Journal of Molecular Biology

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  • Editorial Board
    [Jun 2014]

    Publication date: 26 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 13









    Categories: Journal Articles
  • Contents List
    [Jun 2014]

    Publication date: 26 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 13









    Categories: Journal Articles
  • Ultrafast Redistribution of E. coli SSB along Long Single-Stranded DNA via Intersegment Transfer
    [Jun 2014]

    Publication date: 26 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 13

    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 that 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 600 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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    Categories: Journal Articles
  • Gly25-Ser26 Amyloid β-Protein Structural Isomorphs Produce Distinct Aβ42 Conformational Dynamics and Assembly Characteristics
    [Jun 2014]

    Publication date: 26 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 13

    Author(s): Robin Roychaudhuri , Aleksey Lomakin , Summer Bernstein , Xueyun Zheng , Margaret M. Condron , George B. Benedek , Michael Bowers , David B. Teplow

    One of the earliest events in amyloid β-protein (Aβ) self-association is nucleation of Aβ monomer folding through formation of a turn at Gly25-Lys28. We report here the effects of structural changes at the center of the turn, Gly25-Ser26, on Aβ42 conformational dynamics and assembly. We used “click peptide” chemistry to quasi-synchronously create Aβ42 from 26-O-acyliso-Aβ42 (iAβ42) through a pH jump from 3 to 7.4. We also synthesized Nα-acetyl-Ser26-iAβ42 (Ac-iAβ42), which cannot undergo O→N acyl chemistry, to study the behavior of this ester form of Aβ42 itself at neutral pH. Data from experiments monitoring increases in β-sheet formation (thioflavin T, CD), hydrodynamic radius (R H), scattering intensity (quasielastic light scattering spectroscopy), and extent of oligomerization (ion mobility spectroscopy–mass spectrometry) were quite consistent. A rank order of Ac-iAβ42>iAβ42>Aβ42 was observed. Photochemically cross-linked iAβ42 displayed an oligomer distribution with a prominent dimer band that was not present with Aβ42. These dimers also were observed selectively in iAβ42 in ion mobility spectrometry experiments. The distinct biophysical behaviors of iAβ42 and Aβ42 appear to be due to the conversion of iAβ42 into “pure” Aβ42 monomer, a nascent form of Aβ42 that does not comprise the variety of oligomeric and aggregated states present in pre-existent Aβ42. These results emphasize the importance of the Gly25-Ser26 dipeptide in organizing Aβ42 monomer structure and thus suggest that drugs altering the interactions of this dipeptide with neighboring side-chain atoms or with the peptide backbone could be useful in therapeutic strategies targeting formation of Aβ oligomers and higher-order assemblies.
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    Categories: Journal Articles
  • Connectivity between Catalytic Landscapes of the Metallo-β-Lactamase Superfamily
    [Jun 2014]

    Publication date: 26 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 13

    Author(s): Florian Baier , Nobuhiko Tokuriki

    The expansion of functions in an enzyme superfamily is thought to occur through recruitment of latent promiscuous functions within existing enzymes. Thus, the promiscuous activities of enzymes represent connections between different catalytic landscapes and provide an additional layer of evolutionary connectivity between functional families alongside their sequence and structural relationships. Functional connectivity has been observed between individual functional families; however, little is known about how catalytic landscapes are connected throughout a highly diverged superfamily. Here, we describe a superfamily-wide analysis of evolutionary and functional connectivity in the metallo-β-lactamase (MBL) superfamily. We investigated evolutionary connections between functional families and related evolutionary to functional connectivity; 24 enzymes from 15 distinct functional families were challenged against 10 catalytically distinct reactions. We revealed that enzymes of this superfamily are generally promiscuous, as each enzyme catalyzes on average 1.5 reactions in addition to its native one. Catalytic landscapes in the MBL superfamily overlap substantially; each reaction is connected on average to 3.7 other reactions whereas some connections appear to be unrelated to recent evolutionary events and occur between chemically distinct reactions. These findings support the idea that the highly distinct reactions in the MBL superfamily could have evolved from a common ancestor traversing a continuous network via promiscuous enzymes. Several functional connections (e.g., the lactonase/phosphotriesterase and phosphonatase/phosphodiesterase/arylsulfatase reactions) are also observed in structurally and evolutionary distinct superfamilies, suggesting that these catalytic landscapes are substantially connected. Our results show that new enzymatic functions could evolve rapidly from the current diversity of enzymes and range of promiscuous activities.
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    Categories: Journal Articles
  • Structural Mechanisms Determining Inhibition of the Collagen Receptor DDR1 by Selective and Multi-Targeted Type II Kinase Inhibitors
    [Jun 2014]

    Publication date: 26 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 13

    Author(s): Peter Canning , Li Tan , Kiki Chu , Sam W. Lee , Nathanael S. Gray , Alex N. Bullock

    The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC50 of 9nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and αD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a β-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer.
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    Categories: Journal Articles
  • Elucidation of the Specific Function of the Conserved Threonine Triad Responsible for Human l-Asparaginase Autocleavage and Substrate Hydrolysis
    [Jun 2014]

    Publication date: 26 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 13

    Author(s): Julian Nomme , Ying Su , Arnon Lavie

    Our long-term goal is the design of a human l-asparaginase (hASNase3) variant, suitable for use in cancer therapy without the immunogenicity problems associated with the currently used bacterial enzymes. Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. The key property allowing for the depletion of blood asparagine by bacterial asparaginases is their low micromolar K M value. In contrast, human enzymes have a millimolar K M for asparagine. Toward the goal of engineering an hASNase3 variant with micromolar K M, we conducted a structure/function analysis of the conserved catalytic threonine triad of this human enzyme. As a member of the N-terminal nucleophile family, to become enzymatically active, hASNase3 must undergo autocleavage between residues Gly167 and Thr168. To determine the individual contribution of each of the three conserved active-site threonines (threonine triad Thr168, Thr186, Thr219) for the enzyme-activating autocleavage and asparaginase reactions, we prepared the T168S, T186V and T219A/V mutants. These mutants were tested for their ability to cleave and to catalyze asparagine hydrolysis, in addition to being examined structurally. We also elucidated the first N-terminal nucleophile plant-type asparaginase structure in the covalent intermediate state. Our studies indicate that, while not all triad threonines are required for the cleavage reaction, all are essential for the asparaginase activity. The increased understanding of hASNase3 function resulting from these studies reveals the key regions that govern cleavage and the asparaginase reaction, which may inform the design of variants that attain a low K M for asparagine.
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    Categories: Journal Articles
  • Effect of Serine Phosphorylation and Ser25 Phospho-Mimicking Mutations on Nuclear Localisation and Ligand Interactions of Annexin A2
    [Jun 2014]

    Publication date: 26 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 13

    Author(s): Ann Kari Grindheim , Hanne Hollås , Juan Ramirez , Jaakko Saraste , Gilles Travé , Anni Vedeler

    Annexin A2 (AnxA2) interacts with numerous ligands, including calcium, lipids, mRNAs and intracellular and extracellular proteins. Different post-translational modifications participate in the discrimination of the functions of AnxA2 by modulating its ligand interactions. Here, phospho-mimicking mutants (AnxA2-S25E and AnxA2-S25D) were employed to investigate the effects of Ser25 phosphorylation on the structure and function of AnxA2 by using AnxA2-S25A as a control. The overall α-helical structure of AnxA2 is not affected by the mutations, since the thermal stabilities and aggregation tendencies of the mutants differ only slightly from the wild-type (wt) protein. Unlike wt AnxA2, all mutants bind the anxA2 3′ untranslated region and β-γ-G-actin with high affinity in a Ca2+-independent manner. AnxA2-S25E is not targeted to the nucleus in transfected PC12 cells. In vitro phosphorylation of AnxA2 by protein kinase C increases its affinity to mRNA and inhibits its nuclear localisation, in accordance with the data obtained with the phospho-mimicking mutants. Ca2+-dependent binding of wt AnxA2 to phosphatidylinositol, phosphatidylinositol-3-phosphate, phosphatidylinositol-4-phosphate and phosphatidylinositol-5-phosphate, as well as weaker but still Ca2+-dependent binding to phosphatidylserine and phosphatidylinositol-3,5-bisphosphate, was demonstrated by a protein–lipid overlay assay, whereas binding of AnxA2 to these lipids, as well as its binding to liposomes, is inhibited by the Ser25 mutations. Thus, introduction of a modification (mutation or phosphorylation) at Ser25 appears to induce a conformational change leading to increased accessibility of the mRNA- and G-actin-binding sites in domain IV independent of Ca2+ levels, while the Ca2+-dependent binding of AnxA2 to phospholipids is attenuated.
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    Categories: Journal Articles
  • A Bacteriophage Capsid Protein Provides a General Amyloid Interaction Motif (GAIM) That Binds and Remodels Misfolded Protein Assemblies
    [Jun 2014]

    Publication date: 26 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 13

    Author(s): Rajaraman Krishnan , Haim Tsubery , Ming Y. Proschitsky , Eva Asp , Michal Lulu , Sharon Gilead , Myra Gartner , Jonathan P. Waltho , Peter J. Davis , Andrea M. Hounslow , Daniel A. Kirschner , Hideyo Inouye , David G. Myszka , Jason Wright , Beka Solomon , Richard A. Fisher

    Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis–trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aβ binding to g3p. NMR studies show that g3p binding to Aβ fibers is predominantly through middle and C-terminal residues of the Aβ subunit, indicating β strand–g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aβ fibers (fAβ) (K D =9.4nM); (2); blocks fAβ assembly (IC50 ~50nM) and (3) dissociates fAβ (EC50 =40–100nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif.
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    Categories: Journal Articles
  • Redefining the Dry Molten Globule State of Proteins
    [Jun 2014]

    Publication date: 26 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 13

    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 (TTET) on the villin headpiece subdomain (HP35) revealed an equilibrium between a locked native state 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 (DMG), 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 TTET measurements. Increasing pressure shifts the equilibrium from the locked toward the unlocked state, with a small negative reaction volume for unlocking (ΔV 0 =−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 DMGs, prompting a broader definition of this state.
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    Categories: Journal Articles
  • Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation
    [Jun 2014]

    Publication date: 26 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 13

    Author(s): Minttu Virkki , Carolina Boekel , Kristoffer Illergård , Christoph Peters , Nanjiang Shu , Konstantinos D. Tsirigos , Arne Elofsson , Gunnar von Heijne , IngMarie Nilsson

    While early structural models of helix-bundle integral membrane proteins posited that the transmembrane α-helices [transmembrane helices (TMHs)] were orientated more or less perpendicular to the membrane plane, there is now ample evidence from high-resolution structures that many TMHs have significant tilt angles relative to the membrane. Here, we address the question whether the tilt is an intrinsic property of the TMH in question or if it is imparted on the TMH during folding of the protein. Using a glycosylation mapping technique, we show that four highly tilted helices found in multi-spanning membrane proteins all have much shorter membrane-embedded segments when inserted by themselves into the membrane than seen in the high-resolution structures. This suggests that tilting can be induced by tertiary packing interactions within the protein, subsequent to the initial membrane-insertion step.
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    Categories: Journal Articles
  • Editorial Board
    [Jun 2014]

    Publication date: 12 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 12









    Categories: Journal Articles
  • Contents List
    [Jun 2014]

    Publication date: 12 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 12









    Categories: Journal Articles
  • Beyond DnaA: The Role of DNA Topology and DNA Methylation in Bacterial Replication Initiation
    [Jun 2014]

    Publication date: 12 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 12

    Author(s): Rafał Donczew , Jolanta Zakrzewska-Czerwińska , Anna Zawilak-Pawlik

    The replication of chromosomal DNA is a fundamental event in the life cycle of every cell. The first step of replication, initiation, is controlled by multiple factors to ensure only one round of replication per cell cycle. The process of initiation has been described most thoroughly for bacteria, especially Escherichia coli, and involves many regulatory proteins that vary considerably between different species. These proteins control the activity of the two key players of initiation in bacteria: the initiator protein DnaA and the origin of chromosome replication (oriC). Factors involved in the control of the availability, activity, or oligomerization of DnaA during initiation are generally regarded as the most important and thus have been thoroughly characterized. Other aspects of the initiation process, such as origin accessibility and susceptibility to unwinding, have been less explored. However, recent findings indicate that these factors have a significant role. This review focuses on DNA topology, conformation, and methylation as important factors that regulate the initiation process in bacteria. We present a comprehensive summary of the factors involved in the modulation of DNA topology, both locally at oriC and more globally at the level of the entire chromosome. We show clearly that the conformation of oriC dynamically changes, and control of this conformation constitutes another, important factor in the regulation of bacterial replication initiation. Furthermore, the process of initiation appears to be associated with the dynamics of the entire chromosome and this association is an important but largely unexplored phenomenon.
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    Categories: Journal Articles
  • A Non-Active-Site SET Domain Surface Crucial for the Interaction of MLL1 and the RbBP5/Ash2L Heterodimer within MLL Family Core Complexes
    [Jun 2014]

    Publication date: 12 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 12

    Author(s): Stephen A. Shinsky , Michael Hu , Valarie E. Vought , Sarah B. Ng , Michael J. Bamshad , Jay Shendure , Michael S. Cosgrove

    The mixed lineage leukemia-1 (MLL1) enzyme is a histone H3 lysine 4 (H3K4) monomethyltransferase and has served as a paradigm for understanding the mechanism of action of the human SET1 family of enzymes that include MLL1–MLL4 and SETd1a,b. Dimethylation of H3K4 requires a sub-complex including WRAD (WDR5, RbBP5, Ash2L, and DPY-30), which binds to each SET1 family member forming a minimal core complex that is required for multiple lysine methylation. We recently demonstrated that WRAD is a novel histone methyltransferase that preferentially catalyzes H3K4 dimethylation in a manner that is dependent on an unknown non-active-site surface from the MLL1 SET domain. Recent genome sequencing studies have identified a number of human disease-associated missense mutations that localize to the SET domains of several MLL family members. In this investigation, we mapped many of these mutations onto the three-dimensional structure of the SET domain and noticed that a subset of MLL2 (KMT2D, ALR, MLL4)-associated Kabuki syndrome missense mutations map to a common solvent-exposed surface that is not expected to alter enzymatic activity. We introduced these mutations into the MLL1 SET domain and observed that all are defective for H3K4 dimethylation by the MLL1 core complex, which is associated with a loss of the ability of MLL1 to interact with WRAD or with the RbBP5/Ash2L heterodimer. Our results suggest that amino acids from this surface, which we term the Kabuki interaction surface or KIS, are required for formation of a second active site within SET1 family core complexes.
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    Categories: Journal Articles
  • Folding Pathways of the Tetrahymena Ribozyme
    [Jun 2014]

    Publication date: 12 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 12

    Author(s): David Mitchell III , Rick Russell

    Like many structured RNAs, the Tetrahymena group I intron ribozyme folds through multiple pathways and intermediates. Under standard conditions in vitro, a small fraction reaches the native state (N) with k obs ≈0.6min−1, while the remainder forms a long-lived misfolded conformation (M) thought to differ in topology. These alternative outcomes reflect a pathway that branches late in folding, after disruption of a trapped intermediate (Itrap). Here we use catalytic activity to probe the folding transitions from Itrap to the native and misfolded states. We show that mutations predicted to weaken the core helix P3 do not increase the rate of folding from Itrap but they increase the fraction that reaches the native state rather than forming the misfolded state. Thus, P3 is disrupted during folding to the native state but not to the misfolded state, and P3 disruption occurs after the rate-limiting step. Interestingly, P3-strengthening mutants also increase native folding. Additional experiments show that these mutants are rapidly committed to folding to the native state, although they reach the native state with approximately the same rate constant as the wild-type ribozyme (~1min−1). Thus, the P3-strengthening mutants populate a distinct pathway that includes at least one intermediate but avoids the M state, most likely because P3 and the correct topology are formed early. Our results highlight multiple pathways in RNA folding and illustrate how kinetic competitions between rapid events can have long-lasting effects because the “choice” is enforced by energy barriers that grow larger as folding progresses.
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    Categories: Journal Articles
  • The Crystal Structure of the Anti-σ Factor CnrY in Complex with the σ Factor CnrH Shows a New Structural Class of Anti-σ Factors Targeting Extracytoplasmic Function σ Factors
    [Jun 2014]

    Publication date: 12 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 12

    Author(s): Antoine P. Maillard , Eric Girard , Widade Ziani , Isabelle Petit-Härtlein , Richard Kahn , Jacques Covès

    Gene expression in bacteria is regulated at the level of transcription initiation, a process driven by σ factors. The regulation of σ factor activity proceeds from the regulation of their cytoplasmic availability, which relies on specific inhibitory proteins called anti-σ factors. With anti-σ factors regulating their availability according to diverse cues, extracytoplasmic function σ factors (σECF) form a major signal transduction system in bacteria. Here, structure:function relationships have been characterized in an emerging class of minimal-size transmembrane anti-σ factors, using CnrY from Cupriavidus metallidurans CH34 as a model. This study reports the 1.75-Å-resolution structure of CnrY cytosolic domain in complex with CnrH, its cognate σECF, and identifies a small hydrophobic knob in CnrY as the major determinant of this interaction in vivo. Unsuspected structural similarity with the molecular switch regulating the general stress response in α-proteobacteria unravels a new class of anti-σ factors targeting σECF. Members of this class carry out their function via a 30-residue stretch that displays helical propensity but no canonical structure on its own.
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    Categories: Journal Articles
  • Alanine Scanning Mutagenesis Identifies an Asparagine–Arginine–Lysine Triad Essential to Assembly of the Shell of the Pdu Microcompartment
    [Jun 2014]

    Publication date: 12 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 12

    Author(s): Sharmistha Sinha , Shouqiang Cheng , Yea Won Sung , Dan E. McNamara , Michael R. Sawaya , Todd O. Yeates , Thomas A. Bobik

    Bacterial microcompartments (MCPs) are the simplest organelles known. They function to enhance metabolic pathways by confining several related enzymes inside an all-protein envelope called the shell. In this study, we investigated the factors that govern MCP assembly by performing scanning mutagenesis on the surface residues of PduA, a major shell protein of the MCP used for 1,2-propanediol degradation. Biochemical, genetic, and structural analysis of 20 mutants allowed us to determine that PduA K26, N29, and R79 are crucial residues that stabilize the shell of the 1,2-propanediol MCP. In addition, we identify two PduA mutants (K37A and K55A) that impair MCP function most likely by altering the permeability of its protein shell. These are the first studies to examine the phenotypic effects of shell protein structural mutations in an MCP system. The findings reported here may be applicable to engineering protein containers with improved stability for biotechnology applications.
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    Categories: Journal Articles
  • Co-Crystallization with Conformation-Specific Designed Ankyrin Repeat Proteins Explains the Conformational Flexibility of BCL-W
    [Jun 2014]

    Publication date: 12 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 12

    Author(s): Johannes Schilling , Jendrik Schöppe , Evelyn Sauer , Andreas Plückthun

    BCL-W is a member of the BCL-2 family of anti-apoptotic proteins. A key event in the regulation of apoptosis is the heterodimerization between anti-apoptotic and pro-apoptotic family members, which involves a conserved surface-exposed groove on the anti-apoptotic proteins. Crystal structures of the ligand binding-competent conformation exist for all anti-apoptotic family members, with the exception of BCL-W, due to the flexibility of the BCL-W groove region. Existing structures had suggested major deviations of the BCL-W groove region from the otherwise structurally highly related remaining anti-apoptotic family members. To capture its ligand binding-competent conformation by counteracting the conformational flexibility of the BCL-W groove, we had selected high-affinity groove-binding designed ankyrin repeat proteins (DARPins) using ribosome display. We now determined two high-resolution crystal structures of human BCL-W in complex with different DARPins at resolutions 1.5 and 1.85Å, in which the structure of BCL-W is virtually identical, and BCL-W adopts a conformation extremely similar to the ligand-free conformation of its closest relative BCL-XL in both structures. However, distinct differences to all previous BCL-W structures are evident, notably in the ligand-binding region. We provide the first structural explanation for the conformational flexibility of the BCL-W groove region in comparison to other BCL-2 family members. Due to the importance of the anti-apoptotic BCL-2 family as drug targets, the presented crystal structure of ligand binding-competent BCL-W may serve as a valuable basis for structure-based drug design in the future and provides a missing piece for the structural characterization of this protein family.
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    Categories: Journal Articles
  • The Nucleolar PICT-1/GLTSCR2 Protein Forms Homo-Oligomers
    [Jun 2014]

    Publication date: 12 June 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 12

    Author(s): Tatyana Borodianskiy-Shteinberg , Inna Kalt , Sarit Kipper , Nofar Nachum , Shiri Katz , Maor H. Pauker , Mira Barda-Saad , Doron Gerber , Ronit Sarid

    The human “protein interacting with carboxyl terminus 1” (PICT-1), also designated as the “glioma tumor suppressor candidate region 2 gene product”, GLTSCR2, is a nucleolar protein whose activity is, as yet, unknown. Contradictory results regarding the role of PICT-1 in cancer have been reported, and PICT-1 has been suggested to function either as a tumor suppressor protein or as an oncogene. In this study, we demonstrate self-association of PICT-1. Through yeast two-hybrid assay, we identified PICT-1 as its own interaction partner. We confirmed the interaction of PICT-1 with itself by direct yeast two-hybrid assay and also showed self-association of PICT-1 in mammalian cells by co-immunoprecipitation and fluorescence resonance energy transfer assays. Furthermore, we confirmed direct self-association of PICT-1 by using in vitro microfluidic affinity binding assays. The later assay also identified the carboxy-terminal domain as mediating self-interaction of PICT-1. Glutaraldehyde cross-linking and gel-filtration assays suggest that PICT-1 forms dimers, though it may form higher-order complexes as well. Our findings add another layer of complexity in understanding the different functions of PICT-1 and may help provide insights regarding the activities of this protein.
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    Categories: Journal Articles