Hot off the presses! Apr 24 J Mol Biol

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Latest Articles Include:

• Editorial Board
  - J Mol Biol 388(1):i (2009)
  
• Contents
  - J Mol Biol 388(1):iv-v (2009)
  
• Solution and Crystal Structures of mRNA Exporter Dbp5p and Its Interaction with Nucleotides
  - J Mol Biol 388(1):1-10 (2009)
  DEAD-box protein 5 (Dbp5p) plays very important roles in RNA metabolism from transcription, to translation, to RNA decay. It is an RNA helicase and functions as an essential RNA export factor from nucleus. Here, we report the solution NMR structures of the N- and C-terminal domains (NTD and CTD, respectively) of Dbp5p from Saccharomyces cerevisiae (ScDbp5p) and X-ray crystal structure of Dbp5p from Schizosaccharomyces pombe (SpDbp5p) in the absence of nucleotides and RNA. The crystal structure clearly shows that SpDbp5p comprises two RecA-like domains that do not interact with each other. NMR results show that the N-terminal flanking region of ScDpbp5 (M1-E70) is intrinsically unstructured and the region Y71–R121 including the Q motif is highly dynamic on millisecond–microsecond timescales in solution. The C-terminal flanking region of ScDbp5p forms a short β-strand and a long helix. This helix is unique for ScDbp5p and has not been observed in other DEAD-box prot! eins. Compared with other DEAD-box proteins, Dbp5p has an extra insert with six residues in the CTD. NMR structure reveals that the insert is located in a solvent-exposed loop capable of interacting with other proteins. ATP and ADP titration experiments show that both ADP and ATP bind to the consensus binding site in the NTD of ScDbp5p but do not interact with the CTD at all. Binding of ATP or ADP to NTD induces significant conformational rearrangement too.
• The 2.1-Å Crystal Structure of Native Neuroserpin Reveals Unique Structural Elements That Contribute to Conformational Instability
  - J Mol Biol 388(1):11-20 (2009)
  Neuroserpin is a selective inhibitor of tissue-type plasminogen activator (tPA) that plays an important role in neuronal plasticity, memory, and learning. We report here the crystal structure of native human neuroserpin at 2.1 Å resolution. The structure has a helical reactive center loop and an omega loop between strands 1B and 2B. The omega loop contributes to the inhibition of tPA, as deletion of this motif reduced the association rate constant with tPA by threefold but had no effect on the kinetics of interaction with urokinase. Point mutations in neuroserpin cause the formation of ordered intracellular polymers that underlie dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type neuroserpin is also unstable and readily forms polymers under near-physiological conditions in vitro. This is, in part, due to the substitution of a conserved alanine for serine at position 340. The replacement of Ser340 by Ala increased the melting temperat! ure by 3 °C and reduced polymerization as compared to wild-type neuroserpin. Similarly, neuroserpin has Asn-Leu-Val at the end of helix F and thus differs markedly from the Gly-X-Ile consensus sequence of the serpins. Restoration of these amino acids to the consensus sequence increased thermal stability and reduced the polymerization of neuroserpin and its transition to the latent conformer. Moreover, introduction of the consensus sequence into S49P neuroserpin that causes FENIB increased the stability and inhibitory activity of the mutant, as well as blocked polymerization and increased the yield of protein during refolding. These data provide a molecular explanation for the inherent instability of neuroserpin and the effect of point mutations that underlie the dementia FENIB.
• A Deoxyribozyme, Sero1C, Uses Light and Serotonin to Repair Diverse Pyrimidine Dimers in DNA
  - J Mol Biol 388(1):21-29 (2009)
  An in vitro selection search for DNAs capable of catalyzing photochemistry yielded two distinctive deoxyribozymes (DNAzymes) with photolyase activity: UV1C, which repaired thymine dimers within DNA using a UV light of > 300 nm wavelength and no extraneous cofactor, and Sero1C, which required the tryptophan metabolite serotonin as cofactor in addition to the UV light. Catalysis by Sero1C conformed to Michaelis–Menten kinetics, and analysis of the action spectrum of Sero1C confirmed that serotonin did indeed serve as a catalytic cofactor rather than as a structural cofactor. Sero1C and UV1C showed strikingly distinct wavelength optima for their respective photoreactivation catalyses. Although the rate enhancements characteristic of the two DNAzymes were similar, the cofactor-requiring Sero1C repaired a substantially broader range of substrates compared to UV1C, including thymine, uracil, and a range of chimeric deoxypyrimidine and ribopyrimidine dimers. Similarities an! d differences in the properties of these two photolyase DNAzymes suggest, first, that the harnessing of less damaging UV light for the repair of photolesions may have been a primordial catalytic activity of nucleic acids, and, second, the broader substrate range of Sero1C may highlight an evolutionary advantage to coopting amino-acid-like cofactors by functionality-poor nucleic acid enzymes.
• Mechanism Analysis Indicates that Recombination Events in HIV-1 Initiate and Complete Over Short Distances, Explaining Why Recombination Frequencies Are Similar in Different Sections of the Genome
  - J Mol Biol 388(1):30-47 (2009)
  Strand transfer drives recombination between the co-packaged genomes of HIV-1, a process that allows rapid viral evolution. The proposed invasion-mediated mechanism of strand transfer during HIV-1 reverse transcription has three steps: (1) invasion of the initial or donor primer template by the second or acceptor template; (2) propagation of the primer–acceptor hybrid; and (3) primer terminus transfer. Invasion occurs at a site at which the reverse transcriptase ribonuclease H (RNase H) has created a nick or short gap in the donor template. We used biochemical reconstitution to determine the distance over which a single invasion site can promote transfer. The DNA-primed RNA donor template used had a single-stranded pre-created invasion site (PCIS). Results showed that the PCIS could influence transfer by 20 or more nucleotides in the direction of synthesis. This influence was augmented by viral nucleocapsid protein and additional reverse transcriptase-RNase H cleavag! e. Strand-exchange assays were performed specifically to assess the distance over which a hybrid interaction initiated at the PCIS could propagate to achieve transfer. Propagation by simple branch migration of strands was limited to 24–32 nt. Additional RNase H cuts in the donor RNA allowed propagation to a maximum distance of 32–64 nt. Overall, results indicate that a specific invasion site has a limited range of influence on strand transfer. Evidently, a series of invasion sites cannot collaborate over a long distance to promote transfer. This result explains why the frequency of recombination events does not increase with increasing distance from the start of synthesis, a characteristic that supports effective mixing of viral mutations.
• The Genome of Bacillus subtilis Bacteriophage SPO1
  - J Mol Biol 388(1):48-70 (2009)
  We report the genome sequence of Bacillus subtilis phage SPO1. The unique genome sequence is 132,562 bp long, and DNA packaged in the virion (the chromosome) has a 13,185-bp terminal redundancy, giving a total of 145,747 bp. We predict 204 protein-coding genes and 5 tRNA genes, and we correlate these findings with the extensive body of investigations of SPO1, including studies of the functions of the 61 previously defined genes and studies of the virion structure. Sixty-nine percent of the encoded proteins show no similarity to any previously known protein. We identify 107 probable transcription promoters; most are members of the promoter classes identified in earlier studies, but we also see a new class that has the same sequence as the host sigma K promoters. We find three genes encoding potential new transcription factors, one of which is a distant homologue of the host sigma factor K. We also identify 75 probable transcription terminator structures. Promoters and t! erminators are generally located between genes and together with earlier data give what appears to be a rather complete picture of how phage transcription is regulated. There are complete genome sequences available for five additional phages of Gram-positive hosts that are similar to SPO1 in genome size and in composition and organization of genes. Comparative analysis of SPO1 in the context of these other phages yields insights about SPO1 and the other phages that would not be apparent from the analysis of any one phage alone. These include assigning identities as well as probable functions for several specific genes and inferring evolutionary events in the phages' histories. The comparative analysis also allows us to put SPO1 into a phylogenetic context. We see a pattern similar to what has been noted in phage T4 and its relatives, in which there is minimal successful horizontal exchange of genes among a "core" set of genes that includes most of the virion structural ! genes and some genes of DNA metabolism, but there is extensive! horizontal transfer of genes over the remainder of the genome. There is a correlation between genes in rapid evolutionary flux through these genomes and genes that are small.
• A Molecular Mechanism of Bacterial Flagellar Motor Switching
  - J Mol Biol 388(1):71-84 (2009)
  The high-resolution structures of nearly all the proteins that comprise the bacterial flagellar motor switch complex have been solved; yet a clear picture of the switching mechanism has not emerged. Here, we used NMR to characterize the interaction modes and solution properties of a number of these proteins, including several soluble fragments of the flagellar motor proteins FliM and FliG, and the response-regulator CheY. We find that activated CheY, the switch signal, binds to a previously unidentified region of FliM, adjacent to the FliM–FliM interface. We also find that activated CheY and FliG bind with mutual exclusivity to this site on FliM, because their respective binding surfaces partially overlap. These data support a model of CheY-driven motor switching wherein the binding of activated CheY to FliM displaces the carboxy-terminal domain of FliG (FliGC) from FliM, modulating the FliGC–MotA interaction, and causing the motor to switch rotational sense as req! uired for chemotaxis.
• The Structure of the Arginine Repressor from Mycobacterium tuberculosis Bound with its DNA Operator and Co-repressor, L-Arginine
  - J Mol Biol 388(1):85-97 (2009)
  The biosynthesis of arginine is an essential function for the metabolism of Mycobacterium tuberculosis (Mtb) and for the metabolism of many other microorganisms. The arginine repressor (ArgR) proteins control the transcription of genes encoding the arginine biosynthetic enzymes; they belong to repressors having one of the most intricate oligomerization patterns. Here, we present the crystal structure of the MtbArgR hexamer bound to three copies of the 20 base-pair DNA operator and to the co-repressor, L-arginine, determined to 3.3 Å resolution. This is the first ternary structure of an intact hexameric ArgR in complex with its DNA operator. The structure reported here is very different from the suggested models of the ternary ArgR–DNA complexes; it has revealed the sophisticated symmetry of the complex and the presence of two remarkably different protomer conformations, folded and extended. Both features provide flexibility to DNA binding and are important for under! standing the detailed function of ArgRs. Two of the 20 base-pair DNA operators align in a unified double-helical structure, suggesting the possible presence of a double ARG box in the promoter region of the Mtb arginine operon. Two pairs of protomers bind to the unified double ARG box so that the two folded protomers bind to the central half-sites of the double ARG box, whereas the two extended protomers bind to the remote half-sites. The protomers of the third pair bound to the single DNA operator also have a folded and an extended conformation. A probable mechanism for arginine repression is suggested on the basis of this structure.
• The Crystal Structure of the Pseudomonas dacunhae Aspartate-β-Decarboxylase Dodecamer Reveals an Unknown Oligomeric Assembly for a Pyridoxal-5′-Phosphate-Dependent Enzyme
  - J Mol Biol 388(1):98-108 (2009)
  The Pseudomonas dacunhae l-aspartate-β-decarboxylase (ABDC, aspartate 4-decarboxylase, aspartate 4-carboxylyase, E.C. 4.1.1.12) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the β-decarboxylation of l-aspartate to produce l-alanine and CO2. This catalytically versatile enzyme is known to form functional dodecamers at its optimal pH and is thought to work in conjunction with an l-Asp/l-Ala antiporter to establish a proton gradient across the membrane that can be used for ATP biosynthesis. We have solved the atomic structure of ABDC to 2.35 Å resolution using single-wavelength anomalous dispersion phasing. The structure reveals that ABDC oligomerizes as a homododecamer in an unknown mode among PLP-dependent enzymes and has highest structural homology with members of the PLP-dependent aspartate aminotransferase subfamily. The structure shows that the ABDC active site is very similar to that of aspartate aminotransferase. However, an additional arg! inine side chain (Arg37) was observed flanking the re-side of the PLP ring in the ABDC active site. The mutagenesis results show that although Arg37 is not required for activity, it appears to be involved in the ABDC catalytic cycle.
• Human Neuroserpin: Structure and Time-Dependent Inhibition
  - J Mol Biol 388(1):109-121 (2009)
  Human neuroserpin (hNS) is a protein serine protease inhibitor expressed mainly in the nervous system, where it plays key roles in neural development and plasticity by primarily targeting tissue plasminogen activator (tPA). Four hNS mutations are associated to a form of autosomal dominant dementia, known as familial encephalopathy with neuroserpin inclusion bodies. The medical interest in and the lack of structural information on hNS prompted us to study the crystal structure of native and cleaved hNS, reported here at 3.15 and 1.85 Å resolution, respectively. In the light of the three-dimensional structures, we focus on the hNS reactive centre loop in its intact and cleaved conformations relative to the current serpin polymerization models and discuss the protein sites hosting neurodegenerative mutations. On the basis of homologous serpin structures, we suggest the location of a protein surface site that may stabilize the hNS native (metastable) form. In parallel, we! present the results of kinetic studies on hNS inhibition of tPA. Our data analysis stresses the instability of the hNS–tPA complex with a dissociation half-life of minutes compared to a half-life of weeks observed for other serpin–cognate protease complexes.
• Contributions of the Transmembrane Domain and a Key Acidic Motif to Assembly and Function of the TatA Complex
  - J Mol Biol 388(1):122-132 (2009)
  The twin-arginine translocase (Tat) pathway transports folded proteins across bacterial and thylakoid membranes. In Escherichia coli, a membrane-bound TatA complex, which oligomerizes to form complexes of less than 100 to more than 500 kDa, is considered essential for translocation. We have studied the contributions of various TatA domains to the assembly and function of this heterogeneous TatA complex. The TOXCAT assay was used to analyze the potential contribution of the TatA transmembrane (TM) domain. We observed relatively weak interactions between TatA TM domains, suggesting that the TM domain is not the sole driving force behind oligomerization. A potential hydrogen-bonding role for a TM domain glutamine was also investigated, and it was found that mutation blocks transport at low expression levels, while assembly is unaffected at higher expression levels. Analysis of truncated TatA proteins instead highlighted an acidic motif directly following the TatA amphipat! hic helix. Mutating these negatively charged residues to apolar uncharged residues completely blocks activity, even at high levels of TatA, and appears to disrupt ordered complex formation.
• Desmin and Vimentin Intermediate Filament Networks: Their Viscoelastic Properties Investigated by Mechanical Rheometry
  - J Mol Biol 388(1):133-143 (2009)
  We have investigated the viscoelastic properties of the cytoplasmic intermediate filament (IF) proteins desmin and vimentin. Mechanical measurements were supported by time-dependent electron microscopy studies of the assembly process under similar conditions. Network formation starts within 2 min, but it takes more than 30 min until equilibrium mechanical network strength is reached. Filament bundling is more pronounced for desmin than for vimentin. Desmin filaments (persistence length lp ≈ 900 nm) are stiffer than vimentin filaments (lp ≈ 400 nm), but both IFs are much more flexible than microfilaments. The concentration dependence of the plateau modulus G0 not, vert, similar cα is much weaker than predicted theoretically for networks of semiflexible filaments. This is more pronounced for vimentin (α = 0.47) than for desmin (α = 0.70). Both networks exhibit strain stiffening at large shear deformations. At the transition from linear to nonlinear viscoelastic re! sponse, only desmin shows characteristics of nonaffine network deformation. Strain stiffening and the maximum modulus occur at strain amplitudes about an order of magnitude larger than those for microfilaments. This is probably attributable to axial slippage within the tetramer building blocks of the IFs. Network deformation beyond a critical strain γmax results in irreversible damage. Strain stiffening sets in at lower concentrations, is more pronounced, and is less sensitive to ionic strength for desmin than for vimentin. Hence, desmin exhibits strain stiffening even at low-salt concentrations, which is not observed for vimentin, and we conclude that the strength of electrostatic repulsion compared to the strength of attractive interactions forming the network junctions is significantly weaker for desmin than for vimentin filaments. These findings indicate that both IFs exhibit distinct mechanical properties that are adapted to their respective cellular surroundings [i.e! ., myocytes (desmin) and fibroblasts (vimentin)].
• PAC-1 Activates Procaspase-3 in Vitro through Relief of Zinc-Mediated Inhibition
  - J Mol Biol 388(1):144-158 (2009)
  The direct induction of apoptosis has emerged as a powerful anticancer strategy, and small molecules that either inhibit or activate certain proteins in the apoptotic pathway have great potential as novel chemotherapeutic agents. Central to apoptosis is the activation of the zymogen procaspase-3 to caspase-3. Caspase-3 is the key "executioner" caspase, catalyzing the hydrolysis of a multitude of protein substrates within the cell. Interestingly, procaspase-3 levels are often elevated in cancer cells, suggesting a compound that directly stimulates the activation of procaspase-3 to caspase-3 could selectively induce apoptosis in cancer cells. We recently reported the discovery of a compound, PAC-1, which enhances procaspase-3 activity in vitro and induces apoptotic death in cancer cells in culture and in mouse xenograft models. Described herein is the mechanism by which PAC-1 activates procaspase-3 in vitro. We show that zinc inhibits the enzymatic activity of procas! pase-3 and that PAC-1 strongly activates procaspase-3 in buffers that contain zinc. PAC-1 and zinc form a tight complex with one another, with a dissociation constant of approximately 42 nM. The combined data indicate that PAC-1 activates procaspase-3 in vitro by sequestering inhibitory zinc ions, thus allowing procaspase-3 to autoactivate itself to caspase-3. The small-molecule-mediated activation of procaspases has great therapeutic potential and thus this discovery of the in vitro mechanism of action of PAC-1 is critical to the development and optimization of other procaspase-activating compounds.
• The Determinants of Stability and Folding in Evolutionarily Diverged Cytochromes c
  - J Mol Biol 388(1):159-167 (2009)
  Cytochrome c has served as a paradigm for the study of protein stability, folding, and molecular evolution, but it remains unclear how these aspects of the protein are related. For example, while the bovine and equine cytochromes c are known to have different stabilities, and possibly different folding mechanisms, it is not known how these differences arise from just three amino acid substitutions introduced during divergence. Using site-selectively incorporated carbon–deuterium bonds, we show that like the equine protein, bovine cytochrome c is induced to unfold by guanidine hydrochloride via a stepwise mechanism, but it does not populate an intermediate as is observed with the equine protein. The increased stability also results in more similar free energies of unfolding observed at different sites within the protein, giving the appearance of a more concerted mechanism. Furthermore, we show that the differences in stability and folding appear to result from a singl! e amino acid substitution that stabilizes a helix by allowing for increased solvation of its N-terminus.
• Fibrillin Microfibrils: A Key Role for the Interbead Region in Elasticity
  - J Mol Biol 388(1):168-179 (2009)
  Fibrillin microfibrils have essential roles in elastic fiber formation and elastic tissue homeostasis, as well as transforming growth factor-β sequestration. A role for fibrillin microfibrils in tissue elasticity has been implied by their ability to increase periodicity from 56 to 150 nm. In this study, we found that microfibril periodicity and structure are dependent on the ionic strength of the buffer and Ca2+ concentration; we then used these properties of the microfibril to trap conformation intermediates. Transmission electron microscopy imaging of microfibrils with a range of periodicities between 56 and 154 nm revealed a gross conformational change in the interbead region that accommodates the length change. At periodicities below 85 nm, four thin filaments are visualized in the interbead region, but at periodicities greater than 85 nm, two thick filaments are seen. The diameter of the bead remains almost constant at all periodicities, but there is a decrease i! n stain-exclusion above 85 nm periodicity, which is likely to correspond to a decrease in bead mass. Additionally, we identified eight molecules in cross-section through a microfibril, allowing us to understand microfibril organization in three dimensions. In conclusion, when microfibrils extend, there is a large molecular rearrangement within the interbead region, and this highlights a possible role for Ca2+ in stabilizing the microfibril architecture and moderating extension in vivo.
• Inferential Optimization for Simultaneous Fitting of Multiple Components into a CryoEM Map of Their Assembly
  - J Mol Biol 388(1):180-194 (2009)
  Models of macromolecular assemblies are essential for a mechanistic description of cellular processes. Such models are increasingly obtained by fitting atomic-resolution structures of components into a density map of the whole assembly. Yet, current density-fitting techniques are frequently insufficient for an unambiguous determination of the positions and orientations of all components. Here, we describe MultiFit, a method used for simultaneously fitting atomic structures of components into their assembly density map at resolutions as low as 25 Å. The component positions and orientations are optimized with respect to a scoring function that includes the quality-of-fit of components in the map, the protrusion of components from the map envelope, and the shape complementarity between pairs of components. The scoring function is optimized by our exact inference optimizer DOMINO (Discrete Optimization of Multiple INteracting Objects) that efficiently finds the global min! imum in a discrete sampling space. MultiFit was benchmarked on seven assemblies of known structure, consisting of up to seven proteins each. The input atomic structures of the components were obtained from the Protein Data Bank, as well as by comparative modeling based on a 16–99% sequence identity to a template structure. A near-native configuration was usually found as the top-scoring model. Therefore, MultiFit can provide initial configurations for further refinement of many multicomponent assembly structures described by electron microscopy.
• Threshold Occupancy and Specific Cation Binding Modes in the Hammerhead Ribozyme Active Site are Required for Active Conformation
  - J Mol Biol 388(1):195-206 (2009)
  The relationship between formation of active in-line attack conformations and monovalent (Na+) and divalent (Mg2+) metal ion binding in hammerhead ribozyme (HHR) has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of the threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence and in the absence of a Mg2+ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the HHR folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of HHR catalysis and support the supposition that Mg2+, in addition to stabilizing active con! formations, plays a specific chemical role in catalysis.
• Corrigendum to "Transcriptional Stability of Cultured Cells upon Prion Infection" [J. Mol. Biol. 375 (2008) 1222–1233]
  - J Mol Biol 388(1):207 (2009)
  
• Erratum to "β-Lactoglobulin Assembles into Amyloid through Sequential Aggregated Intermediates" [J. Mol. Biol. 381 (2008) 1332–1348]
  - J Mol Biol 388(1):208 (2009)