Crystal Structure-Based Exploration of Arginine-Containing Peptide Binding in the ADP-Ribosyltransferase Domain of the Type III Effector XopAI Protein.

Plant pathogens secrete proteins called effectors into the cells of their host to modulate the host immune response against colonization. Effectors can either modify or arrest host target proteins to sabotage the signaling pathway, and therefore are considered potential drug targets for crop disease control. In earlier research, the Xanthomonas type III effector XopAI was predicted to be a member of the arginine-specific mono-ADP-ribosyltransferase family. However, the crystal structure of XopAI revealed an altered active site that is unsuitable to bind the cofactor NAD+, but with the capability to capture an arginine-containing peptide from XopAI itself. The arginine peptide consists of residues 60 through 69 of XopAI, and residue 62 (R62) is key to determining the protein-peptide interaction. The crystal structure and the molecular dynamics simulation results indicate that specific arginine recognition is mediated by hydrogen bonds provided by the backbone oxygen atoms from residues W154, T155, and T156, and a salt bridge provided by the E265 sidechain. In addition, a protruding loop of XopAI adopts dynamic conformations in response to arginine peptide binding and is probably involved in target protein recognition. These data suggest that XopAI binds to its target protein by the peptide-binding ability, and therefore, it promotes disease progression. Our findings reveal an unexpected and intriguing function of XopAI and pave the way for further investigation on the role of XopAI in pathogen invasion.

Susceptibility of amphibians to chytridiomycosis is associated with MHC class II conformation.

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) can cause precipitous population declines in its amphibian hosts. Responses of individuals to infection vary greatly with the capacity of their immune system to respond to the pathogen. We used a combination of comparative and experimental approaches to identify major histocompatibility complex class II (MHC-II) alleles encoding molecules that foster the survival of Bd-infected amphibians. We found that Bd-resistant amphibians across four continents share common amino acids in three binding pockets of the MHC-II antigen-binding groove. Moreover, strong signals of selection acting on these specific sites were evident among all species co-existing with the pathogen. In the laboratory, we experimentally inoculated Australian tree frogs with Bd to test how each binding pocket conformation influences disease resistance. Only the conformation of MHC-II pocket 9 of surviving subjects matched those of Bd-resistant species. This MHC-II conformation thus may determine amphibian resistance to Bd, although other MHC-II binding pockets also may contribute to resistance. Rescuing amphibian biodiversity will depend on our understanding of amphibian immune defence mechanisms against Bd. The identification of adaptive genetic markers for Bd resistance represents an important step forward towards that goal.

Molecular basis for inhibition of adhesin-mediated bacterial-host interactions through a peptide-binding domain.

Infections typically begin with pathogens adhering to host cells. For bacteria, this adhesion can occur through specific ligand-binding domains. We identify a 20-kDa peptide-binding domain (PBD) in a 1.5-MDa RTX adhesin of a Gram-negative marine bacterium that colonizes diatoms. The crystal structure of this Ca2+-dependent PBD suggests that it may bind the C termini of host cell-surface proteins. A systematic peptide library analysis reveals an optimal tripeptide sequence with 30-nM affinity for the PBD, and X-ray crystallography details its peptide-protein interactions. Binding of the PBD to the diatom partner of the bacteria can be inhibited or competed away by the peptide, providing a molecular basis for inhibiting bacterium-host interactions. We further show that this PBD is found in other bacteria, including human pathogens such as Vibrio cholerae and Aeromonas veronii. Here, we produce the PBD ortholog from A. veronii and demonstrate, using the same peptide inhibitor, how pathogens may be prevented from adhering to their hosts.

Polymorphic analysis of peptide binding domain of major histocompatibility complex class I in domestic ducks.

Function of duck (Anas platyrhynchos) major histocompatibility complex class I (Anpl-MHC I) molecules in binding peptides is through the peptide binding groove (PBG), which is thought to be influenced by the high polymorphism of α1 and α2 domains. However, little is known about the polymorphism of Anpl-MHC I peptide binding domain (PBD), especially in the domestic duck. Here, we analyzed the polymorphism of forty-eight Anpl-MHC I α1 and α2 domains from domestic duck breeds previously reported. All sequences were analyzed through multiple sequence alignment and a phylogenetic tree was constructed. The coefficient of variance of the peptide binding domains (PBDs) from WS, CV, JD, and SX duck breeds was estimated based on the Wu-Kabat variability index, followed by the location of the highly variable sites (HVSs) on reported crystal structure models. Analysis of α1 and α2 domains showed common features of classical MHC class I and high polymorphism, especially in α1 domain. The constructed phylogenetic tree showed that PBDs of domestic ducks did not segregate based on breeds and had a close phylogenetic relationship, even with wild ducks. In each breed, HVSs were mostly located in the PBG, suggesting that they might determine peptide-binding characteristics and subsequently influence peptide presentation and recognition. The combined results of sequence data and crystal structure provide novel valuable insights into the polymorphism and diversity of Anpl-MHC I PBDs that will facilitate further studies on disease resistance differences between duck breeds and the development of cytotoxic T-lymphocyte (CTL) epitope vaccines suited for preventing diseases in domestic ducks.