THE C-TERMINAL SH3 DOMAIN OF P67PH(PHOX) BINDS ITS NATURAL LIGAND IN A REVERSE ORIENTATION

Src-homology 3 (SH3) domains are small protein modules that bind to pr oline-rich motifs and mediate the formation of signalling complexes. S H3 domains have been implicated in the assembly of the phagocyte NADPH oxidase complex, a multicomponent enzyme responsible for the producti on of antimicrobial oxidants. Two components of the NADPH oxidase, p67 (phax) and p47(phax), each contain two SH3 domains and we have previou sly shown that the SH3 domain near the carboxyl terminus of p67(phax) interacts with a proline-rich region of p47(phax). In order to gain an insight into the specificity of this interaction, a structural model of the p67(phax) SH3 domain has been produced using the known structur e of the c-abl SH3 domain as a template. The model suggests that the p roline-rich ligand of p47(phax) can bind to the SH3 domain in either o f two orientations. In each orientation, the key residues of the SH3 d omain that contact the ligand have been identified and altered by site -directed mutagenesis. The ability of the mutated SH3 domains to assoc iate with p47(phax) from cell lysates was tested and the results provi de the first evidence for the binding of a full-length protein to an S H3 domain in a reversed orientation. (C) 1996 Academic Press Limited