Structure-function analysis of the NB-ARC domain of plant disease resistance proteins

Resistance (R) proteins in plants are involved in pathogen recognitionand subsequent activation of innate immune responses. Most resistanceproteins contain a central nucleotide-binding domain. This so-calledNB-ARC domain consists of three subdomains: NB, ARC1, and ARC2.The NB-ARC domain is a functional ATPase domain, and its nucleotide-bindingstate is proposed to regulate activity of the R protein. A highlyconserved methionine–histidine–aspartate (MHD) motifis present at the carboxy-terminus of ARC2. An extensive mutationalanalysis of the MHD motif in the R proteins I-2 and Mi-1 isreported. Several novel autoactivating mutations of the MHDinvariant histidine and conserved aspartate were identified.The combination of MHD mutants with autoactivating hydrolysismutants in the NB subdomain showed that the autoactivation phenotypesare not additive. This finding indicates an important regulatoryrole for the MHD motif in the control of R protein activity.To explain these observations, a three-dimensional model ofthe NB-ARC domain of I-2 was built, based on the APAF-1 templatestructure. The model was used to identify residues importantfor I-2 function. Substitution of the selected residues resultedin the expected distinct phenotypes. Based on the model, itis proposed that the MHD motif fulfils the same function asthe sensor II motif found in AAA+ proteins (ATPases associatedwith diverse cellular activities)—co-ordination of thenucleotide and control of subdomain interactions. The presented3D model provides a framework for the formulation of hypotheseson how mutations in the NB-ARC exert their effects. Key words: Intramolecular interactions, MHD motif, NB-ARC domain, plant disease resistance, protein structure, R proteins, signal transduction, site-directed mutagenesis