Journal of Molecular Biology

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Synthetic biology – toward therapeutic solutions

Tue, 09/01/2015 - 23:35
Publication date: Available online 1 September 2015
Source:Journal of Molecular Biology

Author(s): Viktor Haellman, Martin Fussenegger

Higher multi-cellular organisms have evolved sophisticated intra- and inter-cellular biological networks that enable cell growth and survival to fulfil an organism’s needs. Although such networks allow the assembly of complex tissues and even provide healing and protective capabilities, malfunctioning cells can have severe consequences for an organism’s survival. In humans, such events can result in severe disorders and diseases, including metabolic and immunological disorders [1, 2], as well as cancer [3]. Dominating the therapeutic frontier for these potentially lethal disorders, cell and gene therapies aim to relieve or eliminate patient suffering by restoring the function of damaged, diseased, and aging cells and tissues via the introduction of healthy cells or alternative genes. However, despite recent success, these efforts have yet to achieve sufficient therapeutic effects, and further work is needed to ensure the safe and precise control of transgene expression and cellular processes. In this review, we describe the biological tools and devices that are at the forefront of synthetic biology and discuss their potential to advance the specificity, efficiency, and safety of the current generation of cell and gene therapies, including how they can be used to confer curative effects that far surpass those of conventional therapeutics. We also highlight the current therapeutic delivery tools and the current limitations that hamper their use in human applications.
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Structural insights into KCTD protein assembly and Cullin3 recognition

Tue, 09/01/2015 - 23:35
Publication date: Available online 31 August 2015
Source:Journal of Molecular Biology

Author(s): Alan X. Ji, Anh Chu, Tine Kragh Nielsen, Samir Benlekbir, John L. Rubinstein, Gilbert G. Privé

Cullin3 (Cul3)-based ubiquitin E3 ligase complexes catalyze the transfer of ubiquitin from an E2 enzyme to target substrate proteins. In these assemblies, the C-terminal region of Cul3 binds Rbx1/E2~ubiquitin, while the N-terminal region interacts with various BTB domain proteins that serve as substrate adaptors. Previous crystal structures of the homodimeric BTB proteins KLHL3, KLHL11 and SPOP in complex with the N-terminal domain of Cul3 revealed the features required for Cul3 recognition in these proteins. A second class of BTB-domain containing proteins, the KCTD proteins, are also Cul3 substrate adaptors, but these do not share many of the previously identified determinants for Cul3 binding. We report the pentameric crystal structures of the KCTD1 and KCTD9 BTB domains, and identify plasticity in the KCTD1 rings. We find that the KCTD proteins 5, 6, 9, and 17 bind to Cul3 with high affinity, while the KCTD proteins 1 and 16 do not have detectable binding. Finally, we confirm the 5:5 assembly of KCTD9/Cul3 complexes by electron cryomicroscopy and provide a molecular rationale for BTB-mediated Cul3 binding specificity in the KCTD family.
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Editorial Board

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17









Categories: Journal Articles

Contents List

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17









Categories: Journal Articles

Exposed: The Many and Varied Roles of Phospholipase C γ SH2 Domains

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Paul C. Driscoll







Categories: Journal Articles

Scaffold Protein SLP-76 Primes PLCγ1 for Activation by ITK-Mediated Phosphorylation

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Sujan Devkota, Raji E. Joseph, Lie Min, D. Bruce Fulton, Amy H. Andreotti

Activation of the phospholipase, PLCγ1, is critical for proper T cell signaling following antigen receptor engagement. In T cells, the Tec family kinase, interleukin-2-induced tyrosine kinase (ITK), phosphorylates PLCγ1 at tyrosine 783 (Y783) leading to activation of phospholipase function and subsequent production of the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. In this work, we demonstrate that PLCγ1 can be primed for ITK-mediated phosphorylation on Y783 by a specific region of the adaptor protein, SLP-76. The SLP-76 phosphotyrosine-containing sequence, pY173IDR, does not conform to the canonical recognition motif for an SH2 domain yet binds with significant affinity to the C-terminal SH2 domain of PLCγ1 (SH2C). The SLP-76 pY173 motif competes with the autoinhibited conformation surrounding the SH2C domain of PLCγ1 leading to exposure of the ITK recognition element on the PLCγ1 SH2 domain and release of the target tyrosine, Y783. These data contribute to the evolving model for the molecular events occurring early in the T cell activation process.
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A Regulated, Ubiquitin-Independent Degron in IκBα

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Karen T. Fortmann, Russell D. Lewis, Kim A. Ngo, Riku Fagerlund, Alexander Hoffmann

Whereas ubiquitin-dependent degrons have been characterized in some detail, how proteins may be targeted to ubiquitin-independent proteasomal degradation remains unclear. Here we show that IκBα contains an ubiquitin-independent degron whose activity is portable to heterologous proteins such as the globular protein GFP (green fluorescent protein) via a proteasome-dependent, ubiquitin-independent, non-lysosomal pathway. The ubiquitin-independent degradation signal resides in an 11-amino-acid sequence, which is not only sufficient but also required for IκBα's short half-life. Finally, we show that this degron's activity is regulated by the interaction with NFκB, which controls its solvent exposure, and we demonstrate that this regulation of the degron's activity is critical for IκBα's signaling functions.
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Over-Expression Analysis of All Eight Subunits of the Molecular Chaperone CCT in Mammalian Cells Reveals a Novel Function for CCTdelta

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Matthias Spiess, Meriem Echbarthi, Andreas Svanström, Roger Karlsson, Julie Grantham

Chaperonin containing tailless complex polypeptide 1 (CCT) forms a classical chaperonin barrel structure where two rings of subunits surround a central cavity. Each ring consists of eight distinct subunits, creating a complex binding interface that makes CCT unique among the chaperonins. In addition to acting as a multimeric chaperonin, there is increasing evidence indicating that the CCT subunits, when monomeric, possess additional functions. Here we assess the role of the CCT subunits individually, using a GFP (green fluorescent protein) tagging approach to express each of the subunits in their monomeric form in cultured mammalian cells. Over-expression of CCTdelta, but not the other seven CCT subunits, results in the appearance of numerous protrusions at the cell surface. Two point mutations, one in the apical domain and one in the ATP binding pocket of CCTdelta, that abolish protrusion formation have been identified, consistent with the apical domain containing a novel interaction site that is influenced by the ATPase activity in the equatorial domain. Structured illumination microscopy, together with sub-cellular fractionation, reveals that only the wild-type CCTdelta is associated with the plasma membrane, thus connecting spatial organization with surface protrusion formation. Expression of the equivalent subunit in yeast, GFP-Cct4, rescues growth of the temperature-sensitive strain cct4-1 at the non-permissive temperature, indicative of conserved subunit-specific activities for CCTdelta.
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Congenital Cataract-Causing Mutation G129C in γC-Crystallin Promotes the Accumulation of Two Distinct Unfolding Intermediates That Form Highly Toxic Aggregates

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Yi-Bo Xi, Xiang-Jun Chen, Wei-Jie Zhao, Yong-Bin Yan

Cataract is a lens opacification disease prevalent worldwide. Cataract-causing mutations in crystallins generally lead to the formation of light-scattering particles in the lens. However, it remains unclear for the detailed structural and pathological mechanisms of most mutations. In this study, we showed that the G129C mutation in γC-crystallin, which is associated with autosomal dominant congenital nuclear cataract, perturbed the unfolding process by promoting the accumulation of two distinct aggregation-prone intermediates under mild denaturing conditions. The abnormally accumulated intermediates escaped from the chaperone-like function of αA-crystallin during refolding. Molecular dynamics simulations indicated that the mutation altered domain pairing geometry and allowed the penetration of extra solvent molecules into the domain binding interface, thereby weakening domain binding energy. Under mild denaturation conditions, the increased domain movements may facilitate the formation of non-native oligomers via domain swapping, which further assembled into amyloid-like fibrils. The intermediate that appeared at 1.6M guanidine hydrochloride was more compact and less aggregatory than the one populated at 0.9M guanidine hydrochloride, which was caused by the increased solvation of acidic residues in the ion-pairing network via the competitive binding of guanidinium ions. More importantly, both the amyloid-like fibrils preformed in vitro and intracellular aggresomes formed by exogenously overexpressed mutant proteins significantly inhibited cell proliferation and induced cell death. The combined data from spectroscopic, structural and cellular studies strongly suggest that both the formation of light-scattering aggregates and the toxic effects of the aggregates may contribute to the onset and development of cataract.
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Metavinculin Tunes the Flexibility and the Architecture of Vinculin-Induced Bundles of Actin Filaments

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Zeynep A. Oztug Durer, Rebecca M. McGillivary, Hyeran Kang, W. Austin Elam, Christina L. Vizcarra, Dorit Hanein, Enrique M. De La Cruz, Emil Reisler, Margot E. Quinlan

Vinculin is an abundant protein found at cell–cell and cell–extracellular matrix junctions. In muscles, a longer splice isoform of vinculin, metavinculin, is also expressed. The metavinculin-specific insert is part of the C-terminal tail domain, the actin-binding site of both isoforms. Mutations in the metavinculin-specific insert are linked to heart disease such as dilated cardiomyopathies. Vinculin tail domain (VT) both binds and bundles actin filaments. Metavinculin tail domain (MVT) binds actin filaments in a similar orientation but does not bundle filaments. Recently, MVT was reported to sever actin filaments. In this work, we asked how MVT influences F-actin alone or in combination with VT. Cosedimentation and limited proteolysis experiments indicated a similar actin binding affinity and mode for both VT and MVT. In real-time total internal reflection fluorescence microscopy experiments, MVT's severing activity was negligible. Instead, we found that MVT binding caused a 2-fold reduction in F-actin's bending persistence length and increased susceptibility to breakage. Using mutagenesis and site-directed labeling with fluorescence probes, we determined that MVT alters actin interprotomer contacts and dynamics, which presumably reflect the observed changes in bending persistence length. Finally, we found that MVT decreases the density and thickness of actin filament bundles generated by VT. Altogether, our data suggest that MVT alters actin filament flexibility and tunes filament organization in the presence of VT. Both of these activities are potentially important for muscle cell function. Perhaps MVT allows the load of muscle contraction to act as a signal to reorganize actin filaments.
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Formation of Tertiary Interactions during rRNA GTPase Center Folding

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Michael J. Rau, Robb Welty, W. Tom Stump, Kathleen B. Hall

The 60-nt GTPase center (GAC) of 23S rRNA has a phylogenetically conserved secondary structure with two hairpin loops and a 3-way junction. It folds into an intricate tertiary structure upon addition of Mg2+ ions, which is stabilized by the L11 protein in cocrystal structures. Here, we monitor the kinetics of its tertiary folding and Mg2+-dependent intermediate states by observing selected nucleobases that contribute specific interactions to the GAC tertiary structure in the cocrystals. The fluorescent nucleobase 2-aminopurine replaced three individual adenines, two of which make long-range stacking interactions and one that also forms hydrogen bonds. Each site reveals a unique response to Mg2+ addition and temperature, reflecting its environmental change from secondary to tertiary structure. Stopped-flow fluorescence experiments revealed that kinetics of tertiary structure formation upon addition of MgCl2 are also site specific, with local conformational changes occurring from 5ms to 4s and with global folding from 1 to 5s. Site-specific substitution with 15N-nucleobases allowed observation of stable hydrogen bond formation by NMR experiments. Equilibrium titration experiments indicate that a stable folding intermediate is present at stoichiometric concentrations of Mg2+ and suggest that there are two initial sites of Mg2+ ion association.
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Structure/Function Analysis of Protein–Protein Interactions Developed by the Yeast Pih1 Platform Protein and Its Partners in Box C/D snoRNP Assembly

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Marc Quinternet, Benjamin Rothé, Muriel Barbier, Claude Bobo, Jean-Michel Saliou, Clémence Jacquemin, Régis Back, Marie-Eve Chagot, Sarah Cianférani, Philippe Meyer, Christiane Branlant, Bruno Charpentier, Xavier Manival

In eukaryotes, nucleotide post-transcriptional modifications in RNAs play an essential role in cell proliferation by contributing to pre-ribosomal RNA processing, ribosome assembly and activity. Box C/D small nucleolar ribonucleoparticles catalyze site-specific 2′-O-methylation of riboses, one of the most prevalent RNA modifications. They contain one guide RNA and four core proteins and their in vivo assembly requires numerous factors including (HUMAN/Yeast) BCD1/Bcd1p, NUFIP1/Rsa1p, ZNHIT3/Hit1p, the R2TP complex composed of protein PIH1D1/Pih1p and RPAP3/Tah1p that bridges the R2TP complex to the HSP90/Hsp82 chaperone and two AAA+ ATPases. We show that Tah1p can stabilize Pih1p in the absence of Hsp82 activity during the stationary phase of growth and consequently that the Tah1p:Pih1p interaction is sufficient for Pih1p stability. This prompted us to establish the solution structure of the Tah1p:Pih1p complex by NMR. The C-terminal tail S93-S111 of Tah1p snakes along Pih1p264-344 folded in a CS domain to form two intermolecular β-sheets and one covering loop. However, a thorough inspection of the NMR and crystal structures revealed structural differences that may be of functional importance. In addition, our NMR and isothermal titration calorimetry data revealed the formation of direct contacts between Pih1p257-344 and the Hsp82MC domain in the presence of Tah1p. By co-expression in Escherichia coli, we demonstrate that Pih1p has two other direct partners, the Rsa1p assembly factor and the Nop58p core protein, and in vivo and in vitro experiments mapped the required binding domains. Our data suggest that these two interactions are mutually exclusive. The implication of this finding for box C/D small nucleolar ribonucleoparticle assembly is discussed.
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Structures of DegQ from Legionella pneumophila Define Distinct ON and OFF States

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Alexander Schubert, Robert Wrase, Rolf Hilgenfeld, Guido Hansen

HtrA (high-temperature requirement A) family proteins play important roles in protein-quality control processes in the bacterial periplasm. A common feature of all members of this family is their modular organization comprising a chymotrypsin-like protease domain and at least one PDZ (postsynaptic density of 95kDa, disks large homolog 1 and zonula occludens 1) domain. All characterized HtrA proteins assemble into complex oligomers consisting of typically 3–24 monomers, which allow a tight regulation of proteolytic activity. Here, we provide evidence that the assembly of proteolytically active, higher-order complexes of DegQ from Legionella pneumophila is triggered by the binding of substrate-derived peptides. Crystal structures of inactive 3-mers and active 12-mers of DegQ reveal molecular details of elements of a conserved allosteric activation cascade that defines distinct protease ON and OFF states. Results from DegQLp variants harboring structure-based amino acid substitutions indicate that peptide binding to the PDZ1 domain is critical for proteolytic activity but not for the formation of higher-order oligomers. Combining structural, mutagenesis and biochemical data, we show that, in contrast to the proteolytic activity, the chaperone function of DegQ is not affected by the state of the activation cascade.
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Structures of the Ultra-High-Affinity Protein–Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Amar Joshi, Rhys Grinter, Inokentijs Josts, Sabrina Chen, Justyna A. Wojdyla, Edward D. Lowe, Renata Kaminska, Connor Sharp, Laura McCaughey, Aleksander W. Roszak, Richard J. Cogdell, Olwyn Byron, Daniel Walker, Colin Kleanthous

How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase–Im interaction is a model system for the study of high-affinity protein–protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase–Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase–ImS2 and pyocin AP41 DNase–ImAP41. These structures represent divergent DNase–Im subfamilies and are important in extending our understanding of protein–protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase–Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase–Immunity pairs that appear to underpin the split of this family into two distinct groups.
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Catalytic Role of the Substrate Defines Specificity of Therapeutic l-Asparaginase

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Andriy Anishkin, Juan M. Vanegas, David M. Rogers, Philip L. Lorenzi, Wai Kin Chan, Preeti Purwaha, John N. Weinstein, Sergei Sukharev, Susan B. Rempe

Type II bacterial l-asparaginases (l-ASP) have played an important therapeutic role in cancer treatment for over four decades, yet their exact reaction mechanism remains elusive. l-ASP from Escherichia coli deamidates asparagine (Asn) and glutamine, with an ~104 higher specificity (k cat/K m) for asparagine despite only one methylene difference in length. Through a sensitive kinetic approach, we quantify competition among the substrates and interpret its clinical role. To understand specificity, we use molecular simulations to characterize enzyme interactions with substrates and a product (aspartate). We present evidence that the aspartate product in the crystal structure of l-ASP exists in an unusual α-COOH protonation state. Consequently, the set of enzyme–product interactions found in the crystal structure, which guided prior mechanistic interpretations, differs from those observed in dynamic simulations of the enzyme with the substrates. Finally, we probe the initial nucleophilic attack with ab initio simulations. The unusual protonation state reappears, suggesting that crystal structures (wild type and a T89V mutant) represent intermediate steps rather than initial binding. Also, a proton transfers spontaneously to Asn, advancing a new hypothesis that the substrate's α-carboxyl serves as a proton acceptor and activates one of the catalytic threonines during l-ASP's nucleophilic attack on the amide carbon. That hypothesis explains for the first time why proximity of the substrate α-COO− group to the carboxamide is absolutely required for catalysis. The substrate's catalytic role is likely the determining factor in enzyme specificity as it constrains the allowed distance between the backbone carboxyl and the amide carbon of any l-ASP substrate.
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The Contribution of Missense Mutations in Core and Rim Residues of Protein–Protein Interfaces to Human Disease

Tue, 09/01/2015 - 23:35
Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Alessia David, Michael J.E. Sternberg

Missense mutations at protein–protein interaction sites, called interfaces, are important contributors to human disease. Interfaces are non-uniform surface areas characterized by two main regions, “core” and “rim”, which differ in terms of evolutionary conservation and physicochemical properties. Moreover, within interfaces, only a small subset of residues (“hot spots”) is crucial for the binding free energy of the protein–protein complex. We performed a large-scale structural analysis of human single amino acid variations (SAVs) and demonstrated that disease-causing mutations are preferentially located within the interface core, as opposed to the rim (p <0.01). In contrast, the interface rim is significantly enriched in polymorphisms, similar to the remaining non-interacting surface. Energetic hot spots tend to be enriched in disease-causing mutations compared to non-hot spots (p =0.05), regardless of their occurrence in core or rim residues. For individual amino acids, the frequency of substitution into a polymorphism or disease-causing mutation differed to other amino acids and was related to its structural location, as was the type of physicochemical change introduced by the SAV. In conclusion, this study demonstrated the different distribution and properties of disease-causing SAVs and polymorphisms within different structural regions and in relation to the energetic contribution of amino acid in protein–protein interfaces, thus highlighting the importance of a structural system biology approach for predicting the effect of SAVs.
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Pseudomonas aeruginosa biofilm infections: community structure, antimicrobial tolerance and immune response

Tue, 09/01/2015 - 23:35
Publication date: Available online 28 August 2015
Source:Journal of Molecular Biology

Author(s): Morten Rybtke, Louise Dahl Hultqvist, Michael Givskov, Tim Tolker-Nielsen

Studies of biopsies from infectious sites, explanted tissue and medical devises have provided evidence that biofilms are the underlying cause of a variety of tissue-associated and implant-associated recalcitrant human infections. With a need for novel anti-biofilm treatment strategies research in biofilm infection microbiology, biofilm formation mechanisms, and biofilm-associated antimicrobial tolerance has become an important area in microbiology. Substantial knowledge about biofilm formation mechanisms, biofilm-associated antimicrobial tolerance and immune evasion mechanisms has been obtained through work with biofilms grown in in vitro experimental setups, and the relevance of this information in the context of chronic infections is being investigated by the use of animal models of infection. Because our current in vitro experimental setups and animal models have limitations new advanced in vitro models developed with knowledge about the chemical landscape at infectious sites are needed.
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STAC – a new domain associated with transmembrane solute transport and two-component signal transduction systems

Tue, 09/01/2015 - 23:35
Publication date: Available online 28 August 2015
Source:Journal of Molecular Biology

Author(s): Mateusz Korycinski, Reinhard Albrecht, Astrid Ursinus, Marcus D. Hartmann, Murray Coles, Jörg Martin, Stanislaw Dunin-Horkawicz, Andrei N. Lupas

Transmembrane receptors are integral components of sensory pathways in prokaryotes. These receptors share a common dimeric architecture, consisting in its basic form of an N-terminal extracellular sensor, transmembrane helices, and an intracellular effector. As an exception, we have identified an archaeal receptor family – exemplified by Af1503 from Archaeoglobus fulgidus – that is C-terminally shortened, lacking a recognizable effector module. Instead, a HAMP domain forms the sole extension for signal transduction in the cytosol. Here we examine the gene environment of Af1503-like receptors and find a frequent association with transmembrane transport proteins. Furthermore, we identify and define a closely associated new protein domain family, which we characterize structurally using Af1502 from A. fulgidus. Members of this family are found both as stand-alone proteins and as domains within extant receptors. In general, the latter appear as connectors between solute carrier 5 (SLC5)–like transmembrane domains and two-component signal transduction (TCST) domains. This is seen for example in the histidine kinase CbrA, which is a global regulator of metabolism, virulence, and antibiotic resistance in Pseudomonads. We propose that this newly identified domain family mediates signal transduction in systems regulating transport processes and name it STAC, for SLC and TCST Associated Component.
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When the scaffold can’t be ignored: The role of the hydrophobic core in ligand binding and specificity

Tue, 09/01/2015 - 23:35
Publication date: Available online 21 August 2015
Source:Journal of Molecular Biology

Author(s): Diana A. Koulechova, Katherine W. Tripp, Geoffrey Horner, Susan Marqusee

The traditional view of protein-ligand binding treats a protein as comprising distinct binding epitopes on the surface of a degenerate structural scaffold, largely ignoring the impact of a protein’s energy landscape. To determine the robustness of this simplification, we compared two small helix-turn-helix transcription factors with different energy landscapes. λ-repressor is stable and well folded, while MarA appears to be marginally stable with multiple native conformations (molten). While λ-repressor is known to tolerate any hydrophobic mutation in the core, we find MarA drastically less tolerant to core mutation. Moreover, core mutations in MarA (distant from the DNA-binding interface) change the relative affinities of its binding partners, altering ligand specificity. These results can be explained by taking into account the effects of mutations on the entire energy landscape and not just the native state. Thus, for proteins with multiple conformations that are close in energy, such as many intrinsically disordered proteins, residues distant from the active site can alter both binding affinity and specificity.
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Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22

Tue, 09/01/2015 - 23:35
Publication date: Available online 21 August 2015
Source:Journal of Molecular Biology

Author(s): Reginald McNulty, Ravi Kumar Lokareddy, Ankoor Roy, Yang Yang, Gabriel C. Lander, Albert J.R. Heck, John E. Johnson, Gino Cingolani

Packaging of viral genomes inside empty procapsids is driven by a powerful ATP-hydrolyzing motor, formed in many double-stranded DNA viruses by a complex of a small terminase (S-terminase) subunit and a large terminase (L-terminase) subunit, transiently docked at the portal vertex during genome packaging. Despite recent progress in elucidating the structure of individual terminase subunits and their domains, little is known about the architecture of an assembled terminase complex. Here, we describe a bacterial co-expression system that yields milligram quantities of the S-terminase:L-terminase complex of the Salmonella phage P22. In vivo assembled terminase complex was affinity-purified and stabilized by addition of non-hydrolyzable ATP, which binds specifically to the ATPase domain of L-terminase. Mapping studies revealed that the N-terminus of L-terminase ATPase domain (residues 1–58) contains a minimal S-terminase binding domain sufficient for stoichiometric association with residues 140–162 of S-terminase, the L-terminase binding domain. Hydrodynamic analysis by analytical ultracentrifugation sedimentation velocity and native mass spectrometry revealed that the purified terminase complex consists predominantly of one copy of the nonameric S-terminase bound to two equivalents of L-terminase (1S-terminase:2L-terminase). Direct visualization of this molecular assembly in negative-stained micrographs yielded a three-dimensional asymmetric reconstruction that resembles a “nutcracker” with two L-terminase protomers projecting from the C-termini of an S-terminase ring. This is the first direct visualization of a purified viral terminase complex analyzed in the absence of DNA and procapsid.
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Categories: Journal Articles