Structural and functional analysis of the middle segment of hsp90: implications for ATP hydrolysis and client protein and cochaperone interactions

Activation of client proteins by the Hsp90 molecular chaperone is dependent on binding and hydrolysis of ATP, which drives a molecular clamp via transient dimerization of the N-terminal domains. The crystal structure of the middle segment of yeast Hsp90 reveals considerable evolutionary divergence from the equivalent regions of other GHKL protein family members such as MutL and GyrB, including an additional domain of new fold. Using the known structure of the N-terminal nucleotide binding domain, a model for the Hsp90 dimer has been constructed. From this structure, residues implicated in the ATPase-coupled conformational cycle and in interactions with client proteins and the activating cochaperone Aha1 have been identified, and their roles functionally characterized in vitro and in vivo

Large deviations for polling systems

Related INRIA Research report available at : http://hal.inria.fr/docs/00/07/27/62/PDF/RR-3892.pdfInternational audienceWe aim at presenting in short the technical report, which states a sample path large deviation principle for a resealed process n-1 Qnt, where Qt represents the joint number of clients at time t in a single server 1-limited polling system with Markovian routing. The main goal is to identify the rate function. A so-called empirical generator is introduced, which consists of Q t and of two empirical measures associated with S t the position of the server at time t. The analysis relies on a suitable change of measure and on a representation of fluid limits for polling systems. Finally, the rate function is solution of a meaningful convex program

GRCBox: Extending Smartphone Connectivity in Vehicular Networks

The low penetration of connectivity-enabled OBUs is delaying the deployment of Vehicular Networks (VNs), and therefore the development of Vehicular Delay Tolerant Network (VDTN) applications, among others. In this paper we present GRCBox, an architecture based on RaspberryPi that allows integrating smartphones in VNs. GRCBox is based on a low-cost device that combines several pieces of software to provide ad-hoc and multi-interface connectivity to smartphones. Using GRCBox each application can choose the interface for its data flows, which increases flexibility and will allow developers to easily implement applications based on ad-hoc connectivity, such as VDTN applications.This work was partially supported by the Ministerio de Economia y Competitividad, Spain, under Grants TIN2011-27543-C03-01 and BES-2012-052673, and by the European Commission under Svagata.eu, the Erasmus Mundus Programme, Action 2 (EMA2).Martínez Tornell, S.; Patra, S.; Tavares De Araujo Cesariny Calafate, CM.; Cano Escribá, JC.; Manzoni, P. (2015). GRCBox: Extending Smartphone Connectivity in Vehicular Networks. International Journal of Distributed Sensor Networks. 2015:1-13. doi:10.1155/2015/478064S1132015Hartenstein, H., & Laberteaux, K. P. (2008). A tutorial survey on vehicular ad hoc networks. IEEE Communications Magazine, 46(6), 164-171. doi:10.1109/mcom.2008.4539481Wu, H., Palekar, M., Fujimoto, R., Guensler, R., Hunter, M., Lee, J., & Ko, J. (2005). An empirical study of short range communications for vehicles. Proceedings of the 2nd ACM international workshop on Vehicular ad hoc networks - VANET ’05. doi:10.1145/1080754.1080769Jerbi, M., Senouci, S.-M., & Haj, M. A. (2007). Extensive Experimental Characterization of Communications in Vehicular Ad Hoc Networks within Different Environments. 2007 IEEE 65th Vehicular Technology Conference - VTC2007-Spring. doi:10.1109/vetecs.2007.533Lee, K. C., Lee, S., Cheung, R., Lee, U., & Gerla, M. (2007). First Experience with CarTorrent in a Real Vehicular Ad Hoc Network Testbed. 2007 Mobile Networking for Vehicular Environments. doi:10.1109/move.2007.4300814Giordano, E., Tomatis, A., Ghosh, A., Pau, G., & Gerla, M. (2008). C-VeT An Open Research Platform for VANETs: Evaluation of Peer to Peer Applications in Vehicular Networks. 2008 IEEE 68th Vehicular Technology Conference. doi:10.1109/vetecf.2008.462Cesana, M., Fratta, L., Gerla, M., Giordano, E., & Pau, G. (2010). C-VeT the UCLA campus vehicular testbed: Integration of VANET and Mesh networks. 2010 European Wireless Conference (EW). doi:10.1109/ew.2010.5483535Santa, J., Tsukada, M., Ernst, T., & Gomez-Skarmeta, A. F. (2009). Experimental analysis of multi-hop routing in vehicular ad-hoc networks. 2009 5th International Conference on Testbeds and Research Infrastructures for the Development of Networks & Communities and Workshops. doi:10.1109/tridentcom.2009.4976248Paula, M. C. G., Isento, J. N., Dias, J. A., & Rodrigues, J. J. P. C. (2011). A real-world VDTN testbed for advanced vehicular services and applications. 2011 IEEE 16th International Workshop on Computer Aided Modeling and Design of Communication Links and Networks (CAMAD). doi:10.1109/camad.2011.5941108Campbell, A., & Choudhury, T. (2012). From Smart to Cognitive Phones. IEEE Pervasive Computing, 11(3), 7-11. doi:10.1109/mprv.2012.41Vandenberghe, W., Moerman, I., & Demeester, P. (2011). On the feasibility of utilizing smartphones for vehicular ad hoc networking. 2011 11th International Conference on ITS Telecommunications. doi:10.1109/itst.2011.6060061Sawada, D., Sato, M., Uehara, K., & Murai, J. (2011). iDANS: A platform for disseminating information on a VANET consisting of smartphone nodes. 2011 11th International Conference on ITS Telecommunications. doi:10.1109/itst.2011.6060062Tornell, S. M., Calafate, C. T., Cano, J.-C., Manzoni, P., Fogue, M., & Martinez, F. J. (2013). Evaluating the Feasibility of Using Smartphones for ITS Safety Applications. 2013 IEEE 77th Vehicular Technology Conference (VTC Spring). doi:10.1109/vtcspring.2013.6692553Mitchell, G. (2012). The Raspberry Pi single-board computer will revolutionise computer science teaching. Engineering & Technology, 7(3), 26-26. doi:10.1049/et.2012.0300Fielding R. T.Architectural styles and the design of network-based software architectures [Ph.D. thesis]2000University of Californi

The C-Terminal Domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC Is a Lectin-Like Carbohydrate Binding Module

The D-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbCCT) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbCCT contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbCCT, linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis. Author Summary Top Tuberculosis (TB), an infectious disease caused by the bacillus Mycobacterium tuberculosis, burdens large swaths of the world population. Treatment of active TB typically requires administration of an antibiotic cocktail over several months that includes the drug ethambutol. This front line compound inhibits a set of arabinosyltransferase enzymes, called EmbA, EmbB and EmbC, which are critical for the synthesis of arabinan, a vital polysaccharide in the pathogen's unique cell envelope. How precisely ethambutol inhibits arabinosyltransferase activity is not clear, in part because structural information of its pharmacological targets has been elusive. Here, we report the high-resolution structure of the C-terminal domain of the ethambutol-target EmbC, a 390-amino acid fragment responsible for acceptor substrate recognition. Combining the X-ray crystallographic analysis with structural comparisons, site-directed mutagenesis, activity and ligand binding assays, we identified two regions in the C-terminal domain of EmbC that are capable of binding acceptor substrate mimics and are critical for activity of the full-length enzyme. Our results begin to define structure-function relationships in a family of structurally uncharacterised membrane-embedded glycosyltransferases, which are an important target for tuberculosis therapy

Structural Basis of PP2A Inhibition by Small t Antigen

The SV40 small t antigen (ST) is a potent oncoprotein that perturbs the function of protein phosphatase 2A (PP2A). ST directly interacts with the PP2A scaffolding A subunit and alters PP2A activity by displacing regulatory B subunits from the A subunit. We have determined the crystal structure of full-length ST in complex with PP2A A subunit at 3.1 Å resolution. ST consists of an N-terminal J domain and a C-terminal unique domain that contains two zinc-binding motifs. Both the J domain and second zinc-binding motif interact with the intra-HEAT-repeat loops of HEAT repeats 3–7 of the A subunit, which overlaps with the binding site of the PP2A B56 subunit. Intriguingly, the first zinc-binding motif is in a position that may allow it to directly interact with and inhibit the phosphatase activity of the PP2A catalytic C subunit. These observations provide a structural basis for understanding the oncogenic functions of ST

A Helix Replacement Mechanism Directs Metavinculin Functions

Cells require distinct adhesion complexes to form contacts with their neighbors or the extracellular matrix, and vinculin links these complexes to the actin cytoskeleton. Metavinculin, an isoform of vinculin that harbors a unique 68-residue insert in its tail domain, has distinct actin bundling and oligomerization properties and plays essential roles in muscle development and homeostasis. Moreover, patients with sporadic or familial mutations in the metavinculin-specific insert invariably develop fatal cardiomyopathies. Here we report the high resolution crystal structure of the metavinculin tail domain, as well as the crystal structures of full-length human native metavinculin (1,134 residues) and of the full-length cardiomyopathy-associated ΔLeu954 metavinculin deletion mutant. These structures reveal that an α-helix (H1′) and extended coil of the metavinculin insert replace α-helix H1 and its preceding extended coil found in the N-terminal region of the vinculin tail domain to form a new five-helix bundle tail domain. Further, biochemical analyses demonstrate that this helix replacement directs the distinct actin bundling and oligomerization properties of metavinculin. Finally, the cardiomyopathy associated ΔLeu954 and Arg975Trp metavinculin mutants reside on the replaced extended coil and the H1′ α-helix, respectively. Thus, a helix replacement mechanism directs metavinculin's unique functions

Vaccinia Virus Proteins A52 and B14 Share a Bcl-2–Like Fold but Have Evolved to Inhibit NF-κB rather than Apoptosis

Vaccinia virus (VACV), the prototype poxvirus, encodes numerous proteins that modulate the host response to infection. Two such proteins, B14 and A52, act inside infected cells to inhibit activation of NF-κB, thereby blocking the production of pro-inflammatory cytokines. We have solved the crystal structures of A52 and B14 at 1.9 Å and 2.7 Å resolution, respectively. Strikingly, both these proteins adopt a Bcl-2–like fold despite sharing no significant sequence similarity with other viral or cellular Bcl-2–like proteins. Unlike cellular and viral Bcl-2–like proteins described previously, A52 and B14 lack a surface groove for binding BH3 peptides from pro-apoptotic Bcl-2–like proteins and they do not modulate apoptosis. Structure-based phylogenetic analysis of 32 cellular and viral Bcl-2–like protein structures reveals that A52 and B14 are more closely related to each other and to VACV N1 and myxoma virus M11 than they are to other viral or cellular Bcl-2–like proteins. This suggests that a progenitor poxvirus acquired a gene encoding a Bcl-2–like protein and, over the course of evolution, gene duplication events have allowed the virus to exploit this Bcl-2 scaffold for interfering with distinct host signalling pathways

The dynamic stator stalk of rotary ATPases

Rotary ATPases couple ATP hydrolysis/synthesis with proton translocation across biological membranes and so are central components of the biological energy conversion machinery. Their peripheral stalks are essential components that counteract torque generated by rotation of the central stalk during ATP synthesis or hydrolysis. Here we present a 2.25-Å resolution crystal structure of the peripheral stalk from Thermus thermophilus A-type ATPase/synthase. We identify bending and twisting motions inherent within the structure that accommodate and complement a radial wobbling of the ATPase headgroup as it progresses through its catalytic cycles, while still retaining azimuthal stiffness necessary to counteract rotation of the central stalk. The conformational freedom of the peripheral stalk is dictated by its unusual right-handed coiled-coil architecture, which is in principle conserved across all rotary ATPases. In context of the intact enzyme, the dynamics of the peripheral stalks provides a potential mechanism for cooperativity between distant parts of rotary ATPases

UPF201 Archaeal Specific Family Members Reveal Structural Similarity to RNA-Binding Proteins but Low Likelihood for RNA-Binding Function

We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10–40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel β-sheet and five α-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation

Complete Structural Model of Escherichia coli RNA Polymerase from a Hybrid Approach

A combination of structural approaches yields a complete atomic model of the highly biochemically characterized Escherichia coli RNA polymerase, enabling fuller exploitation of E. coli as a model for understanding transcription