Structure of the chemokine receptor CXCR1 in phospholipid bilayers

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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 growth1,2,3. 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 inflammation2. 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 receptors4,5,6,7. 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 sequences8, as well as addition of stabilizing antibodies and small molecules9 that facilitate crystallization in cubic phase monoolein mixtures10. The intracellular loops of GPCRs are crucial for G-protein interactions11, and activation of CXCR1 involves both amino-terminal residues and extracellular loops2,12,13. 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 activity14. 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.

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Figure 1: Structure determination of CXCR1.
Figure 2: Three-dimensional structure of CXCR1.
Figure 3: Structural comparison of CXCR1 and CXCR4.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates for residues 29–324 of CXCR1 and NMR restraints have been deposited in the Protein Data Bank (PDB) under accession 2LNL. Assigned NMR frequencies have been deposited in the Biological Magnetic Resonance Bank (BMRB) under accession 18170.

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Acknowledgements

This research was supported by grants R01EB005161, R01GM075877, R21GM94727, R21GM075917, P01AI074805 and P41EB002031 from the National Institutes of Health (NIH). Further support came from Cambridge Isotope Laboratories. F.C. was supported by fellowships from the Swiss National Science Foundation (PBBSP3-123151) and the Novartis Foundation.

Author information

S.J.O. designed the study. S.P. optimized CXCR1 purification, refolding and NMR sample preparation. B.B.D. performed the NMR experiments. H.J.N. assisted in NMR data analysis. H.K. developed initial protocols for CXCR1 purification, refolding and functional assays. K.M. assisted in the revision of these methods for NMR experiments. A.A.D. tested samples for their suitability for NMR experiments. F.C., M.C. and K.M. expressed and purified CXCR1. F.M.M. and Y.T. performed the structure calculations. S.J.O., S.P., B.B.D., F.M.M. and Y.T. prepared the figures and wrote the paper.

Correspondence to Stanley J. Opella.

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The authors declare no competing financial interests.

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Supplementary Information

This file contains detailed Supplementary Methods, Supplementary References, Supplementary Table 1, which shows NMR structural statistics, Supplementary Table 2, which lists NMR experimental parameters and Supplementary Figures 1-11, which display additional experimental data. (PDF 3577 kb)

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Park, S., Das, B., Casagrande, F. et al. Structure of the chemokine receptor CXCR1 in phospholipid bilayers. Nature 491, 779–783 (2012) doi:10.1038/nature11580

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