Structural insights into the complex between Arf1 and ASAP1 at the membrane surface by NMR.

Abstract

ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs) are enzymes that need to bind to membranes to catalyze the hydrolysis of GTP bound to the small GTP-binding protein Arf1. Simultaneous binding of the Pleckstrin homology (PH) domain of the ArfGAP ASAP1 to membranes containing the phosphatidylinositol phosphate PIP(4,5)P2 and to Arf1.GTP is key for maximum GTP hydrolysis, but is not fully understood. Recently, we showed that binding of multiple PI(4,5)P2 molecules to the ASAP1 PH domain triggers an allosteric conformational switch (PIP switch) involving regions distant from the membrane interface. Here, we examined the interface between Arf1 and ASAP1 PH at the membrane surface of nanodiscs by Nuclear Magnetic Resonance (NMR) methods. Chemical Shift Perturbations (CSPs) observed upon complex formation and distance restraints obtained from Paramagnetic Relaxation Enhancement (PRE) NMR experiments between multiple MTSL-tagged Arf1 mutants and isotopically labeled ASAP1 PH were combined to build a model of the Arf1:ASAP1 PH complex at the membrane. Interestingly, the PIP switch resides at the interface between Arf1 and ASAP1 PH, shedding light on how PIP binding to ASAP1 PH may affect GTP hydrolysis.