Abstract
The structure of unmodified human CXCR1 was determined within a phospholipid bilayer.
Major finding: The structure of unmodified human CXCR1 was determined within a phospholipid bilayer.
Approach: Rotationally aligned solid-state NMR allowed structure determination at physiologic conditions.
Impact: This approach may facilitate characterization and pharmacologic targeting of CXCR1 and other GPCRs.
Proinflammatory signaling by interleukin-8 (IL-8) through its receptor chemokine (C-X-C motif) receptor 1 (CXCR1) exerts effects on both tumor cells and the tumor microenvironment that promote a malignant phenotype. The development of small-molecule inhibitors of CXCR1, a G-protein coupled receptor (GPCR), and characterization of CXCR1-mediated signal transduction would be facilitated by detailed structural information. Existing 3-dimensional structures of GPCRs have been determined with mutated or truncated constructs or with the addition of stabilizing molecules that promote crystallization but disrupt normal protein function. To determine the structure of CXCR1 in near-native conditions, Park and colleagues expressed full-length, unmodified, active CXCR1 in Escherichia coli and reconstituted the purified protein within liquid crystalline phospholipid bilayers of proteoliposomes. A method called rotationally aligned (RA) solid-state nuclear magnetic resonance (NMR) spectroscopy that takes into account the rotational diffusion of membrane proteins within lipid bilayers was used to calculate the 3-dimensional structure and membrane orientation of CXCR1. Unlike the previous structure of a modified, inactive construct of the related chemokine receptor CXCR4 that was obtained under non-native conditions, the intracellular loops of CXCR1 identified by RA solid-state NMR were structurally well defined, allowing future structural analyses of G-protein binding and activation. Other findings potentially relevant to CXCR1 activity included a well-defined C-terminal intracellular amphipathic helix that aligns with the phospholipid bilayer surface, indicating that the cell membrane may stabilize the conformation of this region and possibly affect G-protein binding, and a distinct cluster of polar residues formed by several transmembrane helices, which may have consequences for ligand specificity and signal transduction. Together, these findings may expedite mechanistic characterization and pharmacologic targeting of CXCR1-mediated chemokine signaling and establish a framework to determine more accurate structures of other GPCRs and membrane proteins.
