Decoding Structural Determinants of Efficacy and Specificity in a GPCR Subfamily

Project Summary G protein–coupled receptors (GPCRs), the largest family of membrane proteins and a major class of drug targets, convert extracellular stimuli into intracellular responses by undergoing conformational changes that enable them to bind and activate signaling proteins. Identifying the mechanisms that underly activation––the atomic-level rearrangements that propagate from the ligand-binding pocket to induce large-scale motions on the intracellular surface––can powerfully impact drug discovery but requires measurement of conformational change at multiple temporal and spatial scales. Thus, activation mechanisms for only a small number of GPCRs have been carefully mapped at the atomic level, hindering the rational design of therapeutics with high selectivity and few side effects. Uncovering these mechanisms for those therapeutically promising GPCRs that operate as multimers, with large extracellular domains, requires biophysical approaches that can bridge multiple scales. From my Ph.D., I have expertise in using physics-based simulations to capture conformational change in membrane proteins with high spatial and temporal resolution, but the computational cost of molecular dynamics (MD) limits accessible timescales and investigation of many members of a protein family. As a postdoctoral fellow at UC-Berkeley, I have pursued experimental studies to fill in the gaps associated with classical MD simulations. I have learned to carry out hydrogen-deuterium exchange–mass spectrometry (HDX-MS) under the supervision of Dr. Susan Marqusee, enabling me to quantify the local stability of structural elements in a protein. More recently, I have pursued an additional strategy, single-molecule Förster Resonance Energy Transfer (smFRET), to quantify the relative populations of states in a protein conformational landscape, under the guidance of Dr. Ehud Isacoff. I will pursue computational and experimental studies of a difficult-to-drug class of GPCRs, the metabotropic glutamate receptors (mGluRs). The mGluRs have a complex topology: the ligand-binding domains (LBD) of these GPCRs transmit a ligand's effects laterally, to the neighboring subunit, and intracellularly, to the transmembrane domain. In Aim 1, I will determine the mechanisms by which ligand binding affects conformational changes within a single subunit; across the dimer interface; and below, to the transmembrane domain. In Aim 2, I will investigate how sequence variation in the mGluR family leads to the strikingly broad range of glutamate affinity and efficacy previously observed for the eight mGluR subtypes. In Aim 3, I will investigate how endogenous extracellular binding partners modulate mGluRs to affect downstream activation. This work will provide a general strategy for investigating mechanisms of conformational change for multi-domain proteins and will enable discovery of allosteric ligands that can selectively target a particular receptor while eliciting a specific signaling output. The rich scientific environment of UC-Berkeley, along with the support of my outstanding mentors, will enable me to train in experimental biophysics while preparing for an academic career.