Solution structure of the Ras binding domain of the protein kinase Byr2 from Schizosaccharomyces pombe

Background: After activation, small GTPases such as Ras transfer the incomi ng signal to effectors by specifically interacting with the binding domain of these proteins. Structural details of the binding domain of different ef fectors determine which pathway is predominantly activated. Byr2 from fissi on yeast is a functional homolog of Raf, which is the direct downstream tar get of Ras in mammalians that initiates a protein kinase cascade. The amino acid sequence of Byr2's Ras binding domain is only weakly related to that of Raf, and Byr2's three-dimensional structure is unknown. Results: We have solved the 3D structure of the Ras binding domain of Byr2 (Byr2RBD) from Schizosaccharomyces pombe in solution. The structure consist s of three a helices and a mixed five-stranded beta pleated sheet arranged in the topology beta beta alpha beta beta alpha beta alpha with the first s even canonic secondary structure elements forming a ubiquitin superfold. N- 15-H-1-TROSY-HSQC spectroscopy of the complex of Byr2RBD with Ras . Mg2+. G ppNHp reveals that the first and second beta strands and the first alpha he lix of Byr2 are mainly involved in the protein-protein interaction as obser ved in other Ras binding domains. Although the putative interaction site of H-Ras from human and Ras1 from S. pombe are identical in sequence, binding to Byr2 leads to small but significant differences in the NMR spectra, ind icating a slightly different binding mode. Conclusions: The ubiquitin superfold appears to be the general structural m otif for Ras binding domains even in cases with vanishing sequence identity . However, details of the 3D structure and the interacting interface are di fferent, thereby determining the specifity of the recognition of Ras and Ra s-related proteins.