Molecular Basis for Cooperative Binding of Anionic Phospholipids to the PH Domain of the Arf GAP ASAP1

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. •PH domain of ASAP1 has canonical and atypical anionic lipid-binding sites•Phospholipids bind cooperatively to the canonical and atypical sites•Occupancy of both sites is necessary for efficient ASAP1 enzymatic activity Jian et al. identify two cooperative lipid-binding sites in the PH domain of ASAP1, which regulate the enzymatic activity of ASAP1. The authors speculate that cooperative lipid binding may be a feature of PH domains that control the activity of signaling proteins.