R. Andrew Byrd, Senior Investigator, Center for Structural Biology, NIH Center for Cancer Research, Frederick, MD, will give a talk titled: "Surface Dancing: peripheral membrane complexes at the surface – challenges for integrated structural biology."

Zoom link: https://gc-cuny.zoom.us/j/91637964386. Meeting ID:  916 3796 4386. Passcode: asrc+ccny

ABSTRACT

ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs) are enzymes that act at the surface of membranes to catalyze the hydrolysis of GTP bound to the small GTP-binding protein ADP-ribosylation factor 1 (Arf1). Arf1 interacts with multiple cellular partners and membranes to regulate intracellular traffic, organelle structure and actin dynamics. In the GDP-bound state, Arf1 is soluble and not membrane associated; however, upon conversion to the GTP-bound state through interaction with a guanine exchange factor (GEF), Arf1 becomes membrane associated. In the membrane bound form, Arf1 has no intrinsic catalytic activity and requires interaction with an ArfGAP protein. The ArfGAP ASAP1 effects GTP hydrolysis by Arf1 and is upregulated in multiple cancers. ASAP1 utilizes a pleckstrin homology domain (PH) to recognize membranes via PI(4,5)P2 lipid molecules prior to complexation with Arf1. The establishment of membrane bound forms, complexation, and hydrolysis are dynamic processes combining multiple, yet specific, weak interactions. Structural and mechanistic investigation of such membrane surface complexes and processes is extremely challenging, as the system is refractory to crystallography and cryoEM. We have employed nanodisc membrane mimetics and complex isotopic labeling with solution NMR, solid-state NMR, molecular dynamics, and neutron reflectometry to explore multiple facets of this system. We have shown that binding of multiple PI(4,5)P2 molecules to the ASAP1 Pleckstrin homology (PH) domain (i) triggers a functionally relevant allosteric conformational switch involving regions distant from the membrane interface, and  (ii) maintains the PH domain in a well-defined orientation, allowing critical contacts with Arf1. Studies of Arf1 in the membrane reveal that, while Arf1 is anchored to the membrane through its N-terminal myristoylated amphipathic helix, the G domain explores a large conformational space, existing in a dynamic equilibrium between membrane-associated and membrane-distal conformations. Subsequent interaction with ASAP1 PH domain restricts motions of the G domain to lock it in a conformation primed for hydrolysis. Through these integrated/hybrid methods, new roles of the PH domain to stimulate and regulate GTP-hydrolysis are revealed, and exploration of the interaction between the GAP domain of ASAP1 and Arf1 becomes possible.