Crystal structure of the ARF-GAP domain and ankyrin repeats of PYK2-associated protein beta

ADP ribosylation factors (ARFs), which are members of the Pas superfamily o f GTP-binding proteins, are critical components of vesicular trafficking pa thways in eukaryotes, Like Pas, ARFs are active in their GTP-bound form, an d their duration of activity is controlled by GTPase-activating proteins (G APs), which assist ARFs in hydrolyzing GTP to GDP. PAP beta, a protein that binds to and is phosphorylated by the non-receptor tyrosine kinase PYK2, c ontains several modular signaling domains including a pleckstrin homology d omain, an SH3 domain, ankyrin repeats and an ARF-GAP domain. Sequences of A RF-GAP domains show no recognizable similarity to those of other GAPs, and contain a characteristic Cys-X-2-Cys-X16-17-Cys-X-2-Cys moth, The crystal s tructure of the PAP beta ARF-GAP domain and the C-terminal ankyrin repeats has been determined at 2.1 Angstrom resolution. The ARF-GAP domain comprise s a central three-stranded beta-sheet flanked by five alpha-helices, with a Zn2+ ion coordinated by the four cysteines of the cysteine-rich moth, Four ankyrin repeats are also present, the first two of which form an extensive interface with the ARF-GAP domain. An invariant arginine and several nearb y hydrophobic residues are solvent exposed and are predicted to be the site of interaction with ARFs, Site-directed mutagenesis of these residues conf irms their importance in ARF-GAP activity.