Structure and Dynamics of Complexes of Interleukin-8 and its Receptor CXCR1 in Phospholipid Bilayers by Solid-State NMR

Abstract

CXCR1, a G protein-coupled receptor for chemokine interleukin-8 (IL-8) is a key mediator of immune and inflammatory responses and is involved in various diseases, including cancer. Here we describe proton-detected fast MAS solid-state NMR studies of IL-8 and CXCR1 complexes in phospholipid bilayers. More than half of the IL-8 backbone residues were immobilized by the receptor and their chemical shifts significantly perturbed, suggesting that both dynamics and conformation of IL-8 are affected by interactions with CXCR1. Comparison of IL-8 spectra interacting with N-terminal domain and first transmembrane domain (1TM1-72), N-terminal truncated (NT39-350), and wild-type (WT1-350) CXCR1 constructs enabled mapping of IL-8 residues involved in interactions with N-terminal and extracellular regions of CXCR1, providing valuable insight into understanding the first step of the CXCR1 mediated signaling cascade. Long-range distance restraints obtained from intermolecular paramagnetic relaxation enhancement of unnatural amino acid HQA-incorporated CXCR1 constructs serve as crucial input for structure determination of IL-8-CXCR1 complex. Progress toward the determination of the CXCR1 structure bound to Gαi protein and the comparison of the structure of CXCR1 and CXCR2 will be presented.