Showing papers by "Raphael Guerois published in 2008"

Structural and functional analysis of SGT1-HSP90 core complex required for innate immunity in plants.

Abstract: SGT1 (Suppressor of G2 allele of skp1), a co-chaperone of HSP90 (Heat-shock protein 90), is required for innate immunity in plants and animals. Unveiling the cross talks between SGT1 and other co-chaperones such as p23, AHA1 (Activator of HSP90 ATPase 1) or RAR1 (Required for Mla12 resistance) is an important step towards understanding the HSP90 machinery. Nuclear magnetic resonance spectroscopy and mutational analyses of HSP90 revealed the nature of its binding with the CS domain of SGT1. Although CS is structurally similar to p23, these domains were found to non-competitively bind to various regions of HSP90; yet, unexpectedly, full-length SGT1 could displace p23 from HSP90. RAR1 partly shares the same binding site with HSP90 as the CS domain, whereas AHA1 does not. This analysis allowed us to build a structural model of the HSP90–SGT1 complex and to obtain a compensatory mutant pair between both partners that is able to restore virus resistance in vivo through Rx (Resistance to potato virus X) immune sensor stabilization.

Coevolution at protein complex interfaces can be detected by the complementarity trace with important impact for predictive docking

Abstract: Protein surfaces are under significant selection pressure to maintain interactions with their partners throughout evolution. Capturing how selection pressure acts at the interfaces of protein–protein complexes is a fundamental issue with high interest for the structural prediction of macromolecular assemblies. We tackled this issue under the assumption that, throughout evolution, mutations should minimally disrupt the physicochemical compatibility between specific clusters of interacting residues. This constraint drove the development of the so-called Surface COmplementarity Trace in Complex History score (SCOTCH), which was found to discriminate with high efficiency the structure of biological complexes. SCOTCH performances were assessed not only with respect to other evolution-based approaches, such as conservation and coevolution analyses, but also with respect to statistically based scoring methods. Validated on a set of 129 complexes of known structure exhibiting both permanent and transient intermolecular interactions, SCOTCH appears as a robust strategy to guide the prediction of protein–protein complex structures. Of particular interest, it also provides a basic framework to efficiently track how protein surfaces could evolve while keeping their partners in contact.

Unveiling Novel RecO Distant Orthologues Involved in Homologous Recombination

Abstract: The generation of a RecA filament on single-stranded DNA is a critical step in homologous recombination. Two main pathways leading to the formation of the nucleofilament have been identified in bacteria, based on the protein complexes mediating RecA loading: RecBCD (AddAB) and RecFOR. Many bacterial species seem to lack some of the components involved in these complexes. The current annotation of the Helicobacter pylori genome suggests that this highly diverse bacterial pathogen has a reduced set of recombination mediator proteins. While it is now clear that homologous recombination plays a critical role in generating H. pylori diversity by allowing genomic DNA rearrangements and integration through transformation of exogenous DNA into the chromosome, no complete mediator complex is deduced from the sequence of its genome. Here we show by bioinformatics analysis the presence of a RecO remote orthologue that allowed the identification of a new set of RecO proteins present in all bacterial species where a RecR but not RecO was previously identified. HpRecO shares less than 15% identity with previously characterized homologues. Genetic dissection of recombination pathways shows that this novel RecO and the remote RecB homologue present in H. pylori are functional in repair and in RecA-dependent intrachromosomal recombination, defining two initiation pathways with little overlap. We found, however, that neither RecOR nor RecB contributes to transformation, suggesting the presence of a third, specialized, RecA-dependent pathway responsible for the integration of transforming DNA into the chromosome of this naturally competent bacteria. These results provide insight into the mechanisms that this successful pathogen uses to generate genetic diversity and adapt to changing environments and new hosts.

Predicting Free Energy Changes of Mutations in Proteins

Abstract: Originally published in: Protein Folding Handbook. Part I. Edited by Johannes Buchner and Thomas Kiefhaber. Copyright © 2005 Wiley-VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3-527-30784-2 The ability to predict with good accuracy the effect of mutations on protein stability or complex formation is needed in order to design new proteins, as well as to understand the effect of single nucleotide polymorphisms on human health. In the following sections we will summarize what is known and can be used for this purpose. We will start by describing the forces that act on proteins, an understanding of which is a prerequisite to understanding the following section dealing with prediction methods. Finally we will briefly summarize other possible unexpected results of making mutants, such as changes in “in vivo” stability or inducing protein aggregation. The sections in this article are Physical Forces that Determine Protein Conformational Stability Protein Conformational Stability Structures of the N and D States [2-6] Studies Aimed at Understanding the Physical Forces that Determine Protein Conformational Stability [1, 2, 8, 19-26] Forces Determining Conformational Stability [1, 2, 8, 19-27] Intramolecular Interactions van der Waals Interactions Electrostatic Interactions Conformational Strain Solvation Intramolecular Interactions and Solvation Taken Together Entropy Cavity Formation Summary Effect of Point Mutations on in vitro Protein Stability General Considerations on Protein Plasticity upon Mutation Predictive Strategies Methods From Sequence and Multiple Sequence Alignment Analysis Statistical Analysis of the Structure Databases Helix/Coil Transition Model Physicochemical Method Based on Protein Engineering Experiments Methods Based only on the Basic Principles of Physics and Thermodynamics Mutation Effects on in vivo Stability The N-terminal Rule The C-terminal Rule PEST Signals Mutation Effects on Aggregation Keywords: protein stability; point mutation; conformational strain; structure prediction