An integrative structural biology approach to understanding metabolite recognition by cellular receptors

Children'S Hosp Of Philadelphia, Philadelphia PA

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Abstract

PROJECT SUMMARY We propose to develop and apply innovative structural biology, NMR, and complementary biophysical techniques to investigate molecular mechanisms of small molecule recognition by intracellular receptors. The MHC-I-related protein 1 (MR1) binds, traffics, and displays endogenous metabolites derived from aberrant metabolism to provide biomarkers of different intracellular states on the cell surface. It has been recently established that several human viruses, including CMV, HSV and SARS-CoV-2, can directly interfere with known components of the MR1 processing and ligand loading machinery. Thus, unravelling MR1's function will not only help us understand fundamental principles of host/pathogen interactions, but also holds great promise for the future development of universal therapies, since MR1 is highly conserved in the human population. Despite a body of functional and structural studies, key barriers remain pertaining to (i) the molecular determinants of specificity, given that MR1 can display a wide repertoire of chemically distinct ligands; (ii) how binding of small molecules on a limited pocket of the MR1 groove induces global structural adaptations on MR1 surfaces, leading to the formation of unique features that can be leveraged for interactions with chaperones and other components of the MR1 processing pathway, and (iii) How molecular chaperones, such as TAPBPR, can catalyze exchange between free and bound ligands. Because of the dynamic nature of ligand and chaperone interactions with MR1, and small size of the system by the standards of cryoEM, there are significant challenges to conventional structural approaches. To address these bottlenecks, I have developed an integrative structural approach, combining nuclear magnetic resonance (NMR) spectroscopy, complementary biophysical techniques and computational modeling to characterize MR1/metabolite complexes and their interactions with chaperones. Our preliminary data show that metabolite binding to MR1 creates unique conformational states, that can be leveraged by chaperones to promote exchange. In this renewal proposal, will further extend our integrative approach to elucidate molecular determinants of small molecule/MR1 assembly and stability, including the contribution of protein dynamics to the selection of ligands with distinct chemical features. Using a combination of methyl-based NMR constraints with structural modeling and in vitro assays, we will delve deeper into MR1/chaperone interactions, and elucidate the mechanism of ligand exchange. Our studies will provide a mechanistic paradigm of a highly malleable protein binding site that can accommodate a wide range of chemically distinct ligands, and further our understanding of how presentation of small molecules by MR1 can provide robust biomarkers for signaling different metabolic states.