Journal of Molecular Biology

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  • Transient Contacts on the Exterior of the HK97 Procapsid That Are Essential for Capsid Assembly
    [May 2014]

    Publication date: 15 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 10

    Author(s): Dan-ju Tso , Roger W. Hendrix , Robert L. Duda

    The G-loop is a 10-residue glycine-rich loop that protrudes from the surface of the mature bacteriophage HK97 capsid at the C-terminal end of the long backbone helix of major capsid protein subunits. The G-loop is essential for assembly, is conserved in related capsid and encapsulin proteins, and plays its role during HK97 capsid assembly by making crucial contacts between the hill-like hexamers and pentamers in precursor proheads. These contacts are not preserved in the flattened capsomers of the mature capsid. Aspartate 231 in each of the ~400 G-loops interacts with lysine 178 of the E-loop (extended loop) of a subunit on an adjacent capsomer. Mutations disrupting this interaction prevented correct assembly and, in some cases, induced abnormal assembly into tubes, or small, incomplete capsids. Assembly remained defective when D231 and K178 were replaced with larger charged residues or when their positions were exchanged. Second-site suppressors of lethal mutants containing substitution D231L replaced the ionic interaction with new interactions between neutral and hydrophobic residues of about the same size: D231L/K178V, D231L/K178I, and D231L/K178N. We conclude that it is not the charge but the size and shape of the side chains of residues 178 and 231 that are important. These two residues control the geometry of contacts between the E-loop and the G-loop, which apparently must be precisely spaced and oriented for correct assembly to occur. We present a model for how the G-loop could control HK97 assembly and identify G-loop-like protrusions in other capsid proteins that may play analogous roles.
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    Categories: Journal Articles
  • Sequence and Membrane Determinants of the Random Coil–Helix Transition of α-Synuclein
    [May 2014]

    Publication date: 15 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 10

    Author(s): Sowmya B. Lokappa , Jae-Eun Suk , Adithya Balasubramanian , Soma Samanta , Alan J. Situ , Tobias S. Ulmer

    A random coil–helix transition underlies the association of the presynaptic protein α-synuclein (αS) with curved vesicle membranes to fold Asp2–Ala89 into a continuous helix. To clarify this transition, we examined αS folding cooperativity, helix nucleation and propagation in relation to membrane stabilization and leakage on diverse small unilamellar vesicles. The sequences centering on Phe4 and Tyr39 initiate lipid interactions and the Phe4 region nucleates the helix irrespective of the order of Ser9–Ala89. However, helix propagation is not the sum of individual αS–membrane interactions; it requires non-uniform but balanced sequence distributions of lipid affinities and helix flexibility. The attained helix propagation, like folding cooperativity, depends distinctly on membrane lipid composition and correlates to the degree of αS-conferred membrane stabilization. Contrary to classical coil–helix folding thermodynamics, helix propagation proceeds with a small gain in free energy relative to helix nucleation indicating that its binding enthalpy is expended to compensate a high entropic cost of reducing lipid-packing defects in the curved membrane. Non-saturating lipid conditions or rigidification of the αS helix triggers an increase in small unilamellar vesicle membrane leakage. Thus, αS folding parameters appear highly optimized and closely matched to stabilize and protect its target membrane. Aging-associated changes in lipid and αS concentrations may therefore alter synaptic plasticity and contribute to αS misfolding that culminates in fatal neurodegeneration.
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  • Specific RNA-Binding Antibodies with a Four-Amino-Acid Code
    [May 2014]

    Publication date: 15 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 10

    Author(s): Eileen M. Sherman , Sean Holmes , Jing-Dong Ye

    Numerous large non-coding RNAs are rapidly being discovered, and many of them have been shown to play vital roles in gene expression, gene regulation, and human diseases. Given their often structured nature, specific recognition with an antibody fragment becomes feasible and may help define the structure and function of these non-coding RNAs. As demonstrated for protein antigens, specific antibodies may aid in RNA crystal structure elucidation or the development of diagnostic tools and therapeutic drugs targeting disease-causing RNAs. Recent success and limitation of RNA antibody development has made it imperative to generate an effective antibody library specifically targeting RNA molecules. Adopting the reduced chemical diversity design and further restricting the interface diversity to tyrosines, serines, glycines, and arginines only, we have constructed a RNA-targeting Fab library. Phage display selection and downstream characterization showed that this library yielded high-affinity Fabs for all three RNA targets tested. Using a quantitative specificity assay, we found that these Fabs are highly specific, possibly due to the alternate codon design we used to avoid consecutive arginines in the Fab interface. In addition, the effectiveness of the minimal Fab library may challenge our view of the protein–RNA binding interface and provide a unique solution for future design of RNA-binding proteins.
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  • Emerging technologies to map the protein methylome
    [May 2014]

    Publication date: Available online 4 May 2014
    Source:Journal of Molecular Biology

    Author(s): Scott M. Carlson , Or Gozani

    Protein methylation plays an integral role in cellular signaling, most notably by modulating proteins bound at chromatin, and increasingly through regulation of non-histone proteins. One central challenge in understanding how methylation acts in signaling is identifying and measuring protein methylation. This includes locus-specific modification of histones, on individual non-histone proteins, and globally across the proteome. Protein methylation has traditionally been studied using candidate approaches such as methylation-specific antibodies, mapping of post-translational modifications by mass spectrometry, and radioactive labeling to characterize methylation on target proteins. Recent developments have provided new approaches to identify methylated proteins, measure methylation levels, identify substrates of methyltransferase enzymes, and match methylated proteins to methyl-specific reader domains. Methyl-binding protein domains and improved antibodies with broad specificity for methylated proteins are being used to characterize the “protein methylome”. They also have the potential to be used in high-throughput assays for inhibitor screens and drug development. These tools are often coupled to improvements in mass spectrometry to quickly identify methylated residues, and to protein microarrays, where they can be used to screen for methylated proteins. Finally, new chemical biology strategies are being used to probe the function of methyltransferases, demethylases and methyl-binding “reader” domains. These tools are creating a “system-level” understanding of protein methylation, and integrating protein methylation into broader signaling processes.
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  • In vivo roles of BamA, BamB and BamD in the biogenesis of BamA, a core protein of the β-barrel assembly machine of Escherichia coli
    [May 2014]

    Publication date: Available online 2 May 2014
    Source:Journal of Molecular Biology

    Author(s): Rajeev Misra , Ryan Stikeleather , Rebecca Gabriele

    Assembly of the β-barrel outer membrane proteins (OMPs) is an essential cellular process in Gram negative bacteria and in the mitochondria and chloroplasts of eukaryotes—two organelles of bacterial origin. Central to this process is the conserved β-barrel OMP that belongs to the Omp85 superfamily. In Escherichia coli, BamA is the core β-barrel OMP, and together with four outer membrane lipoproteins, BamBCDE, constitute the β-barrel assembly machine (BAM). In this paper, we investigated the roles of BamD, an essential lipoprotein, and BamB in BamA biogenesis. Depletion of BamD caused impairment in BamA biogenesis and cessation of cell growth. These defects of BamD depletion were partly reversed by single amino acid substitutions mapping within the β-barrel domain of BamA. However, in the absence of BamB, the positive effects of the β-barrel substitutions on BamA biogenesis under BamD depletion conditions were nullified. By employing a BamA protein bearing one such substitution, F494L, it was demonstrated that the mutant BamA protein could not only assemble without BamD, but it could also facilitate the assembly of wild-type BamA expressed in trans. Based on these data, we propose a model in which the Bam lipoproteins, which are localized to the outer membrane by the BAM-independent Lol pathway, aid in the creation of new BAM complexes by serving as outer membrane receptors and folding factors for nascent BamA molecules. The newly assembled BAM holocomplex then catalyzes the assembly of substrate OMPs and BamA. These in vivo findings are corroborated by recently published in vitro data.
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  • Ultrafast Redistribution of E. coli SSB Along Long Single-Stranded DNA via Intersegment Transfer
    [May 2014]

    Publication date: Available online 2 May 2014
    Source:Journal of Molecular Biology

    Author(s): Kyung Suk Lee , Amanda B. Marciel , Alexander G. Kozlov , Charles M. Schroeder , Timothy M. Lohman , Taekjip Ha

    Single-stranded DNA binding proteins (SSBs) selectively bind single-stranded DNA (ssDNA) and facilitate recruitment of additional proteins and enzymes to their sites of action on DNA. SSB can also locally diffuse on ssDNA, which allows it to quickly reposition itself while remaining bound to ssDNA. In this work, we used a hybrid instrument that combines single-molecule fluorescence and force spectroscopy to directly visualize the movement of Escherichia coli SSB on long polymeric ssDNA. Long ssDNA was synthesized without secondary structure which can hinder quantitative analysis of SSB movement. The apparent diffusion coefficient of E. coli SSB thus determined ranged from 70,000 to 170,000nt2/s, which is at least six hundred times higher than that determined from SSB diffusion on short ssDNA oligomers, and is within the range of values reported for protein diffusion on double stranded DNA. Our work suggests that SSB can also migrate via a long range intersegment transfer on long ssDNA. The force dependence of SSB movement on ssDNA further supports this interpretation.
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  • Redefining the Dry Molten Globule State of Proteins
    [May 2014]

    Publication date: Available online 2 May 2014
    Source:Journal of Molecular Biology

    Author(s): Sabine Neumaier , Thomas Kiefhaber

    Dynamics and function of proteins are governed by the structural and energetic properties of the different states they adopt and the barriers separating them. In earlier work, native-state triplet-triplet-energy-transfer on the villin headpiece subdomain (HP35) revealed an equilibrium between a locked and an unlocked native state, which are structurally similar but have different dynamic properties. The locked state is restricted to low amplitude motions, whereas the unlocked state shows increased conformational flexibility and undergoes local unfolding reactions. This classified the unlocked state as a dry molten globule, which was proposed to represent an expanded native state with loosened side-chain interactions and a solvent-shielded core. To test whether the unlocked state of HP35 is actually expanded compared to the locked state we performed high pressure triplet-triplet-energy-transfer measurements. Increasing pressure shifts the equilibrium from the locked toward the unlocked state, with a small negative reaction volume for unlocking (ΔV0 =−1.6±0.5cm3/mol). Therefore, rather than being expanded, the unlocked state represents an alternatively packed, compact state, demonstrating that native proteins can exist in several compact folded states, an observation with implications for protein function. The transition state for unlocking/locking, in contrast, has a largely increased volume relative to the locked and unlocked state, with respective activation volumes of 7.1±0.4cm3/mol and 8.7±0.9cm3/mol, indicating an expansion of the protein during the locking/unlocking transition. The presented results demonstrate the existence of both compact, low energy and expanded, high energy dry molten globules, prompting a broader definition of this state.
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    Categories: Journal Articles
  • Editorial Board
    [May 2014]

    Publication date: 1 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 9









    Categories: Journal Articles
  • Contents List
    [May 2014]

    Publication date: 1 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 9









    Categories: Journal Articles
  • The Protein Kinase CK2Andante Holoenzyme Structure Supports Proposed Models of Autoregulation and Trans-Autophosphorylation
    [May 2014]

    Publication date: 1 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 9

    Author(s): Alexander Schnitzler , Birgitte Brinkmann Olsen , Olaf-Georg Issinger , Karsten Niefind

    Eukaryotic protein kinases are typically strictly controlled by second messenger binding, protein/protein interactions, dephosphorylations or similar processes. None of these regulatory mechanisms is known to work for protein kinase CK2 (former name “casein kinase 2”), an acidophilic and constitutively active eukaryotic protein kinase. CK2 predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) complexed to a dimer of non-catalytic subunits (CK2β). One model of CK2 regulation was proposed several times independently by theoretical docking of the first CK2 holoenzyme structure. According to this model, the CK2 holoenzyme forms autoinhibitory aggregates correlated with trans-autophosphorylation and driven by the down-regulatory affinity between an acidic loop of CK2β and the positively charged substrate binding region of CK2α from a neighboring CK2 heterotetramer. Circular trimeric aggregates in which one-half of the CK2α chains show the predicted inhibitory proximity between those regions were detected within the crystal packing of the human CK2 holoenzyme. Here, we present further in vitro support of the “regulation-by-aggregation” model by an alternative crystal form in which CK2 tetramers are arranged as approximately linear aggregates coinciding essentially with the early predictions. In this assembly, the substrate binding region of every CK2α chain is blocked by a CK2β acidic loop from a neighboring tetramer. We found these crystals with CK2 Andante that contains a CK2β variant mutated in a CK2α-contact helix and described to be responsible for a prolonged circadian rhythm in Drosophila. The increased propensity of CK2 Andante to form aggregates with completely blocked active sites may contribute to this phenotype.
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  • Context-Dependent Remodeling of Rad51–DNA Complexes by Srs2 Is Mediated by a Specific Protein–Protein Interaction
    [May 2014]

    Publication date: 1 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 9

    Author(s): Anna K. Lytle , Sofia S. Origanti , Yupeng Qiu , Jeffrey VonGermeten , Sua Myong , Edwin Antony

    The yeast Srs2 helicase removes Rad51 nucleoprotein filaments from single-stranded DNA (ssDNA), preventing DNA strand invasion and exchange by homologous recombination. This activity requires a physical interaction between Srs2 and Rad51, which stimulates ATP turnover in the Rad51 nucleoprotein filament and causes dissociation of Rad51 from ssDNA. Srs2 also possesses a DNA unwinding activity and here we show that assembly of more than one Srs2 molecule on the 3′ ssDNA overhang is required to initiate DNA unwinding. When Rad51 is bound on the double-stranded DNA, its interaction with Srs2 blocks the helicase (DNA unwinding) activity of Srs2. Thus, in different DNA contexts, the physical interaction of Rad51 with Srs2 can either stimulate or inhibit the remodeling functions of Srs2, providing a means for tailoring DNA strand exchange activities to enhance the fidelity of recombination.
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  • Structure and Functional Analysis of YcfD, a Novel 2-Oxoglutarate/Fe2+-Dependent Oxygenase Involved in Translational Regulation in Escherichia coli
    [May 2014]

    Publication date: 1 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 9

    Author(s): Laura M. van Staalduinen , Stefanie K. Novakowski , Zongchao Jia

    The 2-oxoglutarate (2OG)/Fe2+-dependent oxygenases (2OG oxygenases) are a large family of proteins that share a similar overall three-dimensional structure and catalyze a diverse array of oxidation reactions. The Jumonji C (JmjC)-domain-containing proteins represent an important subclass of the 2OG oxygenase family that typically catalyze protein hydroxylation; however, recently, other reactions have been identified, such as tRNA modification. The Escherichia coli gene, ycfD, was predicted to be a JmjC-domain-containing protein of unknown function based on primary sequence. Recently, YcfD was determined to act as a ribosomal oxygenase, hydroxylating an arginine residue on the 50S ribosomal protein L-16 (RL-16). We have determined the crystal structure of YcfD at 2.7Å resolution, revealing that YcfD is structurally similar to known JmjC proteins and possesses the characteristic double-stranded β-helix fold or cupin domain. Separate from the cupin domain, an additional globular module termed α-helical arm mediates dimerization of YcfD. We further have shown that 2OG binds to YcfD using isothermal titration calorimetry and identified key binding residues using mutagenesis that, together with the iron location and structural similarity with other cupin family members, allowed identification of the active site. Structural homology to ribosomal assembly proteins combined with GST (glutathione S-transferase)-YcfD pull-down of a ribosomal protein and docking of RL-16 to the YcfD active site support the role of YcfD in regulation of bacterial ribosome assembly. Furthermore, overexpression of YcfD is shown to inhibit cell growth signifying a toxic effect on ribosome assembly.
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  • A DNA Mimic: The Structure and Mechanism of Action for the Anti-Repressor Protein AbbA
    [May 2014]

    Publication date: 1 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 9

    Author(s): Ashley T. Tucker , Benjamin G. Bobay , Allison V. Banse , Andrew L. Olson , Erik J. Soderblom , M. Arthur Moseley , Richele J. Thompson , Kristen M. Varney , Richard Losick , John Cavanagh

    Bacteria respond to adverse environmental conditions by switching on the expression of large numbers of genes that enable them to adapt to unfavorable circumstances. In Bacillus subtilis, many adaptive genes are under the negative control of the global transition state regulator, the repressor protein AbrB. Stressful conditions lead to the de-repression of genes under AbrB control. Contributing to this de-repression is AbbA, an anti-repressor that binds to and blocks AbrB from binding to DNA. Here, we have determined the NMR structure of the functional AbbA dimer, confirmed that it binds to the N-terminal DNA-binding domain of AbrB, and have provided an initial description for the interaction using computational docking procedures. Interestingly, we show that AbbA has structural and surface characteristics that closely mimic the DNA phosphate backbone, enabling it to readily carry out its physiological function.
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  • Extracellular-Regulated Kinase 2 Is Activated by the Enhancement of Hinge Flexibility
    [May 2014]

    Publication date: 1 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 9

    Author(s): Kevin M. Sours , Yao Xiao , Natalie G. Ahn

    Protein motions underlie conformational and entropic contributions to enzyme catalysis; however, relatively little is known about the ways in which this occurs. Studies of the mitogen-activated protein kinase ERK2 (extracellular-regulated protein kinase 2) by hydrogen-exchange mass spectrometry suggest that activation enhances backbone flexibility at the linker between N- and C-terminal domains while altering nucleotide binding mode. Here, we address the hypothesis that enhanced backbone flexibility within the hinge region facilitates kinase activation. We show that hinge mutations enhancing flexibility promote changes in the nucleotide binding mode consistent with domain movement, without requiring phosphorylation. They also lead to the activation of monophosphorylated ERK2, a form that is normally inactive. The hinge mutations bypass the need for pTyr but not pThr, suggesting that Tyr phosphorylation controls hinge motions. In agreement, monophosphorylation of pTyr enhances both hinge flexibility and nucleotide binding mode, measured by hydrogen-exchange mass spectrometry. Our findings demonstrate that regulated protein motions underlie kinase activation. Our working model is that constraints to domain movement in ERK2 are overcome by phosphorylation at pTyr, which increases hinge dynamics to promote the active conformation of the catalytic site.
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    Categories: Journal Articles
  • Structural Analysis and Identification of PhuS as a Heme-Degrading Enzyme from Pseudomonas aeruginosa
    [May 2014]

    Publication date: 1 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 9

    Author(s): Michael J.Y. Lee , Daniel Schep , Brian McLaughlin , Martin Kaufmann , Zongchao Jia

    Bacterial pathogens require iron for proliferation and pathogenesis. Pseudomonas aeruginosa is a prevalent Gram-negative opportunistic human pathogen that takes advantage of immunocompromised hosts and encodes a number of proteins for uptake and utilization of iron. Here we report the crystal structures of PhuS, previously known as the cytoplasmic heme-trafficking protein from P. aeruginosa, in both the apo- and the holo-forms. In comparison to its homologue ChuS from Escherichia coli O157:H7, the heme orientation is rotated 180° across the α-γ axis, which may account for some of the unique functional properties of PhuS. In contrast to previous findings, heme binding does not result in an overall conformational change of PhuS. We employed spectroscopic analysis and CO measurement by gas chromatography to analyze heme degradation, demonstrating that PhuS is capable of degrading heme using ascorbic acid or cytochrome P450 reductase-NADPH as an electron donor and produces five times more CO than ChuS. Addition of catalase slows down but does not stop PhuS-catalyzed heme degradation. Through spectroscopic and mass spectrometry analysis, we identified the enzymatic product of heme degradation to be verdoheme. These data taken together suggest that PhuS is a potent heme-degrading enzyme, in addition to its proposed heme-trafficking function.
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  • Antiparallel Conformation of Knob and Hole Aglycosylated Half-Antibody Homodimers Is Mediated by a CH2–CH3 Hydrophobic Interaction
    [May 2014]

    Publication date: 1 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 9

    Author(s): J.Michael Elliott , Mark Ultsch , Joshua Lee , Raymond Tong , Kentaro Takeda , Christoph Spiess , Charles Eigenbrot , Justin M. Scheer

    Bispecific antibody and antibody-like molecules are of wide interest as potential therapeutics that can recognize two distinct targets. Among the variety of ways such molecules have been engineered is by creating “knob” and “hole” heterodimerization sites in the CH3 domains of two antibody heavy chains. The molecules produced in this manner maintain their biological activities while differing very little from the native human IgG sequence. To better understand the knob-into-hole interface, the molecular mechanism of heterodimerization, and to engineer Fc domains that could improve the assembly and purity of heterodimeric reaction products, we sought crystal structures of aglycosylated heterodimeric and homodimeric “knob” and “hole” Fc fragments derived from bacterial expression. The structure of the knob-into-hole Fc was determined at 2.64Å. Except for the sites of mutation, the structure is very similar to that of the native human IgG1 Fc, consistent with a heterodimer interaction kinetic K D of <1nM. Homodimers of the “knob” and “hole” mutants were also obtained, and their X-ray structures were determined at resolutions 2.5Å and 2.1Å, respectively. Both kinds of homodimers adopt a head-to-tail quaternary structure and thus do not contain direct knob/knob or hole/hole CH3 interactions. The head-to-tail arrangement was disfavored by adding site-directed mutations at F241 and F243 in the CH2 domains, leading to increases in both rate and efficiency of bispecific (heterodimer) assembly.
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    Categories: Journal Articles
  • Crystal Structure of the Carbapenem Intrinsic Resistance Protein CarG
    [May 2014]

    Publication date: 1 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 9

    Author(s): E.M. Tichy , B.F. Luisi , G.P.C. Salmond

    In the Gram-negative enterobacterium Erwinia (Pectobacterium) and Serratia sp. ATCC 39006, intrinsic resistance to the carbapenem antibiotic 1-carbapen-2-em-3-carboxylic acid is mediated by the CarF and CarG proteins, by an unknown mechanism. Here, we report a high-resolution crystal structure for the Serratia sp. ATCC 39006 carbapenem resistance protein CarG. This structure of CarG is the first in the carbapenem intrinsic resistance (CIR) family of resistance proteins from carbapenem-producing bacteria. The crystal structure shows the protein to form a homodimer, in agreement with results from analytical gel filtration. The structure of CarG does not show homology with any known antibiotic resistance proteins nor does it belong to any well-characterised protein structural family. However, it is a close structural homologue of the bacterial inhibitor of invertebrate lysozyme, PliI-Ah, with some interesting structural variations, including the absence of the catalytic site responsible for lysozyme inhibition. Both proteins show a unique β-sandwich fold with short terminal α-helices. The core of the protein is formed by stacked anti-parallel sheets that are individually very similar in the two proteins but differ in their packing interface, causing the splaying of the two sheets in CarG. Furthermore, a conserved cation binding site identified in CarG is absent from the homologue.
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    Categories: Journal Articles
  • The Cleaved N-Terminus of pVI Binds Peripentonal Hexons in Mature Adenovirus
    [May 2014]

    Publication date: 1 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 9

    Author(s): Joost Snijder , Marco Benevento , Crystal L. Moyer , Vijay Reddy , Glen R. Nemerow , Albert J.R. Heck

    Mature human adenovirus particles contain four minor capsid proteins, in addition to the three major capsid proteins (penton base, hexon and fiber) and several proteins associated with the genomic core of the virion. Of the minor capsid proteins, VI plays several crucial roles in the infection cycle of the virus, including hexon nuclear targeting during assembly, activation of the adenovirus proteinase (AVP) during maturation and endosome escape following cell entry. VI is translated as a precursor (pVI) that is cleaved at both N- and C-termini by AVP. Whereas the role of the C-terminal fragment of pVI, pVIc, is well established as an important co-factor of AVP, the role of the N-terminal fragment, pVIn, is currently elusive. In fact, the fate of pVIn following proteolytic cleavage is completely unknown. Here, we use a combination of proteomics-based peptide identification, native mass spectrometry and hydrogen–deuterium exchange mass spectrometry to show that pVIn is associated with mature human adenovirus, where it binds at the base of peripentonal hexons in a pH-dependent manner. Our findings suggest a possible role for pVIn in targeting pVI to hexons for proper assembly of the virion and timely release of the membrane lytic mature VI molecule.
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    Categories: Journal Articles
  • Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS
    [May 2014]

    Publication date: 1 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 9

    Author(s): Sagar V. Kathuria , Can Kayatekin , Raul Barrea , Elena Kondrashkina , Rita Graceffa , Liang Guo , R. Paul Nobrega , Srinivas Chakravarthy , C. Robert Matthews , Thomas C. Irving , Osman Bilsel

    It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59–heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~27μs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency>90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.
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    Categories: Journal Articles
  • Structural and Functional Analysis of the GerD Spore Germination Protein of Bacillus Species
    [May 2014]

    Publication date: 1 May 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 9

    Author(s): Yunfeng Li , Kai Jin , Sonali Ghosh , Parvathimadhavi Devarakonda , Kristina Carlson , Andrew Davis , Kerry-Ann V. Stewart , Elizabeth Cammett , Patricia Pelczar Rossi , Barbara Setlow , Min Lu , Peter Setlow , Bing Hao

    Spore germination in Bacillus species represents an excellent model system with which to study the molecular mechanisms underlying the nutritional control of growth and development. Binding of specific chemical nutrients to their cognate receptors located in the spore inner membrane triggers the germination process that leads to a resumption of metabolism in spore outgrowth. Recent studies suggest that the inner membrane GerD lipoprotein plays a critical role in the receptor-mediated activation of downstream germination events. The 121-residue core polypeptide of GerD (GerD60-180) from Geobacillus stearothermophilus forms a stable α-helical trimer in aqueous solution. The 2.3-Å-resolution crystal structure of the trimer reveals a neatly twisted superhelical rope, with unusual supercoiling induced by parallel triple-helix interactions. The overall geometry comprises three interleaved hydrophobic screws of interacting helices linked by short turns that have not been seen before. Using complementation analysis in a series of Bacillus subtilis gerD mutants, we demonstrated that alterations in the GerD trimer structure have profound effects on nutrient germination. This important structure–function relationship of trimeric GerD is supported by our identification of a dominant negative gerD mutation in B. subtilis. These results and those of others lead us to propose that GerD mediates clustering of germination proteins in the inner membrane of dormant spores and thus promotes the rapid and cooperative germination response to nutrients.
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    Categories: Journal Articles