SUMMARY We have defined the molecular basis for association of the PH domain of the Arf GAP ASAP1 with phospholipid bilayers. Structures of the unliganded and dibutyryl PtdIns(4,5)P2-bound PH domain were solved. PtdIns(4,5)P2 made contact with both a canonical site (C site) and an atypical site (A site). We hypothesized cooperative binding of PtdIns(4,5)P2 to the C site and a nonspecific anionic phospholipid to the A site. PtdIns(4,5)P2 dependence of binding to large unilamellar vesicles and GAP activity was sigmoidal, consistent with cooperative sites. In contrast, PtdIns(4,5) P2 binding to the PH domain of PLC δ1 was hyperbolic. Mutation of amino acids in either the C or A site resulted in decreased PtdIns(4,5)P2-dependent binding to vesicles and decreased GAP activity. The results support the idea of cooperative phospholipid binding to the C and A sites of the PH domain of ASAP1. We propose that the mechanism underlies rapid switching between active and inactive ASAP1.

Adenosine diphosphate–ribosylation factor (Arf) guanosine triphosphatase–activating proteins (GAPs) are enzymes that need to bind to membranes to catalyze the hydrolysis of guanosine triphosphate (GTP) bound to the small GTP-binding protein Arf. Binding of the pleckstrin homology (PH) domain of the ArfGAP With SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) to membranes containing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is key for maximum GTP hydrolysis but not fully understood. By combining nuclear magnetic resonance, neutron reflectometry, and molecular dynamics simulation, we show that binding of multiple PI(4,5)P2 molecules to the ASAP1 PH domain (i) triggers a functionally relevant allosteric conformational switch and (ii) maintains the PH domain in a well-defined orientation, allowing critical contacts with an Arf1 mimic to occur. Our model provides a framework to understand how binding of the ASAP1 PH domain to PI(4,5)P2 at the membrane may play a role in the regulation of ASAP1.

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