Journal of Molecular Biology

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  • INO80-C and SWR-C: Guardians of the Genome
    [Nov 2014]

    Publication date: Available online 30 October 2014
    Source:Journal of Molecular Biology

    Author(s): Christian-Benedikt Gerhold , Michael H. Hauer , Susan M. Gasser

    The double membrane of the eukaryotic nucleus encapsulates the genome, constraining it to a nuclear sphere. Proteins, RNA-protein particles and artificial chromosome rings diffuse rapidly and freely throughout the nucleoplasm, while chromosomal loci move subdiffusively with varying degrees of constraint. In situ biochemical approaches and live imaging studies have revealed the existence of nuclear subcompartments that are enriched for specific chromatin states and/or enzymatic activities. This sequestration is thought to enhance the propagation or efficient establishment of heterochromatin, particularly when factors of limited abundance are involved. Implicit in the concept of compartmentation, is the notion that chromatin is able to move from one compartment to another. Indeed, in budding yeast, gene activation, repression, and the presence of persistent DNA double-strand breaks has each been shown to provoke subnuclear relocalization of chromatin. In some cases movement has been linked to the action of ATP-dependent chromatin remodeling complexes, more specifically to the Snf2-related ATPase containing complexes, SWR-C and INO80-C. Here we examine how these multi-subunit remodelers contribute to chromatin–based processes linked to the DNA damage response. Chromatin remodelers are able to alter the physical mobility of chromatin as they alter the compaction and organization of nucleosomes. Finally we review recent evidence that supports a role for yeast SWR-C and INO80-C in determining the subnuclear position of damaged domains, and recap the multiple ways in which these remodelers contribute to genomic integrity.
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    Categories: Journal Articles
  • Computational Design of Selective Peptides to Discriminate Between Similar PDZ Domains in an Oncogenic Pathway
    [Nov 2014]

    Publication date: Available online 30 October 2014
    Source:Journal of Molecular Biology

    Author(s): Fan Zheng , Heather Jewell , Jeremy Fitzpatrick , Jian Zhang , Dale F. Mierke , Gevorg Grigoryan

    Reagents that target protein-protein interactions to rewire signaling are of great relevance in biological research. Computational protein design may offer a means of creating such reagents on demand, but methods for encoding targeting selectivity are sorely needed. This is especially challenging when targeting interactions with ubiquitous recognition modules—e.g., PDZ domains, which bind C-terminal sequences of partner proteins. Here we consider the problem of designing selective PDZ inhibitor peptides in the context of an oncogenic signaling pathway, in which two PDZ domains (NHERF-2 PDZ2—N2P2 and MAGI-3 PDZ6—M3P6) compete for a receptor C-terminus to differentially modulate oncogenic activities. Because N2P2 increases tumorigenicity and M3P6 decreases it, we sought to design peptides that inhibit N2P2 without affecting M3P6. We developed a structure-based computational design framework that models peptide flexibility in binding, yet is efficient enough to rapidly analyze tradeoffs between affinity and selectivity. Designed peptides showed low-micromolar inhibition constants for N2P2 and no detectable M3P6 binding. Peptides designed for reverse discrimination bound M3P6 tighter than N2P2, further testing our technology. Experimental and computational analysis of selectivity determinants revealed significant indirect energetic coupling in the binding site. Successful discrimination between N2P2 and M3P6, despite their overlapping binding preferences, is highly encouraging for computational approaches to selective PDZ targeting, especially because design relied on a homology model of M3P6. Still, we demonstrate specific deficiencies of structural modeling that must be addressed to enable truly robust design. The presented framework is general and can be applied in many scenarios to engineer selective targeting.
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  • Interplay between E. coli DnaK, ClpB and GrpE during protein disaggregation
    [Nov 2014]

    Publication date: Available online 29 October 2014
    Source:Journal of Molecular Biology

    Author(s): Shannon M. Doyle , Shankar Shastry , Andrea N. Kravats , Yu-Hsuan Shih , Marika Miot , Joel R. Hoskins , George Stan , Sue Wickner

    The DnaK/Hsp70 chaperone system and ClpB/Hsp104 collaboratively disaggregate protein aggregates and reactivate inactive proteins. The teamwork is specific: E. coli DnaK interacts with E. coli ClpB and yeast Hsp70, Ssa1, interacts with yeast Hsp104. This interaction is between the M-domains of hexameric ClpB/Hsp104 and the DnaK/Hsp70 nucleotide-binding domain (NBD). To identify the site on E. coli DnaK that interacts with ClpB, we substituted amino acid residues throughout the DnaK NBD. We found that several variants with substitutions in subdomain IB and IIB of the DnaK NBD were defective in ClpB interaction in vivo in a bacterial two-hybrid assay and in vitro in a fluorescence anisotropy assay. The DnaK subdomain IIB mutants were also defective in the ability to disaggregate protein aggregates with ClpB, DnaJ and GrpE, although they retained some ability to reactivate proteins with DnaJ and GrpE in the absence of ClpB. We observed that GrpE, which also interacts with subdomains IB and IIB, inhibited the interaction between ClpB and DnaK in vitro, suggesting competition between ClpB and GrpE for binding DnaK. Computational modeling of the DnaK-ClpB hexamer complex indicated that one DnaK monomer contacts two adjacent ClpB protomers simultaneously. The model and the experiments support a common and mutually exclusive GrpE and ClpB interaction region on DnaK. Additionally, homologous substitutions in subdomains IB and IIB of Ssa1 caused defects in collaboration between Ssa1 and Hsp104. Altogether, these results provide insight into the molecular mechanism of collaboration between the DnaK/Hsp70 system and ClpB/Hsp104 for protein disaggregation.
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  • Quality Control in Eukaryotic Membrane Protein Overproduction
    [Nov 2014]

    Publication date: Available online 28 October 2014
    Source:Journal of Molecular Biology

    Author(s): Jennifer A. Thomas , Christopher G. Tate

    The overexpression of authentically folded eukaryotic membrane proteins in milligramme quantities is a fundamental prerequisite for structural studies. One of the most commonly used expression systems for the production of mammalian membrane proteins is the baculovirus expression system in insect cells. However, a detailed analysis by radioligand binding and comparative Western blotting of G protein-coupled receptors and a transporter produced in insect cells showed that a considerable proportion of the expressed protein was misfolded and incapable of ligand binding. In contrast, production of the same membrane proteins in stable inducible mammalian cell lines suggested that the majority was folded correctly. It was noted that detergent solubilisation of the misfolded membrane proteins using either digitonin or dodecylmaltoside was considerably less efficient than using sodium dodecyl sulfate or foscholine-12, whilst these detergents were equally efficient at solubilising correctly folded membrane proteins. This provides a simple and rapid test to suggest whether heterologously expressed mammalian membrane proteins are indeed correctly folded, without requiring radioligand binding assays. This will greatly facilitate the high-throughput production of fully functional membrane proteins for structural studies.
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  • Corrigendum to “Statistical Mechanics of Monod–Wyman–Changeux” [J Mol Biol 425 (9) (May 13 2013) 1433-1460]
    [Nov 2014]

    Publication date: Available online 25 October 2014
    Source:Journal of Molecular Biology

    Author(s): Sarah Marzen , Hernan G. Garcia , Rob Phillips







    Categories: Journal Articles
  • Editorial Board
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21









    Categories: Journal Articles
  • Contents List
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21









    Categories: Journal Articles
  • Control of RecBCD Enzyme Activity by DNA Binding- and Chi Hotspot-Dependent Conformational Changes
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Andrew F. Taylor , Susan K. Amundsen , Miklos Guttman , Kelly K. Lee , Jie Luo , Jeffrey Ranish , Gerald R. Smith

    Faithful repair of DNA double-strand breaks by homologous recombination is crucial to maintain functional genomes. The major Escherichia coli pathway of DNA break repair requires RecBCD enzyme, a complex protein machine with multiple activities. Upon encountering a Chi recombination hotspot (5′ GCTGGTGG 3′) during DNA unwinding, RecBCD's unwinding, nuclease, and RecA-loading activities change dramatically, but the physical basis for these changes is unknown. Here, we identify, during RecBCD's DNA unwinding, two Chi-stimulated conformational changes involving RecC. One produced a marked, long-lasting, Chi-dependent increase in protease sensitivity of a small patch, near the Chi recognition domain, on the solvent-exposed RecC surface. The other change was identified by crosslinking of an artificial amino acid inserted in this RecC patch to RecB. Small-angle X-ray scattering analysis confirmed a major conformational change upon binding of DNA to the enzyme and is consistent with these two changes. We propose that, upon DNA binding, the RecB nuclease domain swings from one side of RecC to the other; when RecBCD encounters Chi, the nuclease domain returns to its initial position determined by crystallography, where it nicks DNA exiting from RecC and loads RecA onto the newly generated 3′-ended single-stranded DNA during continued unwinding; a crevice between RecB and RecC increasingly narrows during these steps. This model provides a physical basis for the intramolecular “signal transduction” from Chi to RecC to RecD to RecB inferred previously from genetic and enzymatic analyses, and it accounts for the enzymatic changes that accompany Chi's stimulation of recombination.
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    Categories: Journal Articles
  • Alteration of the C-Terminal Ligand Specificity of the Erbin PDZ Domain by Allosteric Mutational Effects
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Javier Murciano-Calles , Megan E. McLaughlin , Ariel Erijman , Yogesh Hooda , Nishant Chakravorty , Jose C. Martinez , Julia M. Shifman , Sachdev S. Sidhu

    Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity.
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    Categories: Journal Articles
  • A Structural Portrait of the PDZ Domain Family
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Andreas Ernst , Brent A. Appleton , Ylva Ivarsson , Yingnan Zhang , David Gfeller , Christian Wiesmann , Sachdev S. Sidhu

    PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family.
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    Categories: Journal Articles
  • Dynamic and Thermodynamic Response of the Ras Protein Cdc42Hs upon Association with the Effector Domain of PAK3
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Veronica R. Moorman , Kathleen G. Valentine , Sabrina Bédard , Vignesh Kasinath , Jakob Dogan , Fiona M. Love , A. Joshua Wand

    Human cell division cycle protein 42 (Cdc42Hs) is a small, Rho-type guanosine triphosphatase involved in multiple cellular processes through its interactions with downstream effectors. The binding domain of one such effector, the actin cytoskeleton-regulating p21-activated kinase 3, is known as PBD46. Nitrogen-15 backbone and carbon-13 methyl NMR relaxation was measured to investigate the dynamical changes in activated GMPPCP·Cdc42Hs upon PBD46 binding. Changes in internal motion of the Cdc42Hs, as revealed by methyl axis order parameters, were observed not only near the Cdc42Hs–PBD46 interface but also in remote sites on the Cdc42Hs molecule. The binding-induced changes in side-chain dynamics propagate along the long axis of Cdc42Hs away from the site of PBD46 binding with sharp distance dependence. Overall, the binding of the PBD46 effector domain on the dynamics of methyl-bearing side chains of Cdc42Hs results in a modest rigidification, which is estimated to correspond to an unfavorable change in conformational entropy of approximately −10kcalmol−1 at 298K. A cluster of methyl probes closest to the nucleotide-binding pocket of Cdc42Hs becomes more rigid upon binding of PBD46 and is proposed to slow the catalytic hydrolysis of the γ phosphate moiety. An additional cluster of methyl probes surrounding the guanine ring becomes more flexible on binding of PBD46, presumably facilitating nucleotide exchange mediated by a guanosine exchange factor. In addition, the Rho insert helix, which is located at a site remote from the PBD46 binding interface, shows a significant dynamic response to PBD46 binding.
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  • DNA Looping Provides for “Intersegmental Hopping” by Proteins: A Mechanism for Long-Range Site Localization
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Adam J. Pollak , Aaron T. Chin , Frank L.H. Brown , Norbert O. Reich

    Studies on how transcription factors and DNA modifying enzymes passively locate specific sites on DNA have yet to be reconciled with a sufficient set of mechanisms that can adequately account for the efficiency and speed of this process. This is especially true when considering that these DNA binding/modifying proteins have diverse levels of both cellular copy numbers and genomic recognition site densities. The monomeric bacterial DNA adenine methyltransferase (Dam) is responsible for the rapid methylation of the entire chromosome (with only ~100 Dam copies per cell) and the regulated methylation of closely spaced sites that controls the expression of virulence genes in several human pathogens. Provocatively, we find that Dam travels between its recognition sites most efficiently when those sites are ~500bp apart. We propose that this is manifested by Dam moving between distal regions on the same DNA molecule, which is mediated by DNA looping, a phenomenon we designate as intersegmental hopping. Importantly, an intermediate found in other systems including two simultaneously bound, looped DNA strands is not involved here. Our results suggest that intersegmental hopping contributes to enzymatic processivity (multiple modifications), which invoke recent reports demonstrating that DNA looping can assist in site finding. Intersegmental hopping is possibly used by other sequence-specific DNA binding proteins, such as transcription factors and regulatory proteins, given certain biological context. While a general form of this mechanism is proposed by many research groups, our consideration of DNA looping in the context of processive catalysis provides new mechanistic insights and distinctions.
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    Categories: Journal Articles
  • DNA Recognition by a σ54 Transcriptional Activator from Aquifex aeolicus
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Natasha K. Vidangos , Johanna Heideker , Artem Lyubimov , Meindert Lamers , Yixin Huo , Jeffrey G. Pelton , Jimmy Ton , Jay Gralla , James Berger , David E. Wemmer

    Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain (DBD). The structurally characterized DBDs from activators all belong to the Fis (factor for inversion stimulation) family of helix–turn–helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DBD of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ54 activators. Two NtrC4-binding sites were identified upstream (−145 and −85bp) from the start of the lpxC gene, which is responsible for the first committed step in lipid A biosynthesis. This is the first experimental evidence for σ54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the −145-binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homolog, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base-specific contacts contributing to affinity than for Fis.
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    Categories: Journal Articles
  • Phosphorylation- and Nucleotide-Binding-Induced Changes to the Stability and Hydrogen Exchange Patterns of JNK1β1 Provide Insight into Its Mechanisms of Activation
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Gavin R. Owen , Stoyan Stoychev , Ikechukwu Achilonu , Heini W. Dirr

    Many studies have characterized how changes to the stability and internal motions of a protein during activation can contribute to their catalytic function, even when structural changes cannot be observed. Here, unfolding studies and hydrogen–deuterium exchange (HX) mass spectrometry were used to investigate the changes to the stability and conformation/conformational dynamics of JNK1β1 induced by phosphorylative activation. Equivalent studies were also employed to determine the effects of nucleotide binding on both inactive and active JNK1β1 using the ATP analogue, 5ʹ-adenylyl-imidodiphosphate (AMP-PNP). JNK1β1 phosphorylation alters HX in regions involved in catalysis and substrate binding, changes that can be ascribed to functional modifications in either structure and/or backbone flexibility. Increased HX in the hinge between the N- and C-terminal domains implied that it acquires enhanced flexibility upon phosphorylation that may be a prerequisite for interdomain closure. In combination with the finding that nucleotide binding destabilizes the kinase, the patterns of solvent protection by AMP-PNP were consistent with a novel mode of nucleotide binding to the C-terminal domain of a destabilized and open domain conformation of inactive JNK1β1. Solvent protection by AMP-PNP of both N- and C-terminal domains in active JNK1β1 revealed that the domains close around nucleotide upon phosphorylation, concomitantly stabilizing the kinase. This suggests that phosphorylation activates JNK1β1 in part by increasing hinge flexibility to facilitate interdomain closure and the creation of a functional active site. By uncovering the complex interplay that occurs between nucleotide binding and phosphorylation, we present new insight into the unique mechanisms by which JNK1β1 is regulated.
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    Categories: Journal Articles
  • Characterization of the Grp94/OS-9 Chaperone–Lectin Complex
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Paul M. Seidler , Stephen A. Shinsky , Feng Hong , Zihai Li , Michael S. Cosgrove , Daniel T. Gewirth

    Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum (ER) of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER-associated degradation quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins. To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full-length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal mannose recognition homology lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains “mammalian-specific insets” that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate and it additionally hints at a plausible biological role for the Grp94/OS-9 complex.
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    Categories: Journal Articles
  • A Fibrin-Specific Monoclonal Antibody from a Designed Phage Display Library Inhibits Clot Formation and Localizes to Tumors In Vivo
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Alessia Putelli , Jonathan D. Kiefer , Matthias Zadory , Mattia Matasci , Dario Neri

    Fibrin formation from fibrinogen is a rare process in the healthy organism but is a pathological feature of thrombotic events, cancer and a wide range of inflammatory conditions. We have designed and constructed an antibody phage display library (containing 13 billion clones) for the selective recognition of the N-terminal peptide of fibrin alpha chain. The key structural feature for selective fibrin binding was a K94E mutation in the VH domain. From this library, an antibody was isolated (termed AP2), which recognizes the five N-terminal amino acids of fibrin with high affinity (K d =44nM), but does not bind to fibrinogen. The AP2 antibody could be expressed in various formats (scFv, small immune protein and IgG) and inhibited fibrin clot formation in a concentration-dependent manner. Moreover, the AP2 antibody stained the fibrin-rich provisional stroma in solid tumors but did not exhibit any detectable staining toward normal tissues. Using a radioiodinated antibody preparation and quantitative biodistribution studies in tumor-bearing mice, AP2 was shown to selectively localize to fibrin-rich F9 murine teratocarcinomas, but not to SKRC-52 human kidney cancer xenografts. Collectively, the experiments indicate that the AP2 antibody recognizes fibrin in vitro and in vivo. The antibody may facilitate the development of fibrin-specific therapeutic agents.
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  • Coevolution of Specificity Determinants in Eukaryotic Glutamyl- and Glutaminyl-tRNA Synthetases
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Andrew Hadd , John J. Perona

    The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNAGln for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNAGln and tRNAGlu with glutamate. This ancient GluRS also separately differentiated to exclude tRNAGln as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but they play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNAGln and tRNAGlu recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p:GluRS complex upon the diauxic shift to respiratory conditions.
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  • Crystal Structure of a Symmetric Football-Shaped GroEL:GroES2-ATP14 Complex Determined at 3.8Å Reveals Rearrangement between Two GroEL Rings
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Ayumi Koike-Takeshita , Takatoshi Arakawa , Hideki Taguchi , Tatsuro Shimamura

    The chaperonin GroEL is an essential chaperone that assists in protein folding with the aid of GroES and ATP. GroEL forms a double-ring structure, and both rings can bind GroES in the presence of ATP. Recent progress on the GroEL mechanism has revealed the importance of a symmetric 1:2 GroEL:GroES2 complex (the “football”-shaped complex) as a critical intermediate during the functional GroEL cycle. We determined the crystal structure of the football GroEL:GroES2-ATP14 complex from Escherichia coli at 3.8Å, using a GroEL mutant that is extremely defective in ATP hydrolysis. The overall structure of the football complex resembled the GroES-bound GroEL ring of the asymmetric 1:1 GroEL:GroES complex (the “bullet” complex). However, the two GroES-bound GroEL rings form a modified interface by an ~7° rotation about the 7-fold axis. As a result, the inter-ring contacts between the two GroEL rings in the football complex differed from those in the bullet complex. The differences provide a structural basis for the apparently impaired inter-ring negative cooperativity observed in several biochemical analyses.
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    Categories: Journal Articles
  • Functional Suppression of HAMP Domain Signaling Defects in the E. coli Serine Chemoreceptor
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Run-Zhi Lai , John S. Parkinson

    HAMP domains play key signaling roles in many bacterial receptor proteins. The four-helix HAMP bundle of the homodimeric Escherichia coli serine chemoreceptor (Tsr) interacts with an adjoining four-helix sensory adaptation bundle to regulate the histidine autokinase CheA bound to the cytoplasmic tip of the Tsr molecule. The adaptation helices undergo reversible covalent modifications that tune the stimulus-responsive range of the receptor: unmodified E residues promote kinase-off output, and methylated E residues or Q replacements at modification sites promote kinase-on output. We used mutationally imposed adaptational modification states and cells with various combinations of the sensory adaptation enzymes, CheR and CheB, to characterize the signaling properties of mutant Tsr receptors that had amino acid replacements in packing layer 3 of the HAMP bundle and followed in vivo CheA activity with an assay based on Förster resonance energy transfer. We found that an alanine or a serine replacement at HAMP residue I229 effectively locked Tsr output in a kinase-on state, abrogating chemotactic responses. A second amino acid replacement in the same HAMP packing layer alleviated the I229A and I229S signaling defects. Receptors with the suppressor changes alone mediated chemotaxis in adaptation-proficient cells but exhibited altered sensitivity to serine stimuli. Two of the suppressors (S255E and S255A) shifted Tsr output toward the kinase-off state, but two others (S255G and L256F) shifted output toward a kinase-on state. The alleviation of locked-on defects by on-shifted suppressors implies that Tsr-HAMP has several conformationally distinct kinase-active output states and that HAMP signaling might involve dynamic shifts over a range of bundle conformations.
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    Categories: Journal Articles
  • A Conserved Isoleucine Maintains the Inactive State of Bruton's Tyrosine Kinase
    [Nov 2014]

    Publication date: 23 October 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 21

    Author(s): Scott E. Boyken , Nikita Chopra , Qian Xie , Raji E. Joseph , Thomas E. Wales , D. Bruce Fulton , John R. Engen , Robert L. Jernigan , Amy H. Andreotti

    Despite high level of homology among non-receptor tyrosine kinases, different kinase families employ a diverse array of regulatory mechanisms. For example, the catalytic kinase domains of the Tec family kinases are inactive without assembly of the adjacent regulatory domains, whereas the Src kinase domains are autoinhibited by the assembly of similar adjacent regulatory domains. Using molecular dynamics simulations, biochemical assays, and biophysical approaches, we have uncovered an isoleucine residue in the kinase domain of the Tec family member Btk that, when mutated to the closely related leucine, leads to a shift in the conformational equilibrium of the kinase domain toward the active state. The single amino acid mutation results in measureable catalytic activity for the Btk kinase domain in the absence of the regulatory domains. We suggest that this isoleucine side chain in the Tec family kinases acts as a “wedge” that restricts the conformational space available to key regions in the kinase domain, preventing activation until the kinase domain associates with its regulatory subunits and overcomes the energetic barrier to activation imposed by the isoleucine side chain.
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    Categories: Journal Articles