The structural basis of chemokine binding affinity and receptor selectivity: Role of receptor N-terminal domain

Abstract

Chemokines recruit leukocytes by binding and activating 7-transmembrane G-protein coupled receptors present on the cell surface. Ligand-receptor interaction is complex and involves a broad range of affinities and selectivities. Furthermore, receptor binding involves interaction of both monomeric and dimeric ligands. Interactions between the ligand N-loop and the receptor N-domain (site-I), and between the ligand N-terminus and the receptor extracellular loops (site-II) mediate binding affinity, receptor selectivity and activation. However, the structural basis of how these interactions mediate affinity and selectivity is not known. Therefore studies in this dissertation are focused on the structural basis of interaction of chemokines, IL-8 and MGSA monomers and dimers with their receptors CXCR1 and CXCR2. CXCR1 is specific and binds IL-8 alone with high affinity, whereas CXCR2 is promiscuous and binds both ligands with high affinity. The receptor structures are not known, and also due to the intrinsic difficulties of working with receptors, structural studies were carried out to describe site-I interactions using the isolated receptor N-domains. \r\n\r\nChapter I provides an introduction to the chemokine-receptor system. Chapter II of the dissertation focuses on characterizing the structural basis of differential binding of IL-8 monomers and dimers to the CXCR1 N-domain. Using binding induced NMR chemical shift perturbations and line broadening changes, it was shown that a network of extensive direct and indirect coupled interactions mediate site-I binding of the IL-8 monomer and dimer and that these interactions play a fundamental role in determining binding affinity. Chapter III describes the characterization of the structural basis of CXCR2 receptor N-domain binding of IL-8 and MGSA monomers and dimers, and how coupled interactions between site-I and site-II play a key role in determining receptor affinity and selectivity. Chapter IV describes the structural studies and mechanistic description of how the steric bulk of Ile10, a solvent exposed hydrophobe in the IL-8 N-loop, mediates CXCR1 binding affinity. It was observed that mutating Ile10 to Ala does not affect site-I binding affinity, but negatively regulates site-II binding, resulting in reduced overall receptor binding affinity and activity. Finally, Chapter V describes attempts and progress towards designing inhibitors that target site-I interaction, and how this data could be used for future inhibitor design against chemokine-mediated inflammatory and autoimmune diseases.\r\n