Dynamics and assembly of lipid-anchored proteins - A major focus of research in my laboratory is the Ras superfamily of lipid-anchored small GTPases (LsGs) such as Ras, Rab, Rho, Arf families. LsGs operate as molecular switches to mediate the regulating of cell growth, motility and trafficking, with their dysregulation leading to many types of cancer and developmental disorders. An overarching long-term goal of my laboratory is uncovering the roles of membranes and lipids in the regulation of LsG functions. My goals for the next five years include investigating the impact of membrane binding, dynamics and lipid composition on the inhibition and self-assembly of LsGs using Rheb, Rab11A, RhoA, and Arf1 proteins as model systems. Previous work by us and others has demonstrated that targeting of RAS proteins to the plasma membrane (PM) is a complex process involving an interplay between the lipid anchor, the catalytic G- domain, and the identity of lipids in the PM. Similarly, preliminary studies using long-timescale molecular dynamics (MD) simulations suggest major differences among our model systems in membrane engagement, lipid preference, and orientational dynamics. We also found using biophysical assays large differences in ligand binding affinities between soluble and native lipid nanodisc (ND)-bound LsGs, and in pocket features and interactions of inhibitors. Building on these initial observations, we propose to test the hypothesis that membrane binding and orientational dynamics are major modulators of LsG interactions with small molecule ligands. Moreover, it is becoming increasingly clear that LsGs' functions are regulated not only by nucleotide exchange or effector/exchange factor binding but also through the formation of dynamic oligomers on membrane surfaces. However, there is debate about the nature, size and composition of these oligomers, including whether they are comprised of a defined number of subunits or involve a mixture of dimers, trimers, tetramers and higher-order assemblies. There is also controversy about the role of the G-domain versus the lipid anchor in oligomerization. A major obstacle to resolving these issues has been the lack of suitable experimental and computational techniques to study LsG assemblies at the molecular level. New imaging and mass spectroscopic techniques have recently shown the existence of KRAS dimers in both near-native membranes and reconstituted liposomes, respectively, with the former also finding trimers and tetramers. No comparable studies have been conducted on endo-membrane LsGs such as Arf or Rab, despite evidences of their self-assembly. This is in part because previous studies have mostly focused on PM-resident LsGs due to experimental constraints. We have developed capability to isolate both PM- and endomembrane-resident LsGs in NDs that allows us to fill this gap. We propose to combine this capability with state-of-the-art imaging and computational methods to provide insights into the distribution as well as structure and dynamics of LsG oligomers on membrane surfaces. We anticipate the results to establish that, rather than the prevailing protein-centric or lipid-centric views, a proteolipid hypothesis whereby weak protein-protein interactions and lipid-based processes working in concert underlie LsG oligomerization.
