Author ORCID Identifier

0000-0002-1090-4711

Date of Graduation

5-2022

Document Type

Dissertation (PhD)

Program Affiliation

Biochemistry and Molecular Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Alemayehu A. Gorfe, PhD

Committee Member

John F. Hancock, MB, BChir, PhD

Committee Member

James M. Briggs, PhD

Committee Member

Vasanthi Jayaraman, PhD

Committee Member

M. Neal Waxham, PhD

Abstract

KRAS, a 21 kDa small GTPase protein, functions as a molecular switch playing a key role in regulating cell proliferation. Dysregulation of KRAS signaling by oncogenic mutations leads to uncontrolled cell proliferation, a hallmark of cancer cells. Attempts to therapeutically target oncogenic KRAS have led to limited success resulting in a need to identify new mechanisms to targeting KRAS. The interaction of KRAS with its regulators, effectors, and the membrane present one such avenue. In this study, we investigated how post-translational covalent and environmental modifications could modulate these interactions of KRAS. Using computational molecular dynamics simulations, nuclear magnetic resonance spectroscopy and biochemical and cellular assays, we investigated the structural mechanisms by which monoubquitination and membrane depolarization affected KRAS structure and dynamics and its interactions with regulators, effectors, and the membrane.

KRAS undergoes monoubiquitination at two sites, Lys104 and Lys147. We observed that ubiquitin is quite dynamic around KRAS and forms weak non-specific interactions with KRAS. Through these interactions, it is capable of modulating KRAS dynamics to overcome deleterious effects from monoubiquitination. Additionally, these interactions also lead to certain conformations of monoubiquitinated KRAS being sampled more frequently. These different conformations have different occlusion-mediated effects on KRAS binding to its KRAS regulators, effectors and membrane. We propose a mechanism conformational selection of certain conformations and population shift towards those conformations can help overcome the deleterious effects from monoubiquitination.

Proliferating cells have a depolarized membrane, which was recently shown to increase phosphatidylserine(PS)-associated KRAS nanoclustering at the membrane and consequently enhance KRAS-mediated proliferative signaling. Using MD simulations, we investigated the structural basis of this process by applying an electric field across a lipid-bilayer with multiple copies of the C-terminal membrane anchor of KRAS (tK) embedded in it. We saw that the electric field increase the PS-affinity of tK by two mechanisms: population shift to a tK conformation with high-affinity to PS or increasing the PS-affinity of the entire tK population. Further, membrane asymmetry played a role in dictating which mechanism was utilized. Within the limits of our model, the polarizing electric field enhances tK-PS interactions, contrary to expectations. We propose that the PS-tK co-localization on depolarization observed in experiments may involve other factors beyond direct tK-PS interactions.

Keywords

KRAS, Membrane Depolarization, Ubiquitination, MD simulations, Biophysics, Structural Biology, Cell Biology, Cancer Biology

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