Wednesday, December 4, 2024
12/4/2024
It is often assumed that lipid-anchored proteins non-specifically attach to membranes by the hydrophobic lipid- modified moiety. We propose that it takes more than lipid-modification to productively organize lipid-anchored proteins on target membranes. We will test this hypothesis using the Ras superfamily of lipid-anchored small GTPases (laSGs) as model systems. The Ras superfamily consists of the Ras, Rho, Arf and Rab family of molecular switches that mediate a wide variety of cellular processes controlling cell growth, motility and trafficking. The structure of these proteins consists of a conserved catalytic domain, a flexible linker, and a lipid anchor. Little is known about the precise roles of the intrinsically disordered linker region. By contrast, it is well established that cycling between active GTP-bound and inactive GDP-bound conformational states of the catalytic domain regulate function. Disruption of this cycle by mutation or genetic defects causes many diseases including cancer and developmental disorders. The proposed work will lay the foundation for the development of effective therapies that directly target these proteins. Supported by strong preliminary data, we hypothesize that monomeric laSGs engage membranes through one of the following mechanisms: (i) Those with a moderately long (~20aa) flexible linker between the lipid-anchor and the G-domain adopt multiple distinct orientations with respect to the membrane plane, with the catalytic domain ‘swinging’ and ‘rolling’ on the surface. (ii) Those with a long flexible linker keep the G-domain distal from the membrane, with the long linker collapsing on it as spaghetti would on a wall. (iii) Those with a short (and rigid) linker, such as GTP-bound Arf1, engage membranes in a single orientation with the G-domain able to roll but not swing on the membrane surface. (iv) The G-domain of dually lipid-anchored laSGs does not reorient. We will test these hypotheses using state-of- the-art molecular simulations and simulation-guided experiments. In Aim 1 we will define the sequence and structural determinants of membrane binding and reorientation of laSGs using atomistic molecular dynamics simulations to map the conformational and energy landscapes of Rheb, RhoA, Rab11A and Arf1, representing the Ras, Rho, Rab and Arf family proteins, respectively. In Aim 2, we will determine the functional roles of G- domain membrane engagement and reorientation using mutations and cell signaling assays, confocal imaging, electron microscopy (EM), and single molecule FRET in native lipid nanodiscs. Aim 3 will assess the impact of G-domain membrane interaction and reorientation on the druggability of our model systems. The results will elucidate the principles and structural regions responsible for the dynamic membrane interaction of laSGs and define the determinants of their membrane reorientation, and potentially open up novel therapeutic opportunities to treat many intractable diseases.
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