bind to the positioned nucleosome in a similar way as the thyroid nuclear receptor TR on the gene encoding TRβA39 or the glucocorticoid receptor GR on the MMTV gene40. RAR and RXR are shown in blue and cyan, respectively. Right, the structure of RXR–RAR bound to the DNA on the nucleosome suggests that the p160 coactivator (pink oval) would position the HAT catalytic site on the histone tails of H3 and H4. The helical coactivator LXXLL motif bound to RAR is shown in purple. Author information * Abstract * Author information * Supplementary information Affiliations * Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France. * Natacha Rochel, * Fabrice Ciesielski, * Edelmiro Moman, * Carole Peluso-Iltis & * Dino Moras * Institut National de Santé et de Recherche Médicale U964, Illkirch, France. * Natacha Rochel, * Fabrice Ciesielski, * Edelmiro Moman, * Carole Peluso-Iltis & * Dino Moras * Centre National de Recherche Scientifique UMR 7104, Illkirch, France. * Natacha Rochel, * Fabrice Ciesielski, * Edelmiro Moman, * Carole Peluso-Iltis & * Dino Moras * Université de Strasbourg, Illkirch, France. * Natacha Rochel, * Fabrice Ciesielski, * Julien Godet, * Edelmiro Moman, * Carole Peluso-Iltis, * Yves Mély & * Dino Moras * Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, Illkirch, France. * Julien Godet & * Yves Mély * Centre National de Recherche Scientifique UMR 7213, Illkirch, France. * Julien Godet & * Yves Mély * European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany. * Manfred Roessle & * Dmitri I Svergun * Institut Laue-Langevin, Grenoble, France. * Martine Moulin, * Michael Haertlein & * Phil Callow Contributions F.C., C.P.-I. and N.R. purified proteins; F.C. and N.R. conducted SAXS and analytical ultracentrifugation experiments; N.R. conducted SANS experiments; M.M. and M.H. produced deuterated protein; P.C. and M.R. helped during SANS and SAXS data collection; J.G. and Y.M. conducted and analyzed FRET experiments; E.M. built the initial VDR–RXR model; N.R. and D.I.S. analyzed SAXS data and modeled the complexes; N.R. and D.M. planned the project, integrated and analyzed the data and wrote the manuscript; all authors commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Dino Moras or * Natacha Rochel Author Details * Natacha Rochel Contact Natacha Rochel Search for this author in: * NPG journals * PubMed * Google Scholar * Fabrice Ciesielski Search for this author in: * NPG journals * PubMed * Google Scholar * Julien Godet Search for this author in: * NPG journals * PubMed * Google Scholar * Edelmiro Moman Search for this author in: * NPG journals * PubMed * Google Scholar * Manfred Roessle Search for this author in: * NPG journals * PubMed * Google Scholar * Carole Peluso-Iltis Search for this author in: * NPG journals * PubMed * Google Scholar * Martine Moulin Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Haertlein Search for this author in: * NPG journals * PubMed * Google Scholar * Phil Callow Search for this author in: * NPG journals * PubMed * Google Scholar * Yves Mély Search for this author in: * NPG journals * PubMed * Google Scholar * Dmitri I Svergun Search for this author in: * NPG journals * PubMed * Google Scholar * Dino Moras Contact Dino Moras Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (782K) Supplementary Figures 1–5, Supplementary Tables 1–4 and Supplementary Methods Additional data Entities in this article * Glucocorticoid receptor Nr3c1 Mus musculus * View in UniProt * View in Entrez Gene * Thyroid hormone receptor beta-A thrb-a Xenopus laevis * View in UniProt * View in Entrez Gene * Mediator of RNA polymerase II transcription subunit 1 MED1 Homo sapiens * View in UniProt * View in Entrez Gene * Nuclear receptor coactivator 2 NCOA2 Homo sapiens * View in UniProt * View in Entrez Gene * Retinoic acid receptor alpha RARA Homo sapiens * View in UniProt * View in Entrez Gene * Osteocalcin BGLAP Homo sapiens * View in UniProt * View in Entrez Gene * Retinoic acid receptor beta RARB Homo sapiens * View in UniProt * View in Entrez Gene * Vitamin D3 receptor VDR Homo sapiens * View in UniProt * View in Entrez Gene * Nuclear receptor coactivator 1 NCOA1 Homo sapiens * View in UniProt * View in Entrez Gene * Retinoic acid receptor RXR-alpha RXRA Homo sapiens * View in UniProt * View in Entrez Gene * Peroxisome proliferator-activated receptor gamma PPARG Homo sapiens * View in UniProt * View in Entrez Gene
• Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FcÉ›RI
  - Nat Struct Mol Biol 18(5):571-576 (2011)
  Nature Structural & Molecular Biology | Article Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FcɛRI * Mary D Holdom1, 2, 7 * Anna M Davies1, 2, 7 * Joanne E Nettleship3, 4 * Sarah C Bagby5, 6 * Balvinder Dhaliwal1, 2 * Enrico Girardi1, 2 * James Hunt1, 6 * Hannah J Gould1, 2 * Andrew J Beavil1, 2 * James M McDonnell1, 2 * Ray J Owens3, 4 * Brian J Sutton1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:571–576Year published:(2011)DOI:doi:10.1038/nsmb.2044Received21 April 2011Accepted11 February 2011Published online24 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Among antibody classes, IgE has a uniquely slow dissociation rate from, and high affinity for, its cell surface receptor FcɛRI. We show the structural basis for these key determinants of the ability of IgE to mediate allergic hypersensitivity through the 3.4-Å-resolution crystal structure of human IgE-Fc (consisting of the Cɛ2, Cɛ3 and Cɛ4 domains) bound to the extracellular domains of the FcɛRI α chain. Comparison with the structure of free IgE-Fc (reported here at a resolution of 1.9 Å) shows that the antibody, which has a compact, bent structure before receptor engagement, becomes even more acutely bent in the complex. Thermodynamic analysis indicates that the interaction is entropically driven, which explains how the noncontacting Cɛ2 domains, in place of the flexible hinge region of IgG antibodies, contribute together with the conformational changes to the unique binding properties of IgE. View full text Figures at a glance * Figure 1: Overall structure of the IgE-Fc–sFcɛRIα complex, in two approximately orthogonal views. sFcɛRIα is blue, IgE-Fc chain A is yellow and chain B is purple. () The extensive interface with two distinct subsites, one on each Cɛ3 domain, in a space-filled representation of the interacting side chains. The Cɛ4 domains are hidden in this orientation. () The acute bend, with the pair of Cɛ2 domains packed against the Cɛ3 and Cɛ4 domains, can be seen. The connections to the Fab regions are indicated, and the bend in the IgE molecule ensures that they are oriented away from the membrane (see also Supplementary Video 1). * Figure 2: Closure of an interdomain cleft in IgE-Fc upon receptor binding. () In the free IgE-Fc structure, there is a cleft between the first N-acetylglucosamine carbohydrate unit (blue), N-linked to Asn394 (behind) in Cɛ3B (yellow), and residues Asp271 and Asp307 in Cɛ2B (orange). () In the complex, the movement of Cɛ3A (green), Cɛ2B (orange) and Cɛ2A (not shown) as a rigid unit relative to Cɛ3B (yellow) and the Cɛ4 domains (not shown) closes the cleft, and Asp271 and carbohydrate make contact, with the formation of two potential hydrogen bonds (black lines). Both figures are centered on the first N-acetylglucosamine unit. For clarity, other carbohydrate residues are not shown for the unbound form (); all carbohydrate units except for the first N-acetylglucosamine were disordered in the complex (). * Figure 3: Interactions at the two subsites. () Two salt bridges (Arg334-Glu132 and Asp362-Lys117) with three hydrogen bonds (black lines) between residues of the Cɛ3A domain of IgE-Fc (yellow) and the receptor (blue) contribute to subsite 1. () The proline sandwich at subsite 2, with Pro426 in the Cɛ3B domain of IgE-Fc (purple) packed between Trp87 and Trp110 of the receptor (blue). The alternative orientation of Trp87 observed in the Fcɛ3-4–sFcɛRIα complex (light gray) makes fewer contacts and a weaker interaction with Pro426. * Figure 4: Thermodynamics of the IgE-FcɛRI interaction. (–) SPR sensorgrams of Fcɛ2-4 (IgE-Fc) and Fcɛ3-4 binding to immobilized wild-type sFcɛRIα (,) and to immobilized sFcɛRIα W87D (,), over a range of temperatures. A series of analyte concentrations were tested; sensorgrams for a single concentration point (125 nM) are shown for each temperature (see Supplementary Figs. 5 and 6 for full range of concentration data). () van't Hoff plot of temperature dependence of equilibrium binding affinities for Fcɛ2-4–wild-type sFcɛRIα, Fcɛ2-4–sFcɛRIα W87D, Fcɛ3-4–wild-type sFcɛRIα, and Fcɛ3-4–sFcɛRIα W87D. The fits for both Fcɛ2-4 interactions are linear (R > 0.99), whereas the Fcɛ3-4 interactions show small deviations from linearity (R = 0.96–0.98), consistent with a minor contribution from ΔCp. The derived thermodynamic parameters are summarized in Table 2. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 2WQR * 2Y7Q * 2WQR * 2Y7Q Referenced accessions Protein Data Bank * 1F6A * 1O0V * 1FP5 * 1F6A * 1O0V * 1FP5 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Mary D Holdom & * Anna M Davies Affiliations * King's College London, Randall Division of Cell and Molecular Biophysics, London, UK. * Mary D Holdom, * Anna M Davies, * Balvinder Dhaliwal, * Enrico Girardi, * James Hunt, * Hannah J Gould, * Andrew J Beavil, * James M McDonnell & * Brian J Sutton * Medical Research Council & Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK. * Mary D Holdom, * Anna M Davies, * Balvinder Dhaliwal, * Enrico Girardi, * Hannah J Gould, * Andrew J Beavil, * James M McDonnell & * Brian J Sutton * University of Oxford, The Oxford Protein Production Facility, Division of Structural Biology, Oxford, UK. * Joanne E Nettleship & * Ray J Owens * Oxford Protein Production Facility UK, The Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Science & Innovation Centre, Harwell, UK. * Joanne E Nettleship & * Ray J Owens * University of Oxford, Laboratory of Molecular Biophysics, Department of Biochemistry, Oxford, UK. * Sarah C Bagby * Present addresses: University of California, Department of Earth Science, Marine Science Institute, Santa Barbara, California, USA (S.C.B.); Novartis Horsham Research Centre, Horsham, UK (J.H.). * Sarah C Bagby & * James Hunt Contributions M.D.H. and A.M.D. carried out the crystallographic analysis of the complex, and B.D. the crystallographic analysis of IgE-Fc; M.D.H., J.E.N., J.H., A.J.B. and R.J.O. produced the proteins; S.C.B. and J.M.M. carried out the thermodynamic analysis; E.G. contributed to the analysis of the conformational changes; H.J.G., A.J.B. and B.J.S. planned and directed the project; M.D.H., A.M.D., J.M.M. and B.J.S. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Brian J Sutton Author Details * Mary D Holdom Search for this author in: * NPG journals * PubMed * Google Scholar * Anna M Davies Search for this author in: * NPG journals * PubMed * Google Scholar * Joanne E Nettleship Search for this author in: * NPG journals * PubMed * Google Scholar * Sarah C Bagby Search for this author in: * NPG journals * PubMed * Google Scholar * Balvinder Dhaliwal Search for this author in: * NPG journals * PubMed * Google Scholar * Enrico Girardi Search for this author in: * NPG journals * PubMed * Google Scholar * James Hunt Search for this author in: * NPG journals * PubMed * Google Scholar * Hannah J Gould Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew J Beavil Search for this author in: * NPG journals * PubMed * Google Scholar * James M McDonnell Search for this author in: * NPG journals * PubMed * Google Scholar * Ray J Owens Search for this author in: * NPG journals * PubMed * Google Scholar * Brian J Sutton Contact Brian J Sutton Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information Movies * Supplementary Video 1 (3M) Overall structure of the IgE-Fc/sFcɛRIα complex. This video shows the proposed orientation of the complex in relation to the cell membrane and highlights the acute bend in IgE-Fc, with the Cɛ2 domains packed against the Cɛ3 and Cɛ4 domains. The complex is rotated by 90° clockwise, 180° anti-clockwise, and 90° clockwise about an axis orthogonal to the cell membrane. IgE-Fc chains A and B are colored in green and purple respectively, and sFcɛRIα in yellow. * Supplementary Video 2 (12M) Conformational change in IgE-Fc. This video demonstrates the conformational change that takes place in IgE-Fc upon sFcɛRIα binding. The video shows the same conformational change from four views, each 90° apart. The change is demonstrated by morphing the free structure of IgE-Fc into that of the receptor-bound form, and then back to the free form. IgE-Fc chains A and B are colored in green and purple, respectively. * Supplementary Video 3 (2M) Conformational change in Cɛ2 and Cɛ3 domains on receptor binding. This video demonstrates the conformational change that takes place in IgE-Fc upon sFcɛRIα binding (with emphasis on the Cɛ2 domains), and shows how the Cɛ2A, Cɛ2B and Cɛ3A domains move together as a rigid unit. The video also shows the small conformational change within Cɛ3B. The free structure of IgE-Fc is first shown, and then morphed into the receptor-bound form. The receptor is briefly displayed, after which IgE-Fc is morphed back to the free form. IgE-Fc chains A and B are colored in green and purple respectively, and sFcɛRIα in yellow. PDF files * Supplementary Text and Figures (725K) Supplementary Figures 1–6 Additional data Entities in this article * High affinity immunoglobulin epsilon receptor subunit alpha FCER1A Homo sapiens * View in UniProt * View in Entrez Gene
• Min protein patterns emerge from rapid rebinding and membrane interaction of MinE
  - Nat Struct Mol Biol 18(5):577-583 (2011)
  Nature Structural & Molecular Biology | Article Min protein patterns emerge from rapid rebinding and membrane interaction of MinE * Martin Loose1, 2 * Elisabeth Fischer-Friedrich3 * Christoph Herold1 * Karsten Kruse4 * Petra Schwille1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:577–583Year published:(2011)DOI:doi:10.1038/nsmb.2037Received28 July 2010Accepted11 February 2011Published online24 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In Escherichia coli, the pole-to-pole oscillation of the Min proteins directs septum formation to midcell, which is required for symmetric cell division. In vitro, protein waves emerge from the self-organization of MinD, a membrane-binding ATPase, and its activator MinE. For wave propagation, the proteins need to cycle through states of collective membrane binding and unbinding. Although MinD presumably undergoes cooperative membrane attachment, it is unclear how synchronous detachment is coordinated. We used confocal and single-molecule microscopy to elucidate the order of events during Min wave propagation. We propose that protein detachment at the rear of the wave, and the formation of the E-ring, are accomplished by two complementary processes: first, local accumulation of MinE due to rapid rebinding, leading to dynamic instability; and second, a structural change induced by membrane-interaction of MinE in an equimolar MinD–MinE (MinDE) complex, which supports the robu! stness of pattern formation. View full text Figures at a glance * Figure 1: MinD, MinE and eGFP-MinC in traveling waves in vitro. () Confocal fluorescence micrographs showing waves of MinD (0.8 μM with 10 mol % MinD-Cy3), MinE (1.2 μM MinE with 10 mol % MinE-Cy5) and MinC (0.08 μM, with 40 mol % His-eGFP–MinC) on a supported lipid membrane. () The influence of the presence of MinC on velocity and period of the protein waves. Error bars represent s.d., n = 9. () Fluorescence intensity profiles of MinD, MinE and MinC acquired from the rectangular region shown in . Starting from the front of the wave (right), the density of MinC rises at a slope similar to that of MinE and also shows a similar sharp decrease at the rear of the wave. Note that the detachment of MinC is shifted toward the front of the wave. * Figure 2: Dynamics of Min proteins and their relationships. () Typical TIRF microscopy micrographs of surface waves formed by MinD (0.8 μM with 5 mol % MinD–Alexa 488) and MinE (1.2 μM MinE with 5 mol % MinE-Cy5). () Kymographs associated with rectangular regions in . () Normalized intensity plots obtained from the kymographs, representing averaged intensity distributions. Because the waves move at a constant speed v, the spatial profile corresponds to a temporal sequence where the spatial position x is related to time t by the relationship x = vt. () Derivatives of the intensity plots shown in . Inset in : black line is the ratio of MinE and MinD surface densities (gray region represents s.e.m, n = 4); blue line is the derivative of normalized MinD intensity (see Supplementary Fig. 1). (–) As in –, with His-eGFP-MinC (0.02 μM) instead of MinD–Alexa 488. * Figure 3: MinE accumulates at the rear of the wave during wave propagation. () FRET measurements by acceptor photobleaching. Confocal micrographs of MinD (0.65 μM), MinE-Cy5 (acceptor, 0.29 μM, final percentage of MinE-Cy5 = 40.0 mol %) and MinE-Cy3 (donor, 0.326 μM, final ratio of MinE-Cy3 = 40.6 mol %) before (at −10.8 s) and after bleaching of the acceptor (0 s and 6.3 s) (micrograph is pseudocolored to correspond to the fluorescence intensity). () Top, intensity profiles of the donor acquired from the rectangular areas shown in . Bottom, aligned intensity profiles of the donor before and after bleaching (−10.8 s and 0 s). Below, calculated FRET efficiency along the width of the wave. () Intensity profiles for MinD (0.8 μM, doped with 10 mol % MinD–Alexa 488) and MinE (1.2 μM) shortly after addition of 0.015 μM MinE-Cy5 to traveling protein bands. Whereas in the initial profile (after 5 s), the maximum intensity of MinE-Cy5 is located in front of the MinD maximum, at steady state this maximum intensity is seen at the rear of the wave.! () Intensity profiles corresponding to three traveling protein bands for ten successive frames after the addition of MinE-Cy5. () After bleaching MinE in the middle of the wave, the fluorescence intensity also drops in the rear while the wave is progressing forward. Only after the wave has left the previously bleached area is the fluorescence intensity fully recovered. * Figure 4: Single-molecule studies on Min proteins. () Behavior of single membrane-bound MinD dimers in the absence of waves. Kymographs along a 12-μm-thick line of membrane-bound Cy5-labeled MinD at different concentrations of nonlabeled MinD and without MinE. () Diffusion constants and residence times at different concentrations of MinD. With increasing protein concentration, membrane-bound MinD slows down while the average residence time increases. Error bars represent s.e.m. with n = 4. () Typical TIRF micrograph of single fluorescent Min proteins in traveling protein waves (MinE, 1.2 μM with 0.01 mol % MinE-Cy5; MinD, 0.8 μM with 5 mol % MinD–Alexa 488). () Normalized intensity profiles of Min proteins corresponding to Figure 2. The tracked particles were assigned to different segments of the wave, indicated in different shades of gray. () Portions of attachment and detachment events in different segments of the traveling protein band. () Diffusion constants of Min proteins in different segments of the traveling pro! tein band. () Average residence times of Min proteins in different segments of the traveling protein band. The proteins were assigned to the different segments of the wave depending on where they detached from the membrane. R = rear, M = middle, F = front. * Figure 5: Membrane binding is not required for MinE accumulation. () Confocal fluorescence micrographs showing typical pattern formed by MinD and the membrane-binding deficient mutant MinE C1 (1.2 μM MinE C1 with 10 mol % MinE C1-Cy5 and 0.8 μM MinD with 10 mol % MinD–Alexa 488) on a supported lipid membrane. () Fluorescence intensity profiles of MinD and MinE C1 acquired from the rectangular region shown in . Both proteins show a density distribution similar to those of the wild-type proteins. Toward the rear of the wave (left), the density of MinE C1 still rises when MinD is already detaching, as observed for WT MinE. However, MinE C1 does not form a peak at the rear of the wave. () In contrast to wild-type MinE, MinE C1 is not able to stimulate release of MinC and MinD from phospholipid vesicles. * Figure 6: Model of Min-protein wave propagation. () Starting from the front of the protein wave (or at the beginning of an oscillation cycle, right), MinD-ATP starts to bind to the membrane. With increasing density, the MinD dimers bind longer to the membrane and diffuse more slowly (Fig. 4). MinE dimers bind to membrane-bound MinD, but the concentration of MinE is at first too low to result in membrane detachment becoming dominant. At a sufficiently high [MinE]/[MinD] ratio, protein detachment starts to dominate. Because of rapid MinE rebinding to MinD, the [MinE]/[MinD] ratio can continuously increase toward the rear of the wave. This behavior guarantees that eventually all membrane-bound MinD dimers are in complex with MinE. At a [MinE]/[MinD] ratio of about 1, interaction of MinE with the membrane induces a conformational change, which results in the displacement of all MinC (not shown here). Finally, all proteins rapidly leave the membrane. () Illustration of the order of events at the rear of the protein wave. Shown ! are top (left) and side (middle and right) views of Min proteins bound to the membrane. As seen in the side view, before detachment from the membrane, either MinE forms a complex with MinD, which is present in an altered conformation involving membrane binding by MinE (1), or MinE rebinds to a neighboring membrane-bound MinD, if available (2). Because the density of membrane-bound MinD is higher toward the front of the wave, rebinding MinE is biased in this direction, giving rise to the local saturation of MinD with MinE. After detachment of MinC from MinD (3), MinE can occupy the overlapping binding site on MinD. Author information * Abstract * Author information * Supplementary information Affiliations * Biophysics, BIOTEC, Dresden University of Technology, Dresden, Germany. * Martin Loose, * Christoph Herold & * Petra Schwille * Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany. * Martin Loose & * Petra Schwille * Max Planck Institute for the Physics of Complex Systems, Dresden, Germany. * Elisabeth Fischer-Friedrich * Theoretische Physik, Universitaet des Saarlandes, Saarbruecken, Germany. * Karsten Kruse Contributions M.L., P.S. and K.K. designed the research, M.L. conducted the research, E.F.-F. and C.H. wrote the tracking software, C.H. built the single-molecule TIRF setup, M.L. analyzed and interpreted the data with the help of all authors, and M.L., E.F.-F. and P.S. wrote the paper with the help of K.K. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Petra Schwille Author Details * Martin Loose Search for this author in: * NPG journals * PubMed * Google Scholar * Elisabeth Fischer-Friedrich Search for this author in: * NPG journals * PubMed * Google Scholar * Christoph Herold Search for this author in: * NPG journals * PubMed * Google Scholar * Karsten Kruse Search for this author in: * NPG journals * PubMed * Google Scholar * Petra Schwille Contact Petra Schwille Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Video 1 (3.8MB) Confocal fluorescence micrographs showing waves of MinD (0.8 μM with 10 mol % MinDCy3), MinE (1.2 μM MinE with 10 mol % MinE-Cy5) and MinC-eGFP (0.02 μM) on a supported lipid membrane. Scale bar is 50 μm. * Supplementary Video 2 (4.4MB) TIRF micrograph of waves of MinD (0.8 μM, doped with 5 mol % MinD-Alexa 488) and MinE (1.2 μM MinE, doped with 5 mol % MinE-Cy5) middle. The merged channels are shown on the bottom. Scale bar is 10 μm. * Supplementary Video 3 (11.8MB) Single molecule TIRF micrograph of waves of MinE (1.2 μM, doped with 0.01 mol % MinECy5) (top) and MinD (0.8 μM, doped with 10 mol % MinD-Alexa 488) (middle) and overlay (bottom). Scale bar is 20 μm. * Supplementary Video 4 (5.4MB) Matlab based particle tracking allowed us to track and analyze single particles inside the waves. The movie shows the calculated trajectories for single MinE proteins. * Supplementary Video 5 (2.3MB) Confocal fluorescence micrographs showing waves of MinD (blue, 0.8 μM with 10 mol % MinD-Alexa 488), MinE C1 (red, 1.2 μM MinE C1with 10 mol % MinE C1-Cy5). Scale bar is 50 μm. PDF files * Supplementary Text and Figures (5.4MB) Supplementary Figures 1–7, Supplementary Methods and Supplementary Discussion Additional data Entities in this article * Cell division topological specificity factor minE Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Septum site-determining protein minC minC Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Septum site-determining protein minD minD Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Chromosome partitioning protein parA parA Caulobacter crescentus (strain NA1000 / CB15N) * View in UniProt * View in Entrez Gene * Cell division protein ftsZ ftsZ Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene
• ERK and PDE4 cooperate to induce RAF isoform switching in melanoma
  - Nat Struct Mol Biol 18(5):584-591 (2011)
  Nature Structural & Molecular Biology | Article ERK and PDE4 cooperate to induce RAF isoform switching in melanoma * Amélie Marquette1, 2 * Jocelyne André1, 2 * Martine Bagot1, 2, 3 * Armand Bensussan1, 2, 3 * Nicolas Dumaz1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:584–591Year published:(2011)DOI:doi:10.1038/nsmb.2022Received03 February 2010Accepted26 January 2011Published online10 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Melanocytes use BRAF to activate the MAP kinase (MAPK) pathway because CRAF is inhibited by the cyclic AMP (cAMP) pathway in these cells. By contrast, melanomas harboring Ras mutations use CRAF to activate the MAPK pathway. We describe the molecular mechanism of Raf isoform switching and cAMP pathway disruption, which take place during melanocyte transformation. We show that overactivation of the MAPK pathway, induced by the oncogenic Ras in melanoma, induces constitutive phosphorylation of BRAF on Ser151 by ERK, which inhibits NRAS-BRAF interaction . We also demonstrate that melanoma cells have elevated cAMP phosphodiesterase activity owing to overexpression of the cAMP-specific phosphodiesterase-4 enzymes; this activity inhibits cAMP signaling and allows CRAF reactivation in these cells. Reactivating the cAMP pathway inhibits proliferation and induces apoptosis of Ras-mutated melanoma cells, suggesting a new therapeutic approach for treating melanomas harboring Ras mutatio! ns. View full text Figures at a glance * Figure 1: NRAS G12V is oncogenic in melanocytes and induces Raf isoform switching. () Immunoblots of BRAF, CRAF, phosphorylated ERK (ppERK) or total ERK (ERK) expression in control (SCR) and NRAS G21V Melan-a cells depleted of BRAF or CRAF with siRNA. () Immunoblots of BRAF or CRAF in Melan-a cells mock transfected (–) or transfected with the empty pG418 vector (pG418), pG418 expressing a shRNA control (pG418-SCR) or a shRNA targeting CRAF (pG418-CRAF-A and pG418-CRAF-B). Graphs on right in and , mean ± s.d. of three independent experiments. () Counting of colonies of Melan-a cells transfected with a vector expressing NRAS G12V in combination with the pG418 vectors described above and selected with G418 with (shRNA alone) or without (shRNA + NRAS) TPA. Graph, mean ± s.d. of three independent experiments. * Figure 2: Mek inhibition rescues NRAS-BRAF binding in melanoma. () Melan-a cells expressing NRAS G12V were untreated (–) or treated with DMSO, U0126 (10 μM) or PD98059 (20 μM). NRAS G12V was immunoprecipitated (IP) and probed for BRAF or CRAF. Lysates were directly probed for BRAF, CRAF, phosphorylated ERK (ppERK) or total ERK (ERK). () GST pulldown assay with either GST or GST-NRAS on HM11 and WM1361 human melanoma cell lines treated with DMSO or U0126 (10 μM) and probed for BRAF or GST. Lysates were directly probed for BRAF, ppERK or ERK. See also Supplementary Figure 2. * Figure 3: BRAF is phosphorylated on Ser151. (,) NRAS G12V Melan-a or human melanoma cell lines were treated with DMSO, U0126 (10 μM) or PD98059 (20 μM). BRAF was immunoprecipitated (IP) and probed with a phospho-serine/proline (pBRAF) or a BRAF antibody. Lysates were directly probed for phosphorylated ERK (ppERK) or total ERK (ERK). () NHEMs were transfected with an empty vector (–), Myc-tagged wild-type (WT) BRAF, BRAF S335A or BRAF S151A. BRAF was immunoprecipitated and probed with a phospho-SP (pBRAF) or a BRAF antibody. * Figure 4: Disruption of the cAMP pathway in melanoma. () Immunoblots of phosphorylated CREB (pCREB) or total CREB (CREB) in Melan-a, NRAS G12V Melan-a, WM1361 and WM1791c human melanoma cell lines treated for 15 min with IBMX (100 μM), α-Msh (1 μM), forskolin (10 μM) or a combination of α-Msh (1 μM) + IBMX (100 μM) or forskolin (10 μM) + IBMX (100 μM). () Immunoblots of phosphorylated CREB (pCREB) or total CREB (CREB) in Melan-a, NRAS G12V Melan-a, C8161 and HM11 human melanoma cell lines treated as described above but using rolipram (Rol) (10 μM) instead of IBMX. * Figure 5: Increased PDE4 activity in melanoma compared with melanocytes. (,) Protein extracts from NHEM or melanoma cell lines were assayed for cAMP-PDE activity in the presence of DMSO (D) or PDE inhibitors as indicated: BRL50481 (BRL), dipyridamole (Dyp), rolipram (Rol or R) and zaprinast (Zap). Values are mean ± s.d. of two independent experiments in duplicate. () The mRNA level of PDE4A, PDE4B, PDE4C and PDE4D was evaluated in NHEM and melanoma cell lines by reverse transcription followed by real-time PCR using primers able to detect all species within a family. See also Supplementary Figures 6 and 7. * Figure 6: Increased PDE4B2 expression in melanoma compared with melanocytes. () Immunoblots of PDE4A, PDE4B, PDE4D, phosphorylated ERK (ppERK) and total ERK (ERK) in NHEM, Ras-mutated melanoma, Melan-a and NRAS G12V Melan-a cell lines. () Immunoblots of PDE4B and actin in Melan-a cells mock transfected (−) or transfected with the empty pG418 vector (pG418), pG418 expressing an shRNA control (pG418-SCR) or an shRNA targeting all splice variants of PDE4B (pG418-PDE4B-A and pG418-PDE4B-B). Graph on right, mean ± s.d. of three independent experiments. () Counting of colonies of Melan-a cells transfected with a vector expressing NRAS G12V in combination with the pG418 vectors described above and selected with G418 with (shRNA alone) or without (shRNA + NRAS) TPA. Right, cells transfected with NRAS G12V and the indicated shRNA were selected in the presence of DMSO or rolipram (10 μM). Values are mean ± s.d. of three independent experiments. * Figure 7: Reactivation of the cAMP pathway in melanoma inhibits the MAPK pathway and induces apoptosis. (,) Human melanoma cell lines were treated with DMSO or a combination of forskolin (1 μM) and rolipram (10 μM) for 1 h (F1 + R10). () CRAF activity was measured in vitro; values are mean ± s.d. of three independent experiments. () Lysates were probed for phosphorylated ERK (ppERK) or total ERK (ERK). () DNA synthesis in NHEM or Ras-mutated melanoma cell lines untreated (–) or treated with DMSO, forskolin (1 μM) (F1), rolipram (10 μM) (R10) or a combination of both (F1 + R10). Values are mean ± s.d. of two experiments assayed in triplicate. () Immunoblots of cleaved PARP, cleaved caspase 3 and cleaved caspase 7 in melanoma cell lines treated with DMSO or a combination of forskolin (1 μM) and rolipram (10 μM) for 24 h (F1 + R10 24 h) or 48 h (F1 + R10 48 h). See also Supplementary Figures 8 and 9. Author information * Abstract * Author information * Supplementary information Affiliations * INSERM U976, Hôpital Saint Louis, Paris, France. * Amélie Marquette, * Jocelyne André, * Martine Bagot, * Armand Bensussan & * Nicolas Dumaz * Université Paris 7–Denis Diderot, Hôpital Saint Louis, Paris, France. * Amélie Marquette, * Jocelyne André, * Martine Bagot, * Armand Bensussan & * Nicolas Dumaz * Department of Dermatology, Assistance Public—Hôpitaux de Paris, Hôpital Saint Louis, Paris, France. * Martine Bagot & * Armand Bensussan Contributions A.M., J.A. and N.D. carried out research; M.B., A.B. and N.D. designed and directed the project; N.D. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Nicolas Dumaz Author Details * Amélie Marquette Search for this author in: * NPG journals * PubMed * Google Scholar * Jocelyne André Search for this author in: * NPG journals * PubMed * Google Scholar * Martine Bagot Search for this author in: * NPG journals * PubMed * Google Scholar * Armand Bensussan Search for this author in: * NPG journals * PubMed * Google Scholar * Nicolas Dumaz Contact Nicolas Dumaz Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–11 and Supplementary Methods Additional data Entities in this article * Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 PIN1 Homo sapiens * View in UniProt * View in Entrez Gene * High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A PDE9A Homo sapiens * View in UniProt * View in Entrez Gene * cGMP-specific 3',5'-cyclic phosphodiesterase PDE5A Homo sapiens * View in UniProt * View in Entrez Gene * cAMP-specific 3',5'-cyclic phosphodiesterase 4B PDE4B Homo sapiens * View in UniProt * View in Entrez Gene * Dual 3',5'-cyclic-AMP and -GMP phosphodiesterase 11A PDE11A Homo sapiens * View in UniProt * View in Entrez Gene * cAMP-specific 3',5'-cyclic phosphodiesterase 4A PDE4A Homo sapiens * View in UniProt * View in Entrez Gene * cAMP-specific 3',5'-cyclic phosphodiesterase 4D PDE4D Homo sapiens * View in UniProt * View in Entrez Gene * Caspase-3 CASP3 Homo sapiens * View in UniProt * View in Entrez Gene * cAMP-specific 3',5'-cyclic phosphodiesterase 4C PDE4C Homo sapiens * View in UniProt * View in Entrez Gene * Kinase suppressor of Ras 1 KSR1 Homo sapiens * View in UniProt * View in Entrez Gene * Caspase-9 CASP9 Homo sapiens * View in UniProt * View in Entrez Gene * Mitogen-activated protein kinase 3 MAPK3 Homo sapiens * View in UniProt * View in Entrez Gene * Dual specificity mitogen-activated protein kinase kinase 2 MAP2K2 Homo sapiens * View in UniProt * View in Entrez Gene * Mast/stem cell growth factor receptor KIT Homo sapiens * View in UniProt * View in Entrez Gene * Mitogen-activated protein kinase 1 MAPK1 Homo sapiens * View in UniProt * View in Entrez Gene * GTPase NRas NRAS Homo sapiens * View in UniProt * View in Entrez Gene * RAF proto-oncogene serine/threonine-protein kinase RAF1 Homo sapiens * View in UniProt * View in Entrez Gene * Dual specificity mitogen-activated protein kinase kinase 1 MAP2K1 Homo sapiens * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase A-Raf ARAF Homo sapiens * View in UniProt * View in Entrez Gene * Microphthalmia-associated transcription factor MITF Homo sapiens * View in UniProt * View in Entrez Gene * Melanocyte-stimulating hormone receptor MC1R Homo sapiens * View in UniProt * View in Entrez Gene * Pro-opiomelanocortin POMC Homo sapiens * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase B-raf BRAF Homo sapiens * View in UniProt * View in Entrez Gene * Ras-related protein Rab-27A RAB27A Homo sapiens * View in UniProt * View in Entrez Gene * L-dopachrome tautomerase DCT Homo sapiens * View in UniProt * View in Entrez Gene * 5,6-dihydroxyindole-2-carboxylic acid oxidase TYRP1 Homo sapiens * View in UniProt * View in Entrez Gene * Tyrosinase TYR Homo sapiens * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase B-raf Braf Mus musculus * View in UniProt * View in Entrez Gene * RAF proto-oncogene serine/threonine-protein kinase Raf1 Mus musculus * View in UniProt * View in Entrez Gene * G-protein coupled receptor 143 GPR143 Homo sapiens * View in UniProt * View in Entrez Gene * Caspase-7 CASP7 Homo sapiens * View in UniProt * View in Entrez Gene
• Single-molecule paleoenzymology probes the chemistry of resurrected enzymes
  - Nat Struct Mol Biol 18(5):592-596 (2011)
  Nature Structural & Molecular Biology | Article Single-molecule paleoenzymology probes the chemistry of resurrected enzymes * Raul Perez-Jimenez1 * Alvaro Inglés-Prieto2 * Zi-Ming Zhao3 * Inmaculada Sanchez-Romero2 * Jorge Alegre-Cebollada1 * Pallav Kosuri1, 4 * Sergi Garcia-Manyes1 * T Joseph Kappock5 * Masaru Tanokura6 * Arne Holmgren7 * Jose M Sanchez-Ruiz2 * Eric A Gaucher3, 8, 9 * Julio M Fernandez1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:592–596Year published:(2011)DOI:doi:10.1038/nsmb.2020Received09 August 2010Accepted24 January 2011Published online03 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg It is possible to travel back in time at the molecular level by reconstructing proteins from extinct organisms. Here we report the reconstruction, based on sequence predicted by phylogenetic analysis, of seven Precambrian thioredoxin enzymes (Trx) dating back between ~1.4 and ~4 billion years (Gyr). The reconstructed enzymes are up to 32 °C more stable than modern enzymes, and the oldest show markedly higher activity than extant ones at pH 5. We probed the mechanisms of reduction of these enzymes using single-molecule force spectroscopy. From the force dependency of the rate of reduction of an engineered substrate, we conclude that ancient Trxs use chemical mechanisms of reduction similar to those of modern enzymes. Although Trx enzymes have maintained their reductase chemistry unchanged, they have adapted over 4 Gyr to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth. View full text Figures at a glance * Figure 1: Phylogenetic analysis of Trx enzymes and ancestral sequence reconstruction. () Schematic phylogenetic tree showing the geological time in which different extinct organisms lived (see text). Dashed lines represent further bifurcations. Divergence times are compiled from multiple sources and are summarized in ref. 17. The figure indicates the global environment, although both aerobic and anaerobic organisms are found in modern environments. () Posterior probability distribution of the inferred amino acids across 106 sites for the interested internal nodes. The inferred amino acid at each site for the interested internal node is the residue with the highest posterior probability. () Denaturation temperatures (Tm) versus geological time for ancestral Trx enzymes. Modern E. coli and human Trx enzymes are also indicated. The dashed line represents a lineal fit to the data. Inset, experimental DSC thermograms for E. coliTrx and LBCA Trx. The instrumental uncertainty of DSC measurements is <0.5 °C. * Figure 2: Single-molecule disulfide reduction assay. () Schematic representation of the single-molecule disulfide reduction assay. A first pulse of force rapidly unfolds the I27G32C–A75C domains (11-nm step). When the disulfide bond is exposed to the solvent a single Trx molecule can reduce it (14-nm step). () Experimental force-clamp trace showing single disulfide reductions of a (I27G32C–A75C)8 polyprotein. The unfolding pulse was set at 185 pN for 0.2 s and the test-pulse force at 500 pN. () Probability of reduction (Pred(t)) resulted from summing and normalizing the reduction test pulse at different forces for AECA Trx (3.5 μM). () Force dependency of disulfide reduction by AECA Trx; human TRX is also shown for comparison. Both Trx enzymes show a similar pattern: a negative force dependency of the reduction rate, from 30–200 pN, consistent with a Michaelis-Menten mechanism and a force-independent mechanism, from 200 pN and up, described by an electron transfer reaction16. Notice the higher activity for AECA Trx (3.5! μM for AECA Trx versus 10 μM for human TRX). The lines represent fittings to the kinetic model (see Online Methods). The error bars are given by the s.e.m. obtained using the bootstrap method. * Figure 3: Force dependence of disulfide reduction by ancestral Trx enzymes. (–) The reduction rate at a given force is obtained by summing, averaging and fitting to single exponential numerous traces (15–80) like the one shown in Figure 2b. The solid lines are fitting to the kinetic model. The gray circles and dashed lines represent the rate versus force dependence for modern Trxs: Pea Trxm from chloroplast (), P. falciparumTrx (), E. coliTrx (,) and human TRX (; all extracted from ref. 16). These modern Trxs are descendants of the ancestral Trxs in the same plot. The error bars represent the s.e.m. obtained using the bootstrap method. * Figure 4: Rate constants of disulfide bond reduction at pH 5. () High activity for AECA (squares) and LACA (circles) Trxs at pH 5 when the substrate was pulled at low forces (50–150 pN). LBCA Trx (triangles) showed similar activity to that at pH 7.2 with a similar trend (Fig. 3a). The solid lines are exponential fit to the experimental data. () Rate constants for disulfide reduction by ancestral Trx enzymes at pH 5 are higher than for modern E. coli and human Trx. Thioredoxin from the acidophile A. aceti shows activity at pH 5; enzymes from the thermophilic S. tokodaii do not show a detectable rate of reduction at the same pH. All experiments were conducted at a pulling force of 100 pN. Error bars represent s.e.m. obtained using the bootstrap method. () Activity of ancestral Trxs and modern E. coliTrx measured using DTNB as substrate at pH 5 and determined by monitoring spectrophotometrically the formation of TNB at 412 nm. Error bars represent s.d. from three different measurements. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biological Sciences, Columbia University, New York, New York, USA. * Raul Perez-Jimenez, * Jorge Alegre-Cebollada, * Pallav Kosuri, * Sergi Garcia-Manyes & * Julio M Fernandez * Facultad de Ciencias, Departamento de Química-Física, Universidad de Granada, Granada, Spain. * Alvaro Inglés-Prieto, * Inmaculada Sanchez-Romero & * Jose M Sanchez-Ruiz * Georgia Institute of Technology, School of Biology, Atlanta, Georgia, USA. * Zi-Ming Zhao & * Eric A Gaucher * Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA. * Pallav Kosuri * Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA. * T Joseph Kappock * Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan. * Masaru Tanokura * Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. * Arne Holmgren * Georgia Institute of Technology, School of Chemistry, Atlanta, Georgia, USA. * Eric A Gaucher * Georgia Institute of Technology, Parker H. Petit Institute for Bioengineering and Bioscience, Atlanta, Georgia, USA. * Eric A Gaucher Contributions R.P.-J., J.M.S.-R., E.A.G. and J.M.F. designed the research; Z.-M.Z. and E.A.G. conducted the phylogenetic analysis and sequence reconstruction; A.I.-P. and J.M.S.-R. expressed and purified the ancestral enzymes and conducted the calorimetric measurements and analysis; T.J.K. provided A. aceti Trx; M.T. provided S. tokodaii Trx; A.H. provided human TRX; R.P.-J., I.S.-R., J.A.-C., P.K. and S.G.-M. performed AFM experiments; R.P.-J. and I.S.-R. analyzed AFM data; R.P.-J., E.A.G. and J.M.F. wrote the paper; all authors participated in revising the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Raul Perez-Jimenez or * Julio M Fernandez Author Details * Raul Perez-Jimenez Contact Raul Perez-Jimenez Search for this author in: * NPG journals * PubMed * Google Scholar * Alvaro Inglés-Prieto Search for this author in: * NPG journals * PubMed * Google Scholar * Zi-Ming Zhao Search for this author in: * NPG journals * PubMed * Google Scholar * Inmaculada Sanchez-Romero Search for this author in: * NPG journals * PubMed * Google Scholar * Jorge Alegre-Cebollada Search for this author in: * NPG journals * PubMed * Google Scholar * Pallav Kosuri Search for this author in: * NPG journals * PubMed * Google Scholar * Sergi Garcia-Manyes Search for this author in: * NPG journals * PubMed * Google Scholar * T Joseph Kappock Search for this author in: * NPG journals * PubMed * Google Scholar * Masaru Tanokura Search for this author in: * NPG journals * PubMed * Google Scholar * Arne Holmgren Search for this author in: * NPG journals * PubMed * Google Scholar * Jose M Sanchez-Ruiz Search for this author in: * NPG journals * PubMed * Google Scholar * Eric A Gaucher Search for this author in: * NPG journals * PubMed * Google Scholar * Julio M Fernandez Contact Julio M Fernandez Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Note Additional data Entities in this article * Thioredoxin-1 trxA Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Thioredoxin Trx Plasmodium falciparum (isolate 3D7) * View in UniProt * View in Entrez Gene * Thioredoxin reductase trxB Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Thioredoxin Acetobacter aceti * View in UniProt * Thioredoxin M-type, chloroplastic Pisum sativum * View in UniProt * Thioredoxin TXN Homo sapiens * View in UniProt * View in Entrez Gene
• Three-dimensional structure of a viral genome-delivery portal vertex
  - Nat Struct Mol Biol 18(5):597-603 (2011)
  Nature Structural & Molecular Biology | Article Three-dimensional structure of a viral genome-delivery portal vertex * Adam S Olia1 * Peter E Prevelige Jr2 * John E Johnson3 * Gino Cingolani4 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:597–603Year published:(2011)DOI:doi:10.1038/nsmb.2023Received18 October 2010Accepted31 January 2011Published online17 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg DNA viruses such as bacteriophages and herpesviruses deliver their genome into and out of the capsid through large proteinaceous assemblies, known as portal proteins. Here, we report two snapshots of the dodecameric portal protein of bacteriophage P22. The 3.25-Å-resolution structure of the portal-protein core bound to 12 copies of gene product 4 (gp4) reveals a ~1.1-MDa assembly formed by 24 proteins. Unexpectedly, a lower-resolution structure of the full-length portal protein unveils the unique topology of the C-terminal domain, which forms a ~200-Å-long α-helical barrel. This domain inserts deeply into the virion and is highly conserved in the Podoviridae family. We propose that the barrel domain facilitates genome spooling onto the interior surface of the capsid during genome packaging and, in analogy to a rifle barrel, increases the accuracy of genome ejection into the host cell. View full text Figures at a glance * Figure 1: Crystal structure of the bacteriophage P22 portal protein and the portal-protein core–gp4 complex. (,) Ribbon diagram of the portal-protein core bound to 12 copies of gp4 in a side () and bottom () view. The portal protein is colored in blue, red and yellow, whereas gp4 is in green. (,) The side () and top () views of the full-length portal protein, which includes the barrel domain (residues 603–725) color-coded as in , (see also Supplementary Figs. 1, 2 and 4). * Figure 2: Fitting of P22 portal protein and gp4 X-ray models into the cryo-EM reconstructions of the mature virion and isolated portal vertex structure. () The 17-Å asymmetric reconstruction of bacteriophage P22 mature virion (EMD-1220)1 with capsid and tail colored in gray and purple, respectively. () Enlarged view of the five-fold icosahedral axis occupied by the portal protein, with X-ray models of portal protein (red), gp4 (green) and tail spike31, 32 (purple) fit into the EM density. The density for the barrel domain is partially continuous in the EM model and fades toward the C terminus of the barrel domain. () The 9.4-Å symmetrized cryo-EM reconstruction of the P22 portal vertex structure7 (EMD-5051), with the X-ray models of portal-protein core (red), gp4 (green), tail spike31, 32 (purple) and tail needlegp26 (refs. 10,11; blue) fit into the EM density. () Enlarged view of the portal-protein core–gp4 complex showing the excellent fit of the X-ray models inside the cryo-EM density. * Figure 3: The portal-protein fold. () Domain organization of the P22 portal protomer. (–) Ribbon diagrams of the portal-protein monomers from the bacteriophages P22 (), SPP1 ()23 and phi29 ()17, 20, colored according to similarity to the P22 portal protein in . The proteins have molecular masses of ~83 kDa, ~55 kDa and ~33 kDa, respectively. Despite the overall similarity in fold, the sequence similarity is <20%. (,) Monomer organization in the portal dodecamer. Ribbons diagram of the full-length portal protein, in top () and side () view, with only 1 protomer colored according to domain as in Figures 1 and 2, and the other 11 shown in gray. Each monomer of portal is nearly vertical in the hip domain (blue), but has an apparent tilt of ~30° relative to the 12-fold axis in both the leg domain (red) and the barrel domain (yellow). Res, residue. * Figure 4: Three-dimensional structure of the middle ring factor gp4. () Ribbon diagram of monomeric gp4. () Surface representation of portal-protein core (gray) in complex with 12 copies of gp4, shown as a green ribbon. () Enlarged view of the portal-protein core–gp4 binding interface. Portal protein and gp4 are shown as coils in gray and green, respectively. A semitransparent surface for neighboring gp4 protomers is highlighted in different tones of green. () Oligomeric conformation of gp4 extracted from , with 1 protomer of gp4 colored in green and the other 11 in gray (see also Supplementary Fig. 6). * Figure 5: Structural conservation of the middle ring factor gp4. (–) Ribbon diagrams of P22–gp4 (), SPP1–gp15 (, ref. 37) and HK97–gp6 (, ref. 38). () Superimposition of all three middle ring factors reveals a common, shared protein fold. * Figure 6: Architecture of the DNA-translocating channel. () Side view surface representation of P22 portal protein (gray) with a ~100-nucleotide (nt)-long dsB-DNA (orange) modeled inside the DNA-translocating channel. Four portal-protein protomers have been omitted from the structure to visualize the interior of the DNA-translocating channel. () Tilted view of the model in , revealing an opposite twist between the left-handed portal helices and the right-handed B-DNA helix. () Bottom view, showing the lack of obvious contacts between the portal-protein leg domains and the modeled DNA. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 3LJ4 * 3LJ5 * 3LJ4 * 3LJ5 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA. * Adam S Olia * Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA. * Peter E Prevelige Jr * Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA. * John E Johnson * Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA. * Gino Cingolani Contributions A.S.O. and G.C. crystallized the full-length portal and portal-protein core–gp4 complex, collected the X-ray data and determined the structures. P.E.P. isolated the gene encoding P22 portal protein and helped with data analysis. J.E.J. supervised the crystallization and data collection of full-length portal protein and helped with data analysis. G.C. coordinated the overall project and wrote the manuscript with A.S.O. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Gino Cingolani Author Details * Adam S Olia Search for this author in: * NPG journals * PubMed * Google Scholar * Peter E Prevelige Jr Search for this author in: * NPG journals * PubMed * Google Scholar * John E Johnson Search for this author in: * NPG journals * PubMed * Google Scholar * Gino Cingolani Contact Gino Cingolani Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–6 Additional data Entities in this article * Portal protein 1 Enterobacteria phage P22 * View in UniProt * View in Entrez Gene * Outer membrane protein tolC tolC Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Portal protein SPP1p010 Bacillus phage SPP1 * View in UniProt * View in Entrez Gene * Gp6 HK97p06 Enterobacteria phage HK97 * View in UniProt * View in Entrez Gene * Histone deacetylase 4 HDAC4 Homo sapiens * View in UniProt * View in Entrez Gene * Bifunctional tail protein 9 Enterobacteria phage P22 * View in UniProt * View in Entrez Gene * Packaged DNA stabilization protein gp4 4 Enterobacteria phage P22 * View in UniProt * View in Entrez Gene * Packaged DNA stabilization protein gp10 10 Enterobacteria phage P22 * View in UniProt * View in Entrez Gene * Tail needle protein gp26 26 Enterobacteria phage P22 * View in UniProt * View in Entrez Gene
• Structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase ERAP1
  - Nat Struct Mol Biol 18(5):604-613 (2011)
  Nature Structural & Molecular Biology | Article Structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase ERAP1 * Tina T Nguyen1 * Shih-Chung Chang2, 6 * Irini Evnouchidou3 * Ian A York4, 6 * Christos Zikos3 * Kenneth L Rock5 * Alfred L Goldberg2 * Efstratios Stratikos3 * Lawrence J Stern1, 5 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:604–613Year published:(2011)DOI:doi:10.1038/nsmb.2021Received11 August 2010Accepted26 January 2011Published online10 April 2011Corrected online24 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg ERAP1 trims antigen precursors to fit into MHC class I proteins. To fulfill this function, ERAP1 has unique substrate preferences, trimming long peptides but sparing shorter ones. To identify the structural basis for ERAP1's unusual properties, we determined the X-ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme's catalytic center can accommodate long peptides and has features that explain ERAP1's broad specificity for antigenic peptide precursors. Structural and biochemical analyses suggest a mechanism for ERAP1's length-dependent trimming activity, whereby binding of long rather than short substrates induces a conformational change with reorientation of a key catalytic residue toward the active site. ERAP1's unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antig! enic precursors. View full text Figures at a glance * Figure 1: ERAP1 structure and domain arrangement. () Overall shape of ERAP1 represented as a ribbon diagram and colored according to domains. Dotted lines represent disordered loops. () Structures of M1 aminopeptidases family members and thermolysin, colored by domain: Bacillus thermoproteolyticusthermolysin (PDB 3FV4), LTA4H (PDB 3FUH), ColAP (PDB 3CIA), ePepN (PDB 2HPT), PfAM1 (PDB 3EBG) and TIFF3 (PDB 1Z5H). () Schematic diagram of C-terminal domain of ERAP1 with helices represented as cylinders showing the ARM- or HEAT-motif spiral forming a large cavity lined by the even-numbered helices. () Large cavity formed by the catalytic domain and C-terminal domain shown in a surface representation. Inset shows how the N-terminal domain was removed above the dashed black line to provide a clear view of the cavity. Orange dashed lines represent estimated distances across the cavity. * Figure 2: ERAP1 active site. () Structural elements forming the Loops and helices, shown as cylinders, are colored as in Figure 1a. Bestatin is shown with cyan bonds, zinc as a black sphere. ERAP1 residues in the active site are shown with white bonds. Inset at right shows the entire protein for orientation; in the view shown, helices 19 and 21 that occlude the active site were removed for clarity. () ERAP1 interactions with bound aminopeptidase inhibitor bestatin. Side chains of conserved catalytic residues of ERAP1 are shown with carbon atoms colored by domain. Black arc indicates S1 site. () Conservation of catalytic residues among M1-family aminopeptidase structures, shown as an overlay of the active sites of LTA4H (cyan), COLAP (yellow), ePepN (pink), PfAM1 (purple) and ERAP1 (green). ERAP1 has an altered orientation of Tyr438 relative to the other aminopeptidases (arrows). Residue numbers correspond to ERAP1. () ERAP1 enzymatic activity is dependent on the side chain hydroxyl of Tyr438. L-AMC hydr! olysis activity of wild-type and Y438F ERAP1. Error bars show s.d. of three measurements. () The substrate binding cavity extends from the active site into domain IV. Surface representation of ERAP1 near the active site, with the catalytic zinc shown as a dark gray sphere and bestatin shown with cyan-colored bonds. Expected positions of peptide side chain binding pockets S1 and S1′ are indicated. Arrows represent possible paths of peptides extending from the active site into the cavity. () Electrostatic surface map of ERAP1. Surface colored from red to blue represents negatively charged to positively charged regions. Dotted lines represent indicated distances from zinc ion (gray sphere). Bestatin is represented with cyan-colored bonds. * Figure 3: Open and closed conformations of ERAP1. (–) ERAP1–bestatin in an open conformation as reported here (PDB 3MDJ). (–) ERAP1 in closed conformation as reported in a recent crystal structure (PDB 2XDT). Panels and contain ribbon diagrams, aligned as in Figure 1b, showing domain reorientation. Panels and provide a view of the active site, with zinc atom shown in gray, model tripeptide substrate shown with white bonds and Tyr438 with yellow bonds. ERAP1 helices are shown as cylinders and colored according to domain. Motion of helices H18–H22, reorientation of H5 and folding of an additional region H5′ N-terminal to H5 are apparent in the closed conformation. Panels and show a cutaway view of peptide-binding cavity, with surface of cavity colored according to domain, model peptide substrate with white bonds and position of zinc atom outlined in yellow. Although the cavity is isolated from solvent in the closed conformation, its length is not substantially changed. Panels and give a close-up view of the S1 site,! looking from the cavity toward toward the S1 site. A phenylalanine side chain has been modeled at the P1 position. * Figure 4: Length-dependent peptide cleavage and allosteric activation by ERAP1. () ERAP1 processing of a series of truncated and extended SIINFEKL variants. () ERAP1 processing of a series of LGnL peptides. () Activation of L-AMC hydrolysis by the same SIINFEKL length variants as in . L-AMC hydrolysis in the absence of added peptide is indicated by the open bar and the dotted line. () Activation of L-AMC hydrolysis by LGnL peptides. () ERAP1 processing of LGnL peptides as in panel but in the presence of 50 μM INFEKL peptide. The change in processing by ERAP1 is the ratio of processing with the presence of INFEKL over the processing by ERAP1 without INFEKL. Standard deviations of three separate experiments performed in parallel are indicated by error bars. * Figure 5: Kinetic analysis. () Initial rate of removal of the N-terminal residue of an internally quenched WRVYEKCdnpALK peptide as a function of peptide concentration. Solid line shows best fit to an equation describing simple Michaelis-Menten behavior with Km = 14.8 ± 1.7 μM and Vmax = 62.3 ± 2.9 μmol min−1 nmol−1. () Initial rate of hydrolysis of the fluorogenic L-AMC substrate as a function of concentration. Lines show best fit to simple Michaelis-Menten (dashed line) and allosteric activation (solid line) equations, which include a correction for inner filter effects and reduced apparent activity at high concentrations (see Online Methods for details). () Initial rate of hydrolysis of colorimetric L-pNA substrate as a function of concentration shows sigmoidal kinetics behavior. Lines show best fit to simple Michaelis-Menten (dashed line) and allosteric activation (solid line) equations. () Dynamic light-scattering analysis of ERAP1 in PBS buffer. Hydrodynamic radius estimated for a 105-kDa! protein using a spherical model is 4.35 nm. () Gel-filtration analysis shows enzymatically active monomeric population. Top, elution profile for ERAP1 in PBS followed using absorbance at 280 nm in absorbance units (AU), with elution position of molecular-weight standards shown above plot. Bottom, fractions were tested for L-AMC hydrolysis as a measurement of ERAP1 activity. () L-AMC hydrolysis is linearly dependent on ERAP1 concentration. Standard deviations of 3 trials are indicated by error bars. * Figure 6: Non-hydrolyzable peptide activates L-AMC hydrolysis but inhibits full-length peptide hydrolysis. () An optimized ERAP1 substrate, peptide L (LVAFKARAF), is processed efficiently by ERAP1. Reverse-phase chromatograms of peptides incubated in the presence or absence of ERAP1 are shown. Arrow indicates processed product VAFKARAF. () N-methylation of the first amide bond in LMe peptide prevents degradation by ERAP1. () ERAP1 hydrolysis of L-AMC is activated by increasing concentrations of peptide L. Line shows fit to an equation describing simple hyperbolic activation. () ERAP1 hydrolysis of L-AMC is activated by increasing concentrations of LMe, fit as in . () Concentration dependence of initial rate of L-AMC hydrolysis by ERAP1 in the absence (filled black symbols) or presence (open red symbols) of 50 μM LMe. Lines show fits to allosteric activation equation as in Figure 5b. () ERAP1 hydrolysis of a fluorescent peptide substrate is inhibited by increasing concentrations of LMe. Line shows fit to a competitive binding equation. Standard deviations of three trials are indi! cated by error bars. * Figure 7: Model for ERAP1 length-dependent cleavage activity. () A short peptide (pentamer shown) cannot reach from the catalytic site to the regulatory site. ERAP1 remains in the lower-activity open conformation and the peptide is processed inefficiently. () A long peptide (nonamer shown) can reach the regulatory site. ERAP1 adopts the higher-activity closed conformation and the peptide is processed efficiently. () A small fluorogenic substrate (L-AMC shown) cannot reach from the catalytic site to the regulatory site and is processed inefficiently. () A peptide (heptamer shown) can bind to the regulatory site together with a smaller substrate (L-AMC shown), leading to conversion to the closed conformation and increased aminopeptidase activity. * Figure 8: Ankylosing spondylitis–associated mutations mapped on the surface of ERAP1. Surface of ERAP1 in gray, with polymorphisms associated with ankylosing spondylitis shown in red. Underlined residues are hidden in this view. Accession codes * Abstract * Accession codes * Change history * Author information * Supplementary information Primary accessions Referenced accessions Protein Data Bank * 3MDJ * 2XDT * 3FUH * 2HPT * 3B7T * 2ZXG * 1HS6 * 1Z5H * 3FV4 * 3CIA * 3EBG * 3MDJ * 2XDT * 3FUH * 2HPT * 3B7T * 2ZXG * 1HS6 * 1Z5H * 3FV4 * 3CIA * 3EBG Change history * Abstract * Accession codes * Change history * Author information * Supplementary informationErratum 24 April 2011In the version of this article initially published online, nucleotide should have read peptide in a number of places. The error has been corrected for all versions of this article. Author information * Abstract * Accession codes * Change history * Author information * Supplementary information Affiliations * Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Tina T Nguyen & * Lawrence J Stern * Department of Cell Biology, Harvard Medical School, Boston, Massachusetts. * Shih-Chung Chang & * Alfred L Goldberg * National Centre for Scientific Research 'Demokritos', Aghia Paraskevi, Greece. * Irini Evnouchidou, * Christos Zikos & * Efstratios Stratikos * Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA. * Ian A York * Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Kenneth L Rock & * Lawrence J Stern * Present addresses: Institute of Microbiology and Biochemistry, Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan. (S.C.C.); Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA. (I.A.Y.). * Shih-Chung Chang & * Ian A York Contributions T.T.N. determined the crystal structure, designed and conducted enzymatic studies, interpreted results and helped write the paper, S.-C.C. expressed ERAP1 in insect cells and designed and conducted peptide trimming and allosteric activation experiments, I.E. designed and conducted peptide trimming and allosteric activation and inhibition experiments and interpreted results, I.A.Y. interpreted data and helped write the paper, C.Z. synthesized the LMe peptide, K.L.R. and A.L.G. designed experiments, interpreted data and helped write the paper, and E.S. and L.J.S. designed and conducted experiments, interpreted data and helped write the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Lawrence J Stern Author Details * Tina T Nguyen Search for this author in: * NPG journals * PubMed * Google Scholar * Shih-Chung Chang Search for this author in: * NPG journals * PubMed * Google Scholar * Irini Evnouchidou Search for this author in: * NPG journals * PubMed * Google Scholar * Ian A York Search for this author in: * NPG journals * PubMed * Google Scholar * Christos Zikos Search for this author in: * NPG journals * PubMed * Google Scholar * Kenneth L Rock Search for this author in: * NPG journals * PubMed * Google Scholar * Alfred L Goldberg Search for this author in: * NPG journals * PubMed * Google Scholar * Efstratios Stratikos Search for this author in: * NPG journals * PubMed * Google Scholar * Lawrence J Stern Contact Lawrence J Stern Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Change history * Author information * Supplementary information Movies * Supplementary Movie 1 (2M) Normal mode analysis PDF files * Supplementary Text and Figures (10M) Supplementary Figures 1–4 and Supplementary Tables 1 and 2 Additional data Entities in this article * Leukotriene A-4 hydrolase LTA4H Homo sapiens * View in UniProt * View in Entrez Gene * Thermolysin Bacillus thermoproteolyticus * View in UniProt * Endoplasmic reticulum aminopeptidase 2 ERAP2 Homo sapiens * View in UniProt * View in Entrez Gene * Tricorn protease-interacting factor F3 Ta0815 Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165) * View in UniProt * View in Entrez Gene * Endoplasmic reticulum aminopeptidase 1 Erap1 Mus musculus * View in UniProt * View in Entrez Gene * Interferon gamma IFNG Homo sapiens * View in UniProt * View in Entrez Gene * Antigen peptide transporter 2 TAP2 Homo sapiens * View in UniProt * View in Entrez Gene * Antigen peptide transporter 1 TAP1 Homo sapiens * View in UniProt * View in Entrez Gene * M1 family aminopeptidase Plasmodium falciparum (isolate FcB1 / Columbia) * View in UniProt * Cold-active aminopeptidase CPS_3470 Colwellia psychrerythraea (strain 34H / ATCC BAA-681) * View in UniProt * View in Entrez Gene * Aminopeptidase N pepN Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Endoplasmic reticulum aminopeptidase 1 ERAP1 Homo sapiens * View in UniProt * View in Entrez Gene * Astacin Astacus fluviatilis * View in UniProt * Peptidyl-dipeptidase dcp dcp Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Angiotensin-converting enzyme 2 ACE2 Homo sapiens * View in UniProt * View in Entrez Gene * Ribonuclease pancreatic RNASE1 Bos taurus * View in UniProt * View in Entrez Gene * HLA class I histocompatibility antigen, B-27 alpha chain HLA-B Homo sapiens * View in UniProt * View in Entrez Gene * Insulin-degrading enzyme IDE Homo sapiens * View in UniProt * View in Entrez Gene
• Cryo-EM structure of the ribosome–SecYE complex in the membrane environment
  - Nat Struct Mol Biol 18(5):614-621 (2011)
  Nature Structural & Molecular Biology | Article Cryo-EM structure of the ribosome–SecYE complex in the membrane environment * Jens Frauenfeld1, 2 * James Gumbart3 * Eli O van der Sluis1, 2 * Soledad Funes4 * Marco Gartmann1, 2 * Birgitta Beatrix1, 2 * Thorsten Mielke5 * Otto Berninghausen1, 2 * Thomas Becker1, 2 * Klaus Schulten3 * Roland Beckmann1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:614–621Year published:(2011)DOI:doi:10.1038/nsmb.2026Received21 September 2010Accepted03 February 2011Published online17 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The ubiquitous SecY–Sec61 complex translocates nascent secretory proteins across cellular membranes and integrates membrane proteins into lipid bilayers. Several structures of mostly detergent-solubilized Sec complexes have been reported. Here we present a single-particle cryo-EM structure of the SecYEG complex in a membrane environment, bound to a translating ribosome, at subnanometer resolution. Using the SecYEG complex reconstituted in a so-called Nanodisc, we could trace the nascent polypeptide chain from the peptidyltransferase center into the membrane. The reconstruction allowed for the identification of ribosome–lipid interactions. The rRNA helix 59 (H59) directly contacts the lipid surface and appears to modulate the membrane in immediate vicinity to the proposed lateral gate of the protein-conducting channel (PCC). On the basis of our map and molecular dynamics simulations, we present a model of a signal anchor–gated PCC in the membrane. View full text Figures at a glance * Figure 1: Reconstitution and cryo-EM reconstruction of a 70S RNC–Nd–SecYEG complex. () Binding assay using purified RNCs (RNC) with an excess of reconstituted Nd–E and Nd–SecYEG (Nd-Sec). Supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE and SYPRO Orange staining. Nd–SecYEG binds stably to RNCs, whereas Nd–E does not. () Cryo-EM reconstruction of the active 70S–RNC–Nd–SecYEG complex at 7.1-Å resolution. The ribosomal 30S subunit is shown in yellow, the 50S subunit in blue, SecY in orange, SecE in purple and the Nanodisc in white. () Density as in , but cut perpendicularly to the plane of the membrane along the polypeptide exit tunnel; colors as in with P-site tRNA, signal anchor (SA) and nascent polypeptide chain (NC) in green. () All-atom model of the active 70S–RNC–Nd–SecYEG complex. View and colors as in , proteins and RNA in ribbon representation, and phospholipids in ball-and-stick representation with phospholipid head groups (LH) in red-orange, acyl chains in white and apo-A1 in light purple. * Figure 2: Structure of the Nanodisc. () Left, side view cut perpendicularly to the plane of the membrane of the isolated electron density of the Nanodisc–SecYEG complex (Nd–SecYEG), showing the lateral gate of SecY. The electron density is represented as a transparent gray mesh with the ribbon representation of the fitted model of a SecY (orange), SecE (purple) and the signal anchor (SA) sequence (green). Two layers of density are visible (upper membrane interface, UMI, and lower membrane interface, LMI), separated by a region of lower density (hydrophobic core, HC) containing transmembrane (TM) helices. Dimensions are indicated. Right, same view, with the fitted Nanodisc model containing lipids in ball-and-stick representation. Phospholipid head groups (LH) are in red (oxygen) and orange (phosphate), acyl chains in white (AC, carbon-hydrogen groups). () Left, horizontal section, sliced within the plane of the membrane within the hydrophobic core of the lipid bilayer. Rod-like features are visible in the in! terior of the Nanodisc and account for density of a monomeric SecYEG complex. Right, horizontal section with fitted lipids. * Figure 3: Structure and connections of the membrane-embedded open SecYEG–RNC complex. () Cryo-EM reconstruction of the 70S–RNC–Nd–SecYEG complex; colors and abbreviations as in Figure 1. Insert, molecular model of the 50S subunit with electron density (left) and molecular model for SecYE (right). NC, nascent chain; SA, signal anchor; TM, transmembrane. () Model of the open SecYE complex with SA (green) residing within the lateral gate, view cut perpendicular to the plane of the membrane. Numbers denote transmembrane domains. () Close-up of the interaction area of universal ribosomal adaptor site and SecYE. () Molecular model of the ribosome–SecYE–membrane interface with transparent density filtered at 9–10 Å. Lipid head groups (LH) are indicated. () Cytoplasmic view of the molecular model of the Nd–SecYE complex with contacts to the 50S subunit indicated by circles. () View of the ribosomal tunnel exit site; contact sites as in . * Figure 4: Path of the nascent chain and signal anchor. () Section through molecular models of the ribosomal exit tunnel and Nd–SecYE. The nascent chain (NC) with the signal anchor (SA) is shown in green. The dotted line indicates the cytoplasm–membrane interface. () Conformational changes of L23. Comparison of the model of L23 (gray) of an inactive ribosome (PDB: 2i2v) and of L23 (pink) in the presence of a nascent chain (NC; green), SecY (orange), SecE (purple) and lipid head groups. The intra-tunnel loop of L23 bends toward the nascent chain, close to L6/7 of SecY. () Conformational change of the β-hairpin loop of L24. Comparison of the model of L24 (gray) of an inactive ribosome (PDB: 2i2v) and of L24 (green) in the presence of a nascent chain (green), SecY (orange), SecY C terminus (C-term), SecE (purple) and lipid head groups (LH). () View of the lateral gate of SecYE shown as a surface representation. SecY is shown in orange, SecE in purple and the nascent chain in green. Conserved residues of SecY that contribute to ! the central hydrophobic pore ring are indicated in red, and hydrophobic residues of the hydrophobic crevasse that have been found by mutational analysis to be critical for SecY function5 are indicated in pale yellow. () View of the position of the SA from the cytoplasmic side, sliced within the plane of the membrane. Hydrophobic pore ring residues are indicated in red. * Figure 5: Molecular dynamics simulation and membrane insertion. () Plot of surface area formed between lipids and ribosomal helix H59 during the MD simulation. () Surface representation of the Nd–SecYE complex seen from the ribosome after 16 ns MD simulation. Apo-A1 is shown in light purple, SecY in orange, SecE in purple and nascent chain in green, and the atoms of the lipid head groups are colored in orange (phosphate), red (oxygen) and blue (nitrogen), respectively. Note the accumulation of positive charges in the region close to H59 and the disorder of the lipids forming a groove juxtaposed to the signal anchor (SA). () Schematic depiction of the view in using the same color code and indicating the probable path of the nascent TM domain for integration into the bilayer. () Schematic depiction of the bacterial 50S ribosomal subunit (blue) bound to the SRP (red; 4.5S RNA and the N-terminal 54 NG domain) in the presence of a signal anchor sequence as observed before25. The nascent chain (NC) with the signal anchor (SA) is shown in gre! en. () Schematic depiction of a hypothetical TM domain insertion intermediate showing the bacterial 50S ribosomal subunit (blue) bound to the SecYEG complex (orange) in the presence of a signal anchor, accessing the hydrophobic lipid phase through a partially open lateral gate. () Schematic depiction of the observed insertion intermediate with the signal anchor TM domain fully inserted into the lateral gate and exposed to the hydrophobic core of the bilayer. Note the proximity of the SA position as observed in the targeting complex () and in the insertion intermediate (,). AC, acyl chains; LH, lipid head groups. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 3J00 * 3J01 * 3J00 * 3J01 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Gene Center, Department for Biochemistry, University of Munich, Munich, Germany. * Jens Frauenfeld, * Eli O van der Sluis, * Marco Gartmann, * Birgitta Beatrix, * Otto Berninghausen, * Thomas Becker & * Roland Beckmann * Munich Center for Integrated Protein Science (CIPSM), Department of Chemistry and Biochemistry, Munich, Germany. * Jens Frauenfeld, * Eli O van der Sluis, * Marco Gartmann, * Birgitta Beatrix, * Otto Berninghausen, * Thomas Becker & * Roland Beckmann * Department of Physics, Beckman Institute, University of Illinois at Urbana–Champaign, Urbana, Illinois, USA. * James Gumbart & * Klaus Schulten * Departamento de Genética Molecular, Instituto de Fisiología Celular, Circuito Exterior S/N, Ciudad Universitaria, Universidad Nacional Autónoma de México, Mexico City, Mexico. * Soledad Funes * Ultrastrukturnetzwerk, Max Planck Institute for Molecular Genetics, Institut für Medizinische Physik und Biophysik, Charite–Universitätsmedizin Berlin, Berlin, Germany. * Thorsten Mielke Contributions J.F. prepared the sample, collected the EM data, performed the 3D reconstruction and built the molecular model; J.G. did the MDFF and the MD simulations. E.O.v.d.S., S.F. and B.B. contributed to the purification of SecYEG; M.G. contributed to the data processing. T.M. and O.B. contributed to the EM data collection. T.B. contributed to model building and interpretation. All authors contributed to the study design and to writing the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Roland Beckmann Author Details * Jens Frauenfeld Search for this author in: * NPG journals * PubMed * Google Scholar * James Gumbart Search for this author in: * NPG journals * PubMed * Google Scholar * Eli O van der Sluis Search for this author in: * NPG journals * PubMed * Google Scholar * Soledad Funes Search for this author in: * NPG journals * PubMed * Google Scholar * Marco Gartmann Search for this author in: * NPG journals * PubMed * Google Scholar * Birgitta Beatrix Search for this author in: * NPG journals * PubMed * Google Scholar * Thorsten Mielke Search for this author in: * NPG journals * PubMed * Google Scholar * Otto Berninghausen Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Becker Search for this author in: * NPG journals * PubMed * Google Scholar * Klaus Schulten Search for this author in: * NPG journals * PubMed * Google Scholar * Roland Beckmann Contact Roland Beckmann Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information Movies * Supplementary Movie (30M) Molecular dynamics simulation PDF files * Supplementary Text and Figures (3M) Supplementary Methods, Supplementary Figures 1–12 and Supplementary Tables 1–7 Additional data Entities in this article * Cell division protein ftsQ ftsQ Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Apolipoprotein A-I APOA1 Homo sapiens * View in UniProt * View in Entrez Gene * 50S ribosomal protein L23 rplW Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Protein translocase subunit secA secA Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Protein transport protein Sec61 subunit gamma SEC61G Homo sapiens * View in UniProt * View in Entrez Gene * Preprotein translocase subunit SecE secE Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Protein transport protein Sec61 subunit beta SEC61B Homo sapiens * View in UniProt * View in Entrez Gene * Protein-export membrane protein SecG secG Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * 50S ribosomal protein L22 rplV Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * 50S ribosomal protein L29 rpmC Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * 50S ribosomal protein L24 rplX Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Preprotein translocase subunit SecY secY Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Signal recognition particle protein ffh Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Inner membrane protein oxaA yidC Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene
• A conserved 20S proteasome assembly factor requires a C-terminal HbYX motif for proteasomal precursor binding
  - Nat Struct Mol Biol 18(5):622-629 (2011)
  Nature Structural & Molecular Biology | Article A conserved 20S proteasome assembly factor requires a C-terminal HbYX motif for proteasomal precursor binding * Andrew R Kusmierczyk1, 3 * Mary J Kunjappu2 * Roger Y Kim1 * Mark Hochstrasser1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:622–629Year published:(2011)DOI:doi:10.1038/nsmb.2027Received11 June 2010Accepted07 February 2011Published online17 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Dedicated chaperones facilitate the assembly of the eukaryotic proteasome, but how they function remains largely unknown. Here we show that a yeast 20S proteasome assembly factor, Pba1–Pba2, requires a previously overlooked C-terminal hydrophobic-tyrosine-X (HbYX) motif for function. HbYX motifs in proteasome activators open the 20S proteasome entry pore, but Pba1–Pba2 instead binds inactive proteasomal precursors. We discovered an archaeal ortholog of this factor, here named PbaA, that also binds preferentially to proteasomal precursors in a HbYX motif–dependent fashion using the same proteasomal α-ring surface pockets as are bound by activators. PbaA and the related PbaB protein can be induced to bind mature 20S proteasomes if the active sites in the central chamber are occupied by inhibitors. Our data are consistent with an allosteric mechanism in which the maturation of the proteasome active sites determines the binding of assembly chaperones, potentially shieldin! g assembly intermediates or misassembled complexes from nonproductive associations until assembly is complete. View full text Figures at a glance * Figure 1: An HbYX motif in the yeast Pba1–Pba2 assembly factor and its archaeal orthologs. () Distribution of DUF75 superfamily members among the eukarya and archaea. In eukaryotes, Pba2 and PAC2 proteins belong to the DUF75 superfamily; PAC1 proteins are also related to this superfamily. All archaeal species encode at least two DUF75 proteins, which cluster into two conserved orthologous groups (COGs). Crenarchaeota contain two proteins from the same COG. () A C-terminal HbYX motif (underline) is conserved in eukaryotic Pba1 and PAC1 (top) as well as archaeal DUF75 superfamily members (middle, bottom). Hsap, Homo sapiens; Mmus, Mus musculus; Ggal, Gallus gallus; Xlae, Xenopus laevis; Drer, Danio rerio; Dmel, Drosophila melanogaster; Atha, Arabidopsis thaliana; Scer, Saccharomyces cerevisiae; Mmar, Methanococcus maripaludis; Mjan, Methanocaldococcus jannaschii; Aful, Archaeoglobus fulgidus; Taci, Thermoplasma acidophilum; Tkod, Themococcus kodakarensis; Aper, Aeropyrum pernix; Paer, Pyrobaculum aerophilum; Hbut, Hyperthermus butylicus; Ihos, Ignicoccus hospitalis;! Saci, Sulfolobus acidocaldarius. * Figure 2: The HbYX motifs of yeast Pba1–Pba2 are functionally important in vivo. (,) The indicated yeast strains were transformed with either an empty low-copy vector or vector with the indicated PBA genes. Six-fold dilution series of liquid cultures were spotted onto various media and incubated as indicated. Can, canavanine sulfate; WT, wild type. () The rpn4Δ pba1Δ pba2Δ triple mutant was transformed with a plasmid carrying the wild-type (PBA) or mutant (pba) alleles shown in . Lysates were prepared and analyzed by two immunoblots. The first was probed with anti-Flag (Pba1) antibody, and the membrane stained with Ponceau S to compare loading. The second was probed with anti-His (Pba2, arrowhead) antibody and reprobed with anti-Pgk1 to compare loading. Asterisk, nonspecific band. SD-Ura, synthetic dropout medium without uracil. * Figure 3: The HbYX motifs of yeast Pba1–Pba2 are necessary for proteasome precursor binding. () Native PAGE fractionation of yeast lysates. Samples for lanes 2–4 derive from pba1Δ pba2Δ cells transformed with plasmids that lack inserts (lane 2); carry Flag-tagged PBA1 and His-tagged PBA2 (lane 3); or bear tagged versions of HbYX-mutated alleles, pba1-Y275A and pba2-L265A (lane 4). The top two panels show fluorogenic substrate overlays performed as in Figure 4b; 0.02% SDS was added after the initial (top) picture was taken to visualize free 20S proteasomes (CP; middle panel). The lower panel is a native gel anti-Flag immunoblot. An HbYX-dependent intermediate species is indicated by the arrowhead. () Co-precipitation of the 20S proteasome assembly factor Ump1 with Flag-tagged Pba1 is HbYX dependent. Lanes 5–8 are Flag immunoprecipitations of lanes 1–4, respectively. Sizes at left are in kDa. * Figure 4: Archaeal PbaA binds preferentially to a 20S proteasome intermediate. () Archaeal PbaA and PbaB proteins form a complex. Lysates of E. coli expressing the indicated protein combinations were fractionated by Ni-NTA. Rightmost panel shows a single His-PbaA band in the eluate when PbaB is absent, indicating that the lower band in the left panel is not a proteolytic fragment of His-PbaA. L, load; F, flow-through; E, eluate; T, total lysate; S, soluble fraction (equivalent to L); P, pellet fraction; M, size standards (kDa). () Recombinant M. maripaludis wild-type 20S proteasomes (WT, α-His+βΔpro) and preholoproteasomes (PHP, α-His+β(T1A)) were affinity purified on a Ni-NTA resin and electrophoresed on a nondenaturing 4–10% gradient gel. Proteolytic activity was visualized using a Suc-LLVY-AMC fluorogenic substrate (right), and the gel was then stained with GelCode Blue (CBB; left). () PbaA is sufficient to bind PHP. Lysates of E. coli expressing either His-tagged wild-type archaeal proteasomes or PHP were mixed with lysates of E. coli expres! sing the indicated untagged Pba proteins. The mixtures were fractionated by Ni-NTA binding and native PAGE. Proteins were visualized with GelCode blue. Arrowhead denotes position of a 20S–Pba protein complex. Asterisk, nonassembled α-subunit species; bracket, free PbaA and PbaB. Free Pba proteins in the wild-type samples may have coeluted from Ni-NTA with the nonassembled α-subunits or could represent weakly bound species that did not survive native electrophoresis. The gel-shifted species (arrowhead) were excised and analyzed by LC-MS/MS, resulting in the identification of the indicated proteins (bottom). * Figure 5: PbaA HbYX motif mediates binding to the α-ring of 20S proteasome intermediates. () Deletion of the HbYX motif of archaeal PbaA (ΔC) abrogates binding to the PHP (β-T1A) intermediate. Proteins were analyzed as in Figure 4. ΔC denotes Pba protein lacking last three residues. () Mutation of proteasome α-subunit Lys68 (K68) to alanine (A68) prevents PHP binding to PbaA. () Archaeal Pba proteins do not stimulate mature (WT) 20S proteasome activity. Purified recombinant 20S archaeal proteasomes (α+βΔpro) were incubated with the indicated recombinant Pba proteins. Activity in the absence of Pba proteins was set to unity. Error bars represent s.e.m. () Archaeal Pba proteins stimulate peptidase activity of S2 proteasome intermediates (α+βHis+β). Asterisks denote statistically significant differences from the S2-only sample (**P = 0.003; *P = 0.049; n = 3). Activity of S2 in the absence of Pba proteins was set to unity. Error bars represent s.e.m. () PbaA causes gel shift of S1 (α+β(T1A)His+β) but not S2 (α+βHis+β). Lysates of E. coli expressing e! ither His-tagged S1 or S2 were mixed with lysates of E. coli expressing untagged PbaA. The mixtures were fractionated as in Figure 4c, and proteins were visualized with GelCode blue. Arrowheads, PbaA-shifted proteasomal species. * Figure 6: Binding of archaeal Pba proteins to inhibitor-treated mature 20S proteasomes. () Lysates of E. coli expressing wild-type (α-His+βΔpro) proteasomes were mixed with lysates of E. coli expressing untagged PbaA or with buffer. Where indicated, Z-Leu3-vinyl sulfone (VS) or clasto-lactacystin (LC) were added to 50 μM. For the control experiments in the lower panel, no PbaA was added. Samples were fractionated by native PAGE; gels were stained with GelCode blue. Arrowheads denote position of Pba-shifted 20S species. () E. coli lysates containing wild-type proteasomes and lysates with untagged full-length PbaA (WT) or PbaA lacking its last three residues (ΔHbYX) were mixed and analyzed as in . () Lysates of E. coli expressing WT proteasomes were mixed with lysates of E. coli expressing untagged full-length PbaB (WT) or PbaB lacking its last 3 residues (ΔC). Samples were processed as in except a 4–7% gradient gel was employed. () E. coli lysates with WT proteasomes and lysates with the indicated untagged archaeal Pba proteins were mixed and analyzed as! in . Where indicated, LC (50 μM) or MG-262 (5 μM) was added. For MG-262, the inhibitor was also added to all buffers during the Ni-NTA fractionation. * Figure 7: Binding of PbaA to PHP requires the α-subunit N termini. Lysates of E. coli expressing His-tagged PHPs (α+β(T1A)His) containing either wild-type α subunits (PHP) or mutant α subunits lacking their 13-residue N-terminal tails (PHP αΔN) were mixed with buffer (−) or with lysates of E. coli expressing untagged PbaA. The mixtures were fractionated by Ni-NTA and the eluates subjected to native PAGE on 4–10% gradient gels (left gel). Arrowhead, PbaA–PHP species. The input (I) and eluate (E) fractions from the Ni-NTA resin were also separated by 11% SDS-PAGE (right gel). Migration of the 25-kDa protein standard is indicated. For both gels, proteins were stained with GelCode blue. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA. * Andrew R Kusmierczyk, * Roger Y Kim & * Mark Hochstrasser * Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. * Mary J Kunjappu * Present address: Department of Biology, Indiana University–Purdue University Indianapolis, Indianapolis, Indiana, USA. * Andrew R Kusmierczyk Contributions A.R.K. and M.H. developed the experimental approach. A.R.K., M.J.K. and R.Y.K. carried out experiments. A.R.K. and M.H. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Mark Hochstrasser Author Details * Andrew R Kusmierczyk Search for this author in: * NPG journals * PubMed * Google Scholar * Mary J Kunjappu Search for this author in: * NPG journals * PubMed * Google Scholar * Roger Y Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Hochstrasser Contact Mark Hochstrasser Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (557K) Supplementary Figures 1–12, Supplementary Tables 1 and 2, Supplementary Notes and Supplementary Methods Additional data Entities in this article * Proteasome maturation factor UMP1 UMP1 Saccharomyces cerevisiae * View in UniProt * View in Entrez Gene * Proteasome activator BLM10 BLM10 Saccharomyces cerevisiae * View in UniProt * View in Entrez Gene * Proteasome assembly chaperone 2 PSMG2 Homo sapiens * View in UniProt * View in Entrez Gene * Proteasome assembly chaperone 1 PSMG1 Homo sapiens * View in UniProt * View in Entrez Gene * Conserved hypothetical archaeal protein MMP0914 Methanococcus maripaludis * View in UniProt * View in Entrez Gene * Proteasome assembly chaperone 2 ADD66 Saccharomyces cerevisiae * View in UniProt * View in Entrez Gene * Proteasome-activating nucleotidase psmR Methanococcus maripaludis * View in UniProt * View in Entrez Gene * Putative uncharacterized protein MMP1611 Methanococcus maripaludis * View in UniProt * View in Entrez Gene * Proteasome assembly chaperone 3 PSMG3 Homo sapiens * View in UniProt * View in Entrez Gene * Proteasome chaperone 4 POC4 Saccharomyces cerevisiae * View in UniProt * View in Entrez Gene * Proteasome chaperone 3 IRC25 Saccharomyces cerevisiae * View in UniProt * View in Entrez Gene * Proteasome chaperone 1 PBA1 Saccharomyces cerevisiae * View in UniProt * View in Entrez Gene * Proteasome subunit alpha psmA Methanococcus maripaludis * View in UniProt * View in Entrez Gene * Proteasome subunit beta psmB Methanococcus maripaludis * View in UniProt * View in Entrez Gene * Proteasome activator complex subunit 4 PSME4 Homo sapiens * View in UniProt * View in Entrez Gene * Proteasome assembly chaperone 4 PSMG4 Homo sapiens * View in UniProt * View in Entrez Gene * Proteasome inhibitor PI31 subunit PSMF1 Homo sapiens * View in UniProt * View in Entrez Gene * Protein RPN4 RPN4 Saccharomyces cerevisiae * View in UniProt * View in Entrez Gene * Proteasome component PUP2 PUP2 Saccharomyces cerevisiae * View in UniProt * View in Entrez Gene
• Structure of catalytically competent intein caught in a redox trap with functional and evolutionary implications
  - Nat Struct Mol Biol 18(5):630-633 (2011)
  Nature Structural & Molecular Biology | Brief Communication Structure of catalytically competent intein caught in a redox trap with functional and evolutionary implications * Brian P Callahan1 * Natalya I Topilina1 * Matthew J Stanger1 * Patrick Van Roey1 * Marlene Belfort1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:630–633Year published:(2011)DOI:doi:10.1038/nsmb.2041Received04 October 2010Accepted21 January 2011Published online03 April 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Here we describe self-splicing proteins, called inteins, that function as redox-responsive switches in bacteria. Redox regulation was achieved by engineering a disulfide bond between the intein's catalytic cysteine and a cysteine in the flanking 'extein' sequence. This interaction was validated by an X-ray structure, which includes a transient splice junction. A natural analog of the designed system was identified in Pyrococcus abyssi, suggesting an unprecedented form of adaptive, post-translational regulation. View full text Figures at a glance * Figure 1: Engineered redox-responsive intein precursor in vivo and in vitro. () Scheme for intein splicing. Conserved terminal intein residues are indicated, as is the extein C+1 residue. N-extein and C-extein residues are designated by − and +, respectively. () FRET-based intein reporter with the DnaE intein (red) inserted between CFP and YFP. () Biomimetic Cys−3 variant CPGCDnaE displays enhanced redox sensitivity in vivo. Activities of CPGCDnaE and APGCDnaE inteins were quantified as the percentage of precursor (C-Ia-Y) remaining at 7 h and 18 h after induction of protein expression in E. coli DHB4, AD494 and origami. Data are representative of ≥2 independent experiments. Additional bands, observed previously with unboiled samples, were excluded from the quantification9. () Effect of redox conditions on the in vitro cleavage kinetics of APGCDnaE and CPGCDnaE. N-extein cleavage was monitored by FRET loss that occurs upon cleavage by DTT or by hydroxylamine (HA). Each data point is an average of three measurements. * Figure 2: Crystal structure of the CPGCDnaE intein. () Ribbon diagram showing the intein (red) and the N-terminal extein (green). Magnified view of the Cys1-to-Cys–3 disulfide loop is shown in the box. () Conformation of the CPGC disulfide loop. Comparison of the structure of the disulfide loop in the CPGCDnaE intein (red) with the two other observations of this loop in the Protein Data Bank (1JBQ, green; 1M6Y, cyan). Magnified view shows glycine-cysteine peptide bonds, with an upward arrow pointing to the carbonyl oxygen atoms and a downward arrow pointing toward the amide nitrogen atoms. () Active-site interactions in the CPGCDnaE intein (intein, red; extein, green). Thr69 of the conserved TXXH motif contacts the amide nitrogen of Cys1 and the carbonyl oxygen of Pro−2, whereas His72, also of the TXXH motif, does not interact directly with the N-extein. Asp140 makes a water-mediated contact with the −1 carbonyl and a hydrogen bond with His147, also a conserved residue. Suc159 is the C-terminal succinimide. * Figure 3: Protein splicing by the P. abyssiMoaA intein is redox sensitive. () Schematic of CWYCMoaA precursor. Splicing of the MoaA intein (red) with native N-extein residues (circles) ligates the cyan fluorescent protein to a hexahistidine tag (His6). (,) Suppressed activity of the CWYCMoaA intein in the absence of a reducing agent. Intein autoprocessing was monitored in the presence and absence of the reducing agent TCEP at 22 °C (squares) and 55 °C (triangles). Precursor (CFPCWYCMoaA-His6) and splicing product (CFP-His6) were separated by nonreducing SDS-PAGE and detected by in-gel fluorescence (excitation 457 nm, emission 526 nm). Plot of precursor disappearance in , using data of , shows that TCEP is required for efficient CWYCMoaA processing at 22 °C and at 55 °C. Error bars, s.d.; n = 3. () CWYCMoaA shows redox regulation in E. coli. Representative gel images showing CFP-fused MoaA precursor with wild-type Cys-3 (CFPCWYCMoaA-His6), and CFP-products remaining after 3 h of induction in DHB4 (left lane), AD494 (middle lane) and origami (rig! ht lane). () Cys−3 is required for MoaA precursor accumulation in origami. Graph was derived from images as in and shows the percentage of CFPCWYCMoaA-His6 (C, light shaded) and CFPAWYCMoaA-His6 (A, dark shaded) precursors after expression in the indicated strains, averaged over three independent trials. Error bars, s.d. Accession codes * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3NZM * 3NZM Referenced accessions Protein Data Bank * 1QNO * 1QNO Author information * Accession codes * Author information * Supplementary information Affiliations * Wadsworth Center, New York State Department of Health, Albany, New York, USA. * Brian P Callahan, * Natalya I Topilina, * Matthew J Stanger, * Patrick Van Roey & * Marlene Belfort * Department of Biomedical Sciences, University at Albany, State University of New York, Albany, New York, USA. * Marlene Belfort Contributions B.P.C. conceived the study; B.P.C., N.I.T., P.V.R. and M.B. designed research; B.P.C., N.I.T., M.J.S. and P.V.R. performed research; B.P.C., P.V.R., N.I.T. and M.B. analyzed data; and B.P.C., P.V.R., N.I.T. and M.B. wrote the paper. Competing financial interests The authors filed a provisional patent entitled "A redox trap to control intein activity" with the USPTO on 18 January 2011 (application no. 61/433,730). Corresponding author Correspondence to: * Marlene Belfort Author Details * Brian P Callahan Search for this author in: * NPG journals * PubMed * Google Scholar * Natalya I Topilina Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew J Stanger Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick Van Roey Search for this author in: * NPG journals * PubMed * Google Scholar * Marlene Belfort Contact Marlene Belfort Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (188K) Supplementary Methods, Supplementary Figure 1 and Supplementary Table 1 Additional data Entities in this article * Pyruvate-formate lyase-activating enzyme uncultured archaeon GZfos13E1 * View in UniProt * DNA polymerase III subunit alpha dnaE Synechocystis sp. (strain ATCC 27184 / PCC 6803 / N-1) * View in UniProt * View in Entrez Gene * Ribosomal RNA small subunit methyltransferase H mraW Thermotoga maritima * View in UniProt * View in Entrez Gene * Cystathionine beta-synthase CBS Homo sapiens * View in UniProt * View in Entrez Gene * Beta-mannase Trichoderma reesei * View in UniProt * Probable molybdenum cofactor biosynthesis protein A moaA Pyrococcus abyssi * View in UniProt * View in Entrez Gene * Glutathione reductase gor Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Thioredoxin reductase trxB Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * V-type proton ATPase catalytic subunit A TFP1 Saccharomyces cerevisiae * View in UniProt * View in Entrez Gene * DNA gyrase subunit A Mycobacterium xenopi * View in UniProt