No abstract

Biotrophic plant pathogens encounter a postinfection basal resistance layer controlled by the lipase-like protein enhanced disease susceptibility 1 (EDS1) and its sequence-related interaction partners, senescence-associated gene 101 (SAG101) and phytoalexin deficient 4 (PAD4). Maintainance of separate EDS1 family member clades through angiosperm evolution suggests distinct functional attributes. We report the Arabidopsis EDS1-SAG101 heterodimer crystal structure with juxtaposed N-terminal α/β hydrolase and C-terminal α-helical EP domains aligned via a large conserved interface. Mutational analysis of the EDS1-SAG101 heterodimer and a derived EDS1-PAD4 structural model shows that EDS1 signals within mutually exclusive heterocomplexes. Although there is evolutionary conservation of α/β hydrolase topology in all three proteins, a noncatalytic resistance mechanism is indicated. Instead, the respective N-terminal domains appear to facilitate binding of the essential EP domains to create novel interaction surfaces on the heterodimer. Transitions between distinct functional EDS1 heterodimers might explain the central importance and versatility of this regulatory node in plant immunity.

The NRF2 transcription factor regulates a major environmental and oxidative stress response. NRF2 is itself negatively regulated by KEAP1, the adaptor of a Cul3-ubiquitin ligase complex that marks NRF2 for proteasomal degradation by ubiquitination. Electrophilic compounds activate NRF2 primarily by inhibiting KEAP1-dependent NRF2 degradation, through alkylation of specific cysteines. We have examined the impact on KEAP1 of reactive oxygen and nitrogen species, which are also NRF2 inducers. We found that in untreated cells, a fraction of KEAP1 carried a long range disulfide linking Cys 226 and Cys 613 . Exposing cells to hydrogen peroxide, to the nitric oxide donor spermine NONOate, to hypochlorous acid, or to S-nitrosocysteine further increased this disulfide and promoted formation of a disulfide linking two KEAP1 molecules via Cys 151 . None of these oxidants, except S-nitrocysteine, caused KEAP1 S-nitrosylation. A cysteine mutant preventing KEAP1 intermolecular disulfide formation also prevented NRF2 stabilization in response to oxidants, whereas those preventing intramolecular disulfide formation were functionally silent. Further, simultaneously inactivating the thioredoxin and glutathione pathways led both to major constitutive KEAP1 oxidation and NRF2 stabilization. We propose that KEAP1 intermolecular disulfide formation via Cys 151 underlies the activation of NRF2 by reactive oxygen and nitrogen species.The Cap'n'collar bZip transcription factor NRF2 regulates an environmental and oxidative stress response of major physiological importance in mammals. NRF2 is activated by reactive oxygen and nitrogen species, electrophilic xenobiotics, and heavy metals and promotes cytoprotection and survival toward these stresses (for a review, see Refs. 1 and 2). Activation of NRF2 is intricate, engaging controls at the level of subcellular distribution, interaction with other proteins, phosphorylation, and protein stability (reviewed in Ref. 2). Among these, protein stability is a major control determinant, involving KEAP1, the adaptor of a Cul3-ubiquitin ligase complex that ubiquitinates NRF2 and marks it for proteasomal degradation (3-6). Stress signals that activate NRF2, herein named NRF2 inducers, are primarily sensed at the level of KEAP1, causing NRF2 protein stabilization (7-9) by inhibiting KEAP1-mediated NRF2 ubiquitination (10, 11).The large number of NRF2 inducers and their quite different chemical nature have raised the question of how they are specifically sensed by KEAP1. Although NRF2 inducers are chemically very different, they all have electrophilic properties, which has led to the proposal that they must operate by alkylation and/or oxidation of KEAP1 Cys residues (12). The 624-amino acid-long KEAP1 protein has 25 (mouse) or 27 (human) Cys residues and carries a Broad complex, Tramtrack, Bric-à-Brac (BTB) 2 dimerization domain, an intervening region (IVR), and a six-Kelch repeat domain (Kelch) (see Fig. 2). It also binds zinc with a 1:1 stoichiometry, possibly through the IVR residues Cys 254 , Cys 2...

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.

Asf1 is a conserved histone chaperone implicated in nucleosome assembly, transcriptional silencing, and the cellular response to DNA damage. We solved the NMR solution structure of the Nterminal functional domain of the human Asf1a isoform, and we identified by NMR chemical shift mapping a surface of Asf1a that binds the C-terminal helix of histone H3. This binding surface forms a highly conserved hydrophobic groove surrounded by charged residues. Mutations within this binding site decreased the affinity of Asf1a for the histone H3͞H4 complex in vitro, and the same mutations in the homologous yeast protein led to transcriptional silencing defects, DNA damage sensitivity, and thermosensitive growth. We have thus obtained direct experimental evidence of the mode of binding between a histone and one of its chaperones and genetic data suggesting that this interaction is important in both the DNA damage response and transcriptional silencing.Asf1 histone chaperone ͉ chromatin ͉ DNA damage ͉ NMR chemical shift mapping ͉ nucleosome assembly D NA in eukaryotic cells is packaged as nucleosome core particles containing Ϸ145 bp of DNA wrapped around an octamer comprised of two copies each of histones H2A, H2B, H3, and H4 (1). Assembly of histones into nucleosomes is a tightly orchestrated process (2, 3). Asf1 is a highly conserved histone chaperone that has been linked to both nucleosome assembly and disassembly (4-7). Asf1 interacts with two functional classes of protein: chromatin components, including histone H3 (8), the Hir proteins (9, 10), and the second subunit of CAF-I (5, 11, 12), and checkpoint kinases, including the Rad53 checkpoint kinase in budding yeast (13,14) and the Tousled-like kinases in metazoans (15). The function of most of these interactions has not been defined. However, a Hir binding region of Asf1 was implicated in telomeric silencing but not required for resistance to genotoxic stress (16). Further work is necessary to determine the functional role of the remaining interactions and, in particular, for defining which Asf1 partners are required for the DNA damage response and for optimal cell growth. In this work, we present the solution structure of the functional Nterminal domain of human Asf1a, and we identify its histone H3 binding site. We show that Asf1 mutants severely defective in histone H3͞H4 binding are incompetent in silencing and in providing resistance to DNA damage. MethodsProtein Production. pETM30 allowed the production of recombinant (His) 6 -GST-Tev site-fusion proteins in Escherichia coli strain BL21 gold (DE3). Unlabeled and uniformly labeled proteins were obtained as described in ref. 17. After Tev cleavage, the 15 N-labeled-H3 (122-135) peptide was further purified by reverse-phase chromatography. The NMR buffer was described in ref.17. An unlabeled peptide spanning the 122-133 sequence of histone H3 was obtained by chemical synthesis (Epytop, Nîmes, France). The protein concentrations were precisely measured by amino acid analysis.NMR Structure Determination and Binding Expe...

The 20S proteasome is the catalytic core of the 26S proteasome, a central enzyme in the ubiquitin-proteasome system. Its assembly proceeds in a multistep and orderly fashion. Ump1 is the only well-described chaperone dedicated to the assembly of the 20S proteasome in yeast. Here, we report a phenotype related to the DNA damage response that allowed us to isolate four other chaperones of yeast 20S proteasomes, which we named Poc1-Poc4. Poc1/2 and Poc3/4 form two pairs working at different stages in early 20S proteasome assembly. We identify PAC1, PAC2, the recently described PAC3, and an uncharacterized protein that we named PAC4 as functional mammalian homologs of yeast Poc factors. Hence, in yeast as in mammals, proteasome assembly is orchestrated by two pairs of chaperones acting upstream of the half-proteasome maturase Ump1. Our findings provide evidence for a remarkable conservation of a pairwise chaperone-assisted proteasome assembly throughout evolution.

BackgroundThe 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.ResultsWe show that using a combination of iterative profile-profile comparison and gene context analyses, we can identify a set of head, neck and tail proteins in most tailed bacteriophages of our database. Classification of phages based on neck protein sequences delineates 4 Types corresponding to known morphological subfamilies. Further analysis of the most abundant Type 1 yields 10 Clusters characterized by consistent sets of head, neck and tail proteins. We developed Virfam, a webserver that automatically identifies proteins of the phage head-neck-tail module and assign phages to the most closely related cluster of phages. This server was tested against 624 new phages from the NCBI database. 93% of the tailed and unclassified phages could be assigned to our head-neck-tail based categories, thus highlighting the large representativeness of the identified virion architectures. Types and Clusters delineate consistent subgroups of Caudovirales, which correlate with several virion properties.ConclusionsOur method and webserver have the capacity to automatically classify most tailed phages, detect their structural module, assign a function to a set of their head, neck and tail genes, provide their morphologic subtype and localize these phages within a “head-neck-tail” based classification. It should enable analysis of large sets of phage genomes. In particular, it should contribute to the classification of the abundant unknown viruses found on assembled contigs of metagenomic samples.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1027) contains supplementary material, which is available to authorized users.