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Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists

Abstract

CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2–chemokine axis. To aid drug discovery efforts5, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein–protein interactions, receptor–chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

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Figure 1: Structure of a complex between CCR2, BMS-681 and CCR2-RA-[R] and comparison with other allosteric modulators of class A GPCRs.
Figure 2: Ligand binding sites and receptor interactions.
Figure 3: Crystallographic conformation of double-antagonist-bound CCR2 has pronounced structural signatures of an inactive state.
Figure 4: Structural motifs exploited by small molecule antagonists of chemokine receptors.

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Acknowledgements

We thank A. Ishchenko and H. Zhang for help with X-ray data collection, C. Wang and H. X. Wu for suggestions on construct design, F. Li for help with data processing, and M. Galella for assistance with BMS compound data and statistics. We thank C. Ogata, R. Sanishvili, N. Venugopalan, M. Becker, and S. Corcoran at beamline 23ID at GM/CA CAT Advanced Photon Source. Funding for this research was provided by National Institutes of Health grants R01 GM071872, U54 GM094618, R01 AI118985, R21 AI121918, and R21 AI122211. GM/CA@APS has been funded in whole or in part with federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract number DE-AC02-06CH11357.

Author information

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Authors

Contributions

I.K. and T.M.H. designed the study and coordinated all experiments. Y.Z. designed and engineered protein constructs, performed crystallization experiments, collected the diffraction data, and determined the structure. L.Q., M.G., and C.Z. assisted with protein engineering and crystallization. G.W.H. assisted with structure determination and refinement. A.P.I. and L.H.H. designed, and N.V.O.Z. and H.d.V. performed, equilibrium and kinetics binding experiments. I.K. performed computational and bioinformatics analyses. R.J.C., P.C., and A.T. synthesized, characterized, and crystallized the BMS compound analogues. M.D. assisted with compound crystallization. D.S. assisted with the allosteric compound characterization. R.A. assisted with structure analysis. V.C. and R.C.S. assisted with crystallization. Y.Z., N.V.O.Z., A.P.I., L.H.H., I.K., and T.M.H. wrote the paper.

Corresponding authors

Correspondence to Laura H. Heitman or Irina Kufareva or Tracy M. Handel.

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Competing interests

R.A. has an equity interest in Molsoft, LLC. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. R.C., P.C., and A.T. are employees of Bristol-Myers Squibb Company. D.S. is an employee of Vertex Pharmaceuticals, Inc.

Extended data figures and tables

Extended Data Figure 1 CCR2-T4L crystals and crystal packing.

ac, Crystal packing of CCR2-T4L. CCR2 is a blue ribbon with ECL2 coloured red and T4L yellow. The unit cell is shown as a green box. CCR2-T4L molecules are arranged in a type I packing with hydrophilic stacking mediated by T4L and T4L–ECL2 interactions along axis c. a, Crystal packing in the ac plane. CCR2 makes abundant hydrophobic contacts with its neighbour via an interface mediated by antiparallel helix IV–helix VI interactions related by a screw axis along axis a. b, Crystal packing in the bc plane. Contacts between receptors and T4L involve ECL2 and the intracellular surface of CCR2 including helix VIII. Direct contacts between T4L are along axis b. One layer of CCR2-T4L molecules at the very top of the stacking column is omitted for clarity. c, Crystal packing in the ab plane. There are no direct interactions between T4L along axis a. d, Crystals of CCR2-T4L in the LCP bolus. Average crystals grew to 60 μm × 10 μm × 10 μm before harvesting.

Extended Data Figure 2 BMS-681 binding may disrupt a chemokine-recognizing conformation of the CCR2 N terminus and helix I.

a, Model of CCR2–CCL2 built by homology from the structure of CXCR4–vMIP-II11 suggests that a productive chemokine-compatible conformation of the receptor requires re-orientation of the N terminus from almost parallel to almost perpendicular to the membrane plane, and formation of an extra helical turn in helix I to bring it closer to helix VII and ECL3. b, Binding of BMS-681 may disrupt this chemokine-compatible conformation by inserting between helices I and VII.

Extended Data Figure 3 CCR2-RA-[R] directly binds to CCR2 residues that are homologous to those involved in G-protein coupling in other GPCRs.

Partial alignment of intracellular regions of CCR2 and homologous regions in bovine Rho (bRho) and β2 adrenergic receptor (β2AR), alongside profile of contacts that CCR2-RA-[R], the Gαt C-terminal peptide21, and Gαs C terminus22 make with the three respective receptors. Contacts are shown by circles above and below the alignment, with circle area indicative of contact strength. Backbone and side-chain contacts are grey and black, respectively. Assuming structural homology between the CCR2–G-protein interface and at least one of the bRho–Gαt and β2AR–Gαs interfaces, several residue positions seem to be involved in binding both CCR2-RA-[R] and the C terminus of the G protein.

Extended Data Figure 4 Equilibrium binding and binding kinetics of BMS-681 and CCR2-RA-[R] with WT CCR2 and CCR2-T4L.

a, b, Displacement of [3H]INCB-3344 (5 nM, a) and [3H]CCR2-RA-[R] (3 nM, b) from WT CCR2 and CCR2-T4L in CHO cells by increasing concentrations of unlabelled INCB-3344, CCR2-RA-[R] and BMS-681. c, d, Association and (e, f) dissociation of 7 nM [3H]CCR2-RA from CHO cell membranes transiently expressing WT CCR2 (c, e) or CCR2-T4L (d, f) at 25 °C, in the absence or presence of 1 μM BMS-681. Figures represent normalized and combined data from three independent experiments performed in duplicate, with results presented as mean ± s.e.m. percentage of specific [3H]CCR2-RA binding.

Extended Data Figure 5 A Zn2+ binding site was identified by X-ray fluorescence emission analysis of the CCR2-T4L–BMS-681–CCR2-RA-[R] crystals.

a, View of the Zn2+ ion at an interface formed by CCR2 helices III and VI and the N terminus of T4L. The Zn2+ ion is coordinated by side chains of H1443.56 (from WT receptor), E2386.30 (from the engineered part of the receptor), and E1005 (from T4L) as well as a structured water. b, Background fluorescence signal of an empty MiTeGen micromount is low, indicating the absence of metal ion. Excitation at 12 keV results in a peak at 11.7 keV (owing to the incidence beam). c, X-ray fluorescence emission signal from a wide fluorescence scan of the CCR2-T4L crystal. The fluorescence peaks at 8.60 keV and 9.53 keV correspond to X-ray emission lines Kα (8.64 keV) and Kβ (9.57 keV) and indicate the presence of Zn2+ bound to CCR2-T4L. d, A zoomed-in view of the X-ray fluorescence emission signal from c.

Extended Data Table 1 Data collection and refinement statistics (molecular replacement)
Extended Data Table 2 Small-molecule (BMS-681) X-ray data collection and refinement
Extended Data Table 3 Displacement of specific [3H]INCB-3344 (5 nM) and [3H]CCR2-RA (3 nM) binding from CCR2 constructs transiently expressed on CHO cells
Extended Data Table 4 Observed association and dissociation rate constants of [3H]CCR2-RA (7 nM) on membranes from CHO cells transiently expressing WT CCR2 and CCR2-T4L, in the absence or presence of 1 μM BMS-681

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Zheng, Y., Qin, L., Zacarías, N. et al. Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists. Nature 540, 458–461 (2016). https://doi.org/10.1038/nature20605

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