Stabilization of HIV-1 envelope in the CD4-bound conformation through specific cross-linking of a CD4 mimetic.

CD4 binding on gp120 leads to the exposure of highly conserved regions recognized by the HIV co-receptor CCR5 and by CD4-induced (CD4i) antibodies. A covalent gp120-CD4 complex was shown to elicit CD4i antibody responses in monkeys, which was correlated with control of the HIV virus infection (DeVico, A., Fouts, T., Lewis, G. K., Gallo, R. C., Godfrey, K., Charurat, M., Harris, I., Galmin, L., and Pal, R. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 17477-17482). Because the inclusion of CD4 in a vaccine formulation should be avoided, due to potential autoimmune reactions, we engineered small sized CD4 mimetics (miniCD4s) that are poorly immunogenic and do not induce anti-CD4 antibodies. We made covalent complexes between such an engineered miniCD4 and gp120 or gp140, through a site-directed coupling reaction. These complexes were recognized by CD4i antibodies as well as by the HIV co-receptor CCR5. In addition, they elicit CD4i antibody responses in rabbits and therefore represent potential vaccine candidates that mimic an important HIV fusion intermediate, without autoimmune hazard.

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

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.

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

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.

HMM-Kalign: a tool for generating sub-optimal HMM alignments.

Recent development of strategies using multiple sequence alignments (MSA) or profiles to detect remote homologies between proteins has led to a significant increase in the number of proteins whose structures can be generated by comparative modeling methods. However, prediction of the optimal alignment between these highly divergent homologous proteins remains a difficult issue. We present a tool based on a generalized Viterbi algorithm that generates optimal and sub-optimal alignments between a sequence and a Hidden Markov Model. The tool is implemented as a new function within the HMMER package called hmmkalign.

Sequence search methods and scoring functions for the design of protein structures.

This chapter focuses on the methods developed for the automatic or semiautomatic design of protein structures. We present several algorithms for the exploration of the sequence space and scoring of the designed models. There are now several successful designs that have been achieved using these approaches such as the stabilization of a protein fold, the stabilization of a protein-protein complex interface, and the optimization of a protein function. A rapid presentation of the methodologies is followed by a detailed analysis of two test case studies. The first one deals with the redesign of a protein hydrophobic core and the second one with the stabilization of a protein structure through mutations at the surface. The different approaches are compared and the consistency of the predictions with the experimental data are discussed. All the programs tested in these protocols are freely available through the internet and may be applied to a wide range of design issues.

Detection of a tandem BRCT in Nbs1 and Xrs2 with functional implications in the DNA damage response.

MOTIVATION: Human Nbs1 and its homolog Xrs2 in Saccharomyces cerevisiae are part of the conserved MRN complex (MRX in yeast) which plays a crucial role in maintaining genomic stability. NBS1 corresponds to the gene mutated in the Nijmegen breakage syndrome (NBS) known as a radiation hyper-sensitive disease. Despite the conservation and the importance of the MRN complex, the high sequence divergence between Nbs1 and Xrs2 precluded the identification of common domains downstream of the N-terminal Fork-Head Associated (FHA) domain. RESULTS: Using HMM-HMM profile comparisons and structure modelling, we assessed the existence of a tandem BRCT in both Nbs1 and Xrs2 after the FHA. The structure-based conservation analysis of the tandem BRCT in Nbs1 supports its function as a phosphoserine binding domain. Remarkably, the 5 bp deletion observed in 95% of NBS patients cleaves the tandem at the linker region while preserving the structural integrity of each BRCT domain in the resulting truncated gene products.

Molecular dynamics simulations of E. coli MsbA transmembrane domain: formation of a semipore structure.

The human P-glycoprotein (MDR1/P-gp) is an ATP-binding cassette (ABC) transporter involved in cellular response to chemical stress and failures of anticancer chemotherapy. In the absence of a high-resolution structure for P-gp, we were interested in the closest P-gp homolog for which a crystal structure is available: the bacterial ABC transporter MsbA. Here we present the molecular dynamics simulations performed on the transmembrane domain of the open-state MsbA in a bilayer composed of palmitoyl oleoyl phosphatidylethanolamine lipids. The system studied contained more than 90,000 atoms and was simulated for 50 ns. This simulation shows that the open-state structure of MsbA can be stable in a membrane environment and provides invaluable insights into the structural relationships between the protein and its surrounding lipids. This study reveals the formation of a semipore-like structure stabilized by two key phospholipids which interact with the hinge region of the protein during the entire simulation. Multiple sequence alignments of ABC transporters reveal that one of the residues involved in the interaction with these two phospholipids are under a strong selection pressure specifically applied on the bacterial homologs of MsbA. Hence, comparison of molecular dynamics simulation and phylogenetic data appears as a powerful approach to investigate the functional relevance of molecular events occurring during simulations.