Molecular Mechanisms of the Interaction between Arf1 and ASAP1 pH Domain at the Membrane Interface

Abstract

Pleckstrin Homology (PH) domains have dual functions: localizing proteins to specific membrane regions and modulating membrane protein function through interdomain interactions. Here, we focused on the PH domain of ASAP1, which is necessary for phosphatidylinositol 4, 5- bisphosphate (PIP2)- dependent conversion of Arf1, a member of the RAS superfamily, from its GTP to GDP form. The interaction of the PH domain with the lipid surface was studied by 15N, 13C methyl NMR and SPR, using PIP2 and phosphatidylserine (PS) - containing nanodiscs as mimics of the membrane surface. Comparison of the chemical shift perturbations within PH upon interaction with nanodiscs- containing a single PIP2, multiple PIP2 lipids or a single PIP2 and increasing PS mole fraction showed that (i) the two loops between β sheets 1, 2 and 3, 4 delineate two, equivalent and independent PIP2 lipid binding sites and (ii) the PH domain is attracted to the membrane surface predominantly by non- specific electrostatic interactions with the background anionic lipids, before docking to the target lipid PIP2. Constraints obtained from neutron reflectometry and Paramagnetic Relaxation NMR experiments suggest that the PH domain is oriented with its long axis parallel to the membrane normal. Interestingly, deletion of 14 amino acids immediately N term of the first beta strand of the PH domain strongly reduces ASAP1 catalytic efficiency even if the deletion has no effect on ASAP1 equilibrium binding to PIP2. Taken together, this suggests that simultaneous binding to two PIP2 strictly orients the PH domain at the membrane interface, promoting functionally critical contacts between Arf1 and the N-term of ASAP1.