Date of Award

2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Materials Science

First Advisor

Juan M. Vanegas

Abstract

The Angiotensin II Type 1 (AT1) receptor is one of the most widely studied G-protein coupled receptors (GPCRs) within the context of biased signaling. Unlike other GPCRs, the AT1 receptor is activated not only by agonists such as the peptide AngII, but also by mechanical stimuli including membrane stretch and shear in the absence of a ligand. These various chemical and mechanical modes of activation induce different cellular signaling pathways. Despite the importance of mechanical activation of the AT1 receptor in biological processes such as vasoconstriction, little is known about the structural changes induced by external physical stimuli mediated by the surrounding lipid membrane. Here, we present a systematic molecular dynamics (MD) simulation study that characterizes the activation of the AT1 receptor under various membrane environments and mechanical stimuli along with the AT1 receptor complexes with agonists and a G-protein mimicking nanobody. First, we characterized the stability of the active state of the AT1 receptor and its sensitivity to changes in the surrounding membrane environment from variations in the lipid acyl chain length, head-group chemistry, and membrane tension (i.e., mechanical stretch). MD simulations of the apo AT1 receptor in DMPC, POPC, and SOPC show that there is an optimal bilayer thickness that favors activation (POPC), while shorter (DMPC) and longer (SOPC) lipids promote inactivation within a few microseconds. Addition of SOPE lipids, which have a more negative spontaneous curvature, to the SOPC membrane in a 1:1 ratio also stabilizes the active state despite having a larger bilayer thickness compared to SOPC. Similarly to changing the acyl chain length, we found that membrane tension stabilizes the AT1 receptor's active state in a value-dependent manner, where intermediate tensions around 10 mN/m are optimal. Structural comparison of membrane-mediated vs.~agonist-induced activation shows that the AT1 receptor has distinct active conformations. This is supported by long multi-microsecond unbiased simulations and free energy calculations that show unique landscapes for the inactive and various active states. In addition to the mechanical activation of the AT1 receptor, we also explored the role of biased agonists as well as constitutive mutations. Our MD simulations and free energy calculations show that G-protein and β-arrestin biased agonists stabilize active-like states with different structural features. Finally, we explore the constitutively active double mutant F77A/N111G, which has been shown experimentally to increase the basal activity of the AT1 receptor, has a highly dynamic structure that can readily explore active and inactive states. Our modeling results provide structural insights into the mechanical and chemical activation of the AT1 receptor and how it may produce different functional outcomes within the framework of biased agonism.

Language

en

Number of Pages

106 p.

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