Homo-dimerization and ligand binding by the leucine-rich repeat domain at RHG1/RFS2 underlying resistance to two soybean pathogens

BACKGROUND: The protein encoded by GmRLK18-1 (Glyma_18_02680 on chromosome 18) was a receptor like kinase (RLK) encoded within the soybean (Glycine max L. Merr.) Rhg1/Rfs2 locus. The locus underlies resistance to the soybean cyst nematode (SCN) Heterodera glycines (I.) and causal agent of sudden death syndrome (SDS) Fusarium virguliforme (Aoki). Previously the leucine rich repeat (LRR) domain was expressed in Escherichia coli. RESULTS: The aims here were to evaluate the LRRs ability to; homo-dimerize; bind larger proteins; and bind to small peptides. Western analysis suggested homo-dimers could form after protein extraction from roots. The purified LRR domain, from residue 131–485, was seen to form a mixture of monomers and homo-dimers in vitro. Cross-linking experiments in vitro showed the H274N region was close (<11.1 A) to the highly conserved cysteine residue C196 on the second homo-dimer subunit. Binding constants of 20–142 nM for peptides found in plant and nematode secretions were found. Effects on plant phenotypes including wilting, stem bending and resistance to infection by SCN were observed when roots were treated with 50 pM of the peptides. Far-Western analyses followed by MS showed methionine synthase and cyclophilin bound strongly to the LRR domain. A second LRR from GmRLK08-1 (Glyma_08_g11350) did not show these strong interactions. CONCLUSIONS: The LRR domain of the GmRLK18-1 protein formed both a monomer and a homo-dimer. The LRR domain bound avidly to 4 different CLE peptides, a cyclophilin and a methionine synthase. The CLE peptides GmTGIF, GmCLE34, GmCLE3 and HgCLE were previously reported to be involved in root growth inhibition but here GmTGIF and HgCLE were shown to alter stem morphology and resistance to SCN. One of several models from homology and ab-initio modeling was partially validated by cross-linking. The effect of the 3 amino acid replacements present among RLK allotypes, A87V, Q115K and H274N were predicted to alter domain stability and function. Therefore, the LRR domain of GmRLK18-1 might underlie both root development and disease resistance in soybean and provide an avenue to develop new variants and ligands that might promote reduced losses to SCN

Mapping the binding interactions between the ISW2 complex and nucleosomes

The compact structure of the eukaryotic genome dictates the accessibility to genes, and therefore adds an additional layer of regulation for gene expression. A specialized class of proteins called chromatin remodelers facilitates this process in the cell. The imitation switch (ISWI) subfamily of chromatin remodelers is a well studied class of proteins affecting gene expression. Its member ISW2 was recently shown to behave differently from other chromatin remodeling proteins. For instance, the ISW2 complex has been shown to be stimulated by ~5-6 fold in its ATPase activity when bound to a nucleosome rather than to a DNA molecule. Nucleosome remodeling by ISW2 has even been shown to depend on the N-terminal tail of histone H4 and therefore, the octamer of a nucleosome might be playing a significant role in nucleosome remodeling by the ISW2 complex. The aim in this investigation was to delineate the protein-protein interactions that the ISW2 complex establishes with the octamer upon binding to a nucleosome. Several histones with unique cysteines engineered at specific positions were refolded with other wild type histones to produce histone octamers with a single cysteine in one of the four histones. Based on previous reports from site-specific DNA photoaffinity cross-linking and hydroxyl-radical footprinting experiments, it was inferred that the SHL2, entry-exit position and the extranucleosomal linker DNA were contacted by the ISW2 complex on a nucleosome1. Considering these critical regions and taking into account the accessibility of residues in close proximity to these regions, five discrete positions were selected on the octamer surface for scanning the face of the nucleosome. The five sites were residues 19, 89 and 113 of histone H2A (H2A-19, H2A-89, H2A-113), residue 109 of histone H2B (H2B-109), and residue 80 of histone H3 (H3-80). Initially, octamers with cysteine at one position in one of the four histone proteins were reconstituted on a 0N70 DNA (where `N\u27 represents the 147bp 601 DNA sequence, and the lengths of the linker DNA is represented by the numbers 0 and 70). Nucleosomes were modified with the protein-protein cross-linker- MAB (methanethiosulfonate-tetrafluorophenylazide-biotin) reagent. This reagent makes a disulfide bond with the cysteines in the octamer of a nucleosome. The MAB reagent had a distance of ~11.1Aº between its photoreactive tetrafluorophenylazide group and the disulfide forming methanethiosulfonate group. The ISW2 complex was bound to the modified mononucleosomes and cross-linked by irradiating with UV-light. Under reducing conditions the biotin moiety was transferred from the nucleosome to the ISW2 complex. The subunit of the remodeler that was photocross-linked at these positions on the nucleosome was blotted onto nitrocellulose and detected with streptavidin conjugated to horseradish peroxidase (HRP). The catalytic subunit-Isw2 of the ISW2 complex was cross-linked at all five positions but with the following order of intensity from most to least- H2A-89, H3-80, H2B-109, H2A-19, and H2A113. Mass spectrometry was used to decipher these residues, motifs or domains of the catalytic subunit- Isw2 that interacted with the octamer at each position. The ISW2 complex was digested with trypsin, and the biotinylated peptides were enriched using monomeric avidin affinity chromatography. The largest subunit of the ISW2 complex, Itc1, did not get cross-linked at any of the positions