Three-dimensional models of NB-ARC domains of disease resistance proteins in tomato, arabidopsis, and flax

Abstract

Three dimensional models of NB-ARC domains in five different proteins were constructed based on the recently published crystal structure of the apoptotic protease activating factor 1, of which two are for tomato species, one each for flax, Arabidopsis, and nematode. Standard multiple sequence alignment was performed for chosen members of the NB-ARC domains, very divergent from each other in protein sequence, followed by homology model building and structure refinement. In this alignment, amino acid insertions and deletions between members generally fall in loop regions or at ends of alpha helices. Despite the presence of sequence divergence between the species, it is argued that the NB-ARC domains carry out the similar biological functions in the various species, highlighting the ATP binding and ATPase activity. By our comparative study of these models, it is predicted that NB-ARC domains should bind ADP/ATP rather than GDP/GTP. Both natural and induced mutants of Arabidopsis within the RPS2 locus and their phenotypes for disease reaction against Pseudomonas syringae are rationalized from the protein model. Apaf-1 Thr263 and Arg265 positions conserved totally within the NB-ARC domains are predicted to take active part in the catalytic activity of kinase-3 motif, the arginine known as the sensor I motif in AAA+ proteins. This was later verified for the Ced-4 crystal structure in complex with Ced-9. Our model of Ced-4 based on Apaf-1 was also compared with its crystal structure in the Ced-4-Ced-9 complex; the 3 layered alpha/beta domain superposes quite well, helical domain I is shifted by about 5 A but the winged helix domain is rotated away to a new position. Since Apaf-1 was crystallized with ADP and Ced-4-Ced9 with magnesium-ATP, this rotation signifies a change in structure of these NB-ARC domains between the two forms. Further, we hypothesize that certain mutants in the plant R proteins called 'constitutive gain-of-function' or 'autocatalytic' dispose their winged helix domains permanently like the magnesium-ATP form as observed for Ced-4, avoiding the closed ADP conformation. The models are also validated with mutagenesis data for a related tomato protein I-2, tomato prf and flax, including loss of function, wild type and autocatalytic phenotypes, and compared with similar data for potato and tobacco proteins, for which models were not built. These three dimensional models would help us to understand the spatial arrangement, function of R proteins and their conserved motifs.