The genetic diversity observed among bacteriophages remains a major obstacle for the identification of homologs and the comparison of their functional modules. In the structural module, although several classes of homologous proteins contributing to the head and tail structure can be detected, proteins of the head-to-tail connection (or neck) are generally more divergent. Yet, molecular analyses of a few tailed phages belonging to different morphological classes suggested that only a limited number of structural solutions are used in order to produce a functional virion. To challenge this hypothesis and analyze proteins diversity at the virion neck, we developed a specific computational strategy to cope with sequence divergence in phage proteins. We searched for homologs of a set of proteins encoded in the structural module using a phage learning database.

SGT1 (for suppressor of G2 allele of skp1) and RAR1 (for required for Mla12 resistance) are highly conserved eukaryotic proteins that interact with the molecular chaperone HSP90 (for heat shock protein90). In plants, SGT1, RAR1, and HSP90 are essential for disease resistance triggered by a number of resistance (R) proteins. Here, we present structural and functional characterization of plant SGT1 proteins. Random mutagenesis of Arabidopsis thaliana SGT1b revealed that its CS (for CHORD-SGT1) and SGS (for SGT1 specific) domains are essential for disease resistance. NMR-based interaction surface mapping and mutational analyses of the CS domain showed that the CHORD II domain of RAR1 and the N-terminal domain of HSP90 interact with opposite sides of the CS domain. Functional analysis of the CS mutations indicated that the interaction between SGT1 and HSP90 is required for the accumulation of Rx, a potato (Solanum tuberosum) R protein. Biochemical reconstitution experiments suggest that RAR1 may function to enhance the SGT1–HSP90 interaction by promoting ternary complex formation.

We present the results for CAPRI Round 30, the first joint CASP‐CAPRI experiment, which brought together experts from the protein structure prediction and protein–protein docking communities. The Round comprised 25 targets from amongst those submitted for the CASP11 prediction experiment of 2014. The targets included mostly homodimers, a few homotetramers, and two heterodimers, and comprised protein chains that could readily be modeled using templates from the Protein Data Bank. On average 24 CAPRI groups and 7 CASP groups submitted docking predictions for each target, and 12 CAPRI groups per target participated in the CAPRI scoring experiment. In total more than 9500 models were assessed against the 3D structures of the corresponding target complexes. Results show that the prediction of homodimer assemblies by homology modeling techniques and docking calculations is quite successful for targets featuring large enough subunit interfaces to represent stable associations. Targets with ambiguous or inaccurate oligomeric state assignments, often featuring crystal contact‐sized interfaces, represented a confounding factor. For those, a much poorer prediction performance was achieved, while nonetheless often providing helpful clues on the correct oligomeric state of the protein. The prediction performance was very poor for genuine tetrameric targets, where the inaccuracy of the homology‐built subunit models and the smaller pair‐wise interfaces severely limited the ability to derive the correct assembly mode. Our analysis also shows that docking procedures tend to perform better than standard homology modeling techniques and that highly accurate models of the protein components are not always required to identify their association modes with acceptable accuracy. Proteins 2016; 84(Suppl 1):323–348. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

The revolution brought about by AlphaFold2 opens promising perspectives to unravel the complexity of protein-protein interaction networks. The analysis of interaction networks obtained from proteomics experiments does not systematically provide the delimitations of the interaction regions. This is of particular concern in the case of interactions mediated by intrinsically disordered regions, in which the interaction site is generally small. Using a dataset of protein-peptide complexes involving intrinsically disordered regions that are non-redundant with the structures used in AlphaFold2 training, we show that when using the full sequences of the proteins, AlphaFold2-Multimer only achieves 40% success rate in identifying the correct site and structure of the interface. By delineating the interaction region into fragments of decreasing size and combining different strategies for integrating evolutionary information, we manage to raise this success rate up to 90%. We obtain similar success rates using a much larger dataset of protein complexes taken from the ELM database. Beyond the correct identification of the interaction site, our study also explores specificity issues. We show the advantages and limitations of using the AlphaFold2 confidence score to discriminate between alternative binding partners, a task that can be particularly challenging in the case of small interaction motifs.

Heterodimeric complexes containing the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) are regarded as central regulators of plant innate immunity. In this context, a complex of EDS1 with PHYTOALEXIN DEFICIENT4 (PAD4) is required for basal resistance and signaling downstream of immune receptors containing an N-terminal Toll-interleukin-1 receptor-like domain (TNLs) in Arabidopsis (Arabidopsis thaliana). Here we analyze EDS1 functions in the model Solanaceous plant Nicotiana benthamiana (Nb). Stable Nb mutants deficient in EDS1 complexes are not impaired in basal resistance, a finding which contradicts a general role for EDS1 in immunity. In Nb, PAD4 demonstrated no detectable immune functions, but TNL-mediated resistance responses required EDS1 complexes incorporating a SENESCENCE ASSOCIATED GENE101 (SAG101) isoform. Intriguingly, SAG101 is restricted to those genomes also encoding TNL receptors, and we propose it may be required for TNL-mediated immune signaling in most plants, except the Brassicaceae. Transient complementation in Nb was used for accelerated mutational analyses while avoiding complex biotic interactions. We identify a large surface essential for EDS1-SAG101 immune functions that extends from the N-terminal lipase domains to the C-terminal EDS1-PAD4 domains and might mediate interaction partner recruitment. Furthermore, this work demonstrates the value of genetic resources in Nb, which will facilitate elucidation of EDS1 functions.