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

InterEvol database: exploring the structure and evolution of protein complex interfaces

Capturing how the structures of interacting partners evolved at their binding interfaces is a fundamental issue for understanding interactomes evolution. In that scope, the InterEvol database was designed for exploring 3D structures of homologous interfaces of protein complexes. For every chain forming a complex in the protein data bank (PDB), close and remote structural interologs were identified providing essential snapshots for studying interfaces evolution. The database provides tools to retrieve and visualize these structures. In addition, pre-computed multiple sequence alignments of most likely interologs retrieved from a wide range of species can be downloaded to enrich the analysis. The database can be queried either directly by pdb code or keyword but also from the sequence of one or two partners. Interologs multiple sequence alignments can also be recomputed online with tailored parameters using the InterEvolAlign facility. Last, an InterEvol PyMol plugin was developed to improve interactive exploration of structures versus sequence alignments at the interfaces of complexes. Based on a series of automatic methods to extract structural and sequence data, the database will be monthly updated. Structures coordinates and sequence alignments can be queried and downloaded from the InterEvol web interface at http://biodev.cea.fr/interevol/

Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs

Homologous recombination is a key in contributing to bacteriophages genome repair, circularization and replication. No less than six kinds of recombinase genes have been reported so far in bacteriophage genomes, two (UvsX and Gp2.5) from virulent, and four (Sak, Redβ, Erf and Sak4) from temperate phages. Using profile–profile comparisons, structure-based modelling and gene-context analyses, we provide new views on the global landscape of recombinases in 465 bacteriophages. We show that Sak, Redβ and Erf belong to a common large superfamily adopting a shortcut Rad52-like fold. Remote homologs of Sak4 are predicted to adopt a shortcut Rad51/RecA fold and are discovered widespread among phage genomes. Unexpectedly, within temperate phages, gene-context analyses also pinpointed the presence of distant Gp2.5 homologs, believed to be restricted to virulent phages. All in all, three major superfamilies of phage recombinases emerged either related to Rad52-like, Rad51-like or Gp2.5-like proteins. For two newly detected recombinases belonging to the Sak4 and Gp2.5 families, we provide experimental evidence of their recombination activity in vivo. Temperate versus virulent lifestyle together with the importance of genome mosaicism is discussed in the light of these novel recombinases. Screening for these recombinases in genomes can be performed at http://biodev.extra.cea.fr/virfam

XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining

International audienceThe Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to the ends of DNA double-strand breaks and recruits factors of the non-homologous end-joining (NHEJ) repair pathway through molecular interactions that remain unclear. We have determined crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBM motifs bind remote sites of the Ku80 alpha/beta domain. The X-KBM occupies an internal pocket formed by an unprecedented large outward rotation of the Ku80 alpha/beta domain. We observe independent recruitment of the APLF-interacting protein XRCC4 and of XLF to laser-irradiated sites via binding of A- and X-KBMs, respectively, to Ku80. Finally, we show that mutation of the X-KBM and A-KBM binding sites in Ku80 compromises both the efficiency and accuracy of end joining and cellular radiosensitivity. A- and X-KBMs may represent two initial anchor points to build the intricate interaction network required for NHEJ

Evolutionary analysis, structural prediction and inhibition of the interactions protein-protein

Les interactions protéine-protéine sont fondamentales dans la plupart des processus cellulaires. Cette thèse est centrée sur l analyse et la prédiction de ces interactions en utilisant à la fois les données structurales et l information issue de l évolution. A travers l étude de plus de 1000 couples d interfaces homologues, extraits d une base de données développée dans notre équipe, nous avons mis en évidence une plasticité étonnante dans l évolution de la structure des interfaces. Nous avons cependant identifié des propriétés assez conservées qui fournissent des pistes pour l extraction d information à partir des alignements de séquences multiples de deux partenaires en interaction. Nous avons ensuite développé une fonction de score gros grain utilisant un potentiel statistique multi-corps couplé à l information évolutive. Cette fonction améliore les prédictions d interfaces protéiques et a été utilisée dans deux cas concrets d amarrage moléculaire. Enfin, nous avons développé un protocole bio-informatique robuste pour le design d inhibiteurs peptidiques d une interaction protéine-protéine.Protein-protein interactions are of fundamental importance in virtually all cellular processes. This PhD thesis has focused on the analysis and prediction of these interactions through the combined use of structural data and evolutionary information. In a study of over 1,000 couples of homologous interfaces extracted from a database developed in our team, we uncovered astonishing plasticity in the way interface structure evolves, although we identified some rather invariant features which provide tracks for extracting meaningful information from multiple sequence alignments of binding partners. Consequently, we developed a coarse-grained interface scoring function using a multi-body statistical potential coupled to evolution. This scoring function improves the prediction of protein interfaces and was used among other methods on two practical cases of protein docking. Finally, we developed a robust computational protocol to rationalize the design of peptidic interaction inhibitors.PARIS-BIUSJ-Biologie recherche (751052107) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Versatility and Invariance in the Evolution of Homologous Heteromeric Interfaces

<div><p>Evolutionary pressures act on protein complex interfaces so that they preserve their complementarity. Nonetheless, the elementary interactions which compose the interface are highly versatile throughout evolution. Understanding and characterizing interface plasticity across evolution is a fundamental issue which could provide new insights into protein-protein interaction prediction. Using a database of 1,024 couples of close and remote heteromeric structural interologs, we studied protein-protein interactions from a structural and evolutionary point of view. We systematically and quantitatively analyzed the conservation of different types of interface contacts. Our study highlights astonishing plasticity regarding polar contacts at complex interfaces. It also reveals that up to a quarter of the residues switch out of the interface when comparing two homologous complexes. Despite such versatility, we identify two important interface descriptors which correlate with an increased conservation in the evolution of interfaces: apolar patches and contacts surrounding anchor residues. These observations hold true even when restricting the dataset to transiently formed complexes. We show that a combination of six features related either to sequence or to geometric properties of interfaces can be used to rank positions likely to share similar contacts between two interologs. Altogether, our analysis provides important tracks for extracting meaningful information from multiple sequence alignments of conserved binding partners and for discriminating near-native interfaces using evolutionary information.</p> </div

Conservation of the anchor position and its atomic contacts in colicin/IM complexes.

<p>The represented interfaces are colicin E9 (green) in complex with IM 9 (cyan) (PDB id 1fr2) and colicin E7 (pale green) in complex with IM 7 (pale cyan) (PDB id 2jb0). The Phe in 1fr2 (F86, chain B) and the structurally aligned Lys in 2jb0 (K528, chain B) are both anchors (highlighted in purple) and the conservation of atomic contacts involving this position is 76%. The residues in orange are those contacting the anchor residues.</p