Date of Award
Doctor of Philosophy (PhD)
Dr. Brian N. Dominy, Committee Chair
Dr. Steve J. Stuart
Dr. Emil Alexov
Dr. Jason McNeill
G protein-coupled receptors (GPCRs) which are seven-transmembrane allosteric machine constitutes largest and diverse family of membrane proteins. GPCR participate in activating a diverse range of signaling pathways, in response to ligand perturbation which ranges from neurotransmitters, hormones to photons. The role of GPCRs in a wide range of key physiological processes and their ubiquity in mammalian genome makes them attractive pharmaceutical targets. Signal transduction in GPCR occur mainly, via G-proteins and leads to a cascade of signaling. In addition to the orthosteric site, GPCRs also possesses a topographically distinct allosteric site which contributes to allosteric modulation, i.e long distant ligand binding for activating G proteins and trigger GDP release. The mechanism that governs allosteric activation triggering GDP release is yet uncertain. Differential ligands bind to GPCR's orthosteric sites and can modulate allosteric signaling. Ligands that increase or decrease the GPCR signaling are classified as agonists and antagonists respectively. Compared to orthosteric ligand allosteric modulator through electrostatic repulsion, steric hindrance or conformational stability can select subsets of signaling responses. We in this study are trying to understand the basis of ligand-biased signaling or functional selectivity that leads to long-distance signaling in a receptor. Using the information from crystal structures of the receptor, combined with molecular dynamics simulations, we performed a systematic analysis to identify the basis of conformational selectivity for allosteric bias in GPCRs. Our study explores the conformational landscape of GPCRs as a function of the activity of the receptor. Normal modes analysis (NMA) was used to identify low-frequency modes that describe conformational changes due to large-scale domain motions in the receptor. NMA characterized changes in correlated motions of residues in the rest six global modes and revealed conformation shift starting from the inactive structure. We used MD simulations coupled with network analysis to reveal correlated motion between G-protein Coupling site and ligand binding site. Changes in dynamically correlated residue motion in allosteric networks reveals the characteristic feature of receptor activity in GPCRs. Single point mutations studies were aimed to analyze the changes in the structural scaffold of GPCRs as a result of mutations. Mutational studies facilitated in determining the basis of functional selectivity and changes in the allosteric communication as a result of allosteric binding to the receptor. Single point mutations also revealed residues critical for functional activity of GPCRs. Inter residue contact network responsible for biased signaling using microsecond atomic level simulations reveals differential allosteric modulation. Finally, comparative analysis using mutual information in the internal coordinates of mutants and wild types helped to quantify the allosteric modulation and long-range cooperativity between binding sites in GPCRs.
Agrawal, Vibhor, "Understanding Allosteric Modulation of G-Protein Coupled Receptors" (2016). All Dissertations. 1630.