Structure of the chemokine receptor CXCR1 in phospholipid bilayers

Abstract

CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation. CXCR1 is a class A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences, as well as addition of stabilizing antibodies and small molecules that facilitate crystallization in cubic phase monoolein mixtures. The intracellular loops of GPCRs are crucial for G-protein interactions, and activation of CXCR1 involves both amino-terminal residues and extracellular loops. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.

Figure 1. Structure determination of CXCR1

a, CXCR1 topology with four disulfide bonds (gold). b-c, Strip plots from three-dimensional experiments taken at specific ¹⁵N and ¹³CA chemical shifts for three representative regions of CXCR1: N-terminus (residues 31-35) (red), TM2 (residues 78-82) (blue), and ECL2 (residues 175-179) (green). b, NCACX data used for resonance assignments. c, ¹³C-detected ¹H-¹⁵N SLF spectra. d, Dipolar wave plot of the experimentally measured ¹H-¹⁵N DC values as a function of residue number. Sinusoidal fits (cyan) to the data (4.1 kHz RMSD) highlight the transmembrane (TM1-TM7) and C-terminal (H8) helices. e, Ensemble of 10 lowest energy structures of CXCR1 aligned in the membrane (n: bilayer normal). f-g, Correlation plots of experimental and back-calculated ¹H-¹⁵N DC restraints obtained (f) before and (g) after refinement against the experimental data.

Figure 2. Three-dimensional structure of CXCR1

Backbone representation of CXCR1 showing helices (TM1-TM7 and H8) in aqua, extracellular loops (ECL1-ECL3) in gray, and intracellular loops in blue (ICL1), green (ICL2) and red (ICL3). Disulfide bonded Cys pairs (C30-C277; C110-C187) are shown as sticks. a, Side view (n: bilayer normal). b, View from the extracellular side. c, View from the intracellular side.

Figure 3. Structural comparison of CXCR1 (cyan, PDB ID: 2LNL) and CXCR4 (pink, PDB ID: 3ODU)

a, Comparison of TM helices. TM2 (residues 74-101) of CXCR1 (blue) has a kink that changes helix direction at Phe88. The kink is reflected in disruption of the dipolar wave near Phe88. TM2 of CXCR4 (magenta) has a kink at the same location (Phe87). b, Comparison of backbone structures. The third intracellular loop (ICL3) of CXCR4 is replaced by T4 lysozyme (T4L, molecular surface representation). The C-terminus of CXCR1 forms a well-defined amphipathic helix (H8), while that of CXCR4 is only loosely helical.