Understanding Periperal Membrane Protein Interactions | BPS Thematic Meeting

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Wednesday Speaker Abstracts

CHARACTERIZING THE MEMBRANE ORIENTATION DYNAMICS OF RHEB AND ITS FUNCTIONAL CONSEQUENCES Chase M Hutchins 1 ; Cynthia V Pagba 1 ; Jacob L Jakubec 1 ; Geetika V Verma 2 ; Vasanthi Jayaraman 2 ; Alemayehu A. Gorfe 1 ; 1 University of Texas McGovern Medical in Houston , IntegrativeBiology and Pharmacology, Houston , TX, USA 2 University of Texas McGovern Medical School in Houston , Biochemistry and Molecular Biology , Houston, TX, USA Rheb is a Ras family small GTPase that regulates cell growth and proliferation through direct activation of mTORC1. In this study, we utilized molecular dynamics (MD) simulations in an endoplasmic reticulum model bilayer, single molecule FRET (smFRET) in native lipid nanodiscs and cell signaling assays to characterize the membrane dynamics of Rheb and explore functional consequences of this phenomenon. MD simulations revealed that GDP and GTP-bound Rheb sample 4 distinct membrane orientation states. Alignment of membrane-bound Rheb in each state to an experimental structure of mTORC1 showed that all but one state displayed clashing between membrane and mTORC1, suggesting that only one of these states is signaling competent. smFRET validation of Rheb’s membrane dynamics also showed 4 dominant conformational states, with similar mean predicted distances and population sizes to simulation identified states. Measurement of protein-lipid contact frequency revealed distinct, dynamically important residues that stabilized each orientation through sustained membrane interactions. We assessed the functional consequences of disrupting these orientations by mutating these residues to the less reactive alanine and assessed the functional effects by testing the ability of mutants to stimulate mTORC1 activity with immunoblotting. These assays showed orientation-dependent negative or positive modulation of mTORC1 kinase activity, suggesting that membrane reorientation plays a functional role in Rheb. Overall, we show that Rheb has similar dynamics to Ras proteins and importantly that membrane re-orienation plays a role in GTPases other than Ras and may represent a fundamental regulator of small GTPase signaling.

UNRAVELING THE MECHANISTIC DIVERSITY OF PERIPHERAL PROTEINS BINDING TO MEMBRANES THROUGH INTEGRATIVE APPROACHES Matteo Dal Peraro 1 ; 1 EPFL, Lausanne, Switzerland

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