Crystal Structure of CC Chemokine Receptor 2A in Complex with an Orthosteric Antagonist Provides Insights for the Design of Selective Antagonists

Abstract

Graphical AbstractHighlights d Two CCR2A structures in complex with the orthosteric antagonist MK-0812 were solved d IMISX in situ plates facilitated high-quality diffraction data collection d Residue E291 7.39 was confirmed as important in orthosteric antagonist binding d The non-conserved residue H121 3.33 is important for CCR2 selectivity over CCR5 SUMMARYWe determined two crystal structures of the chemokine receptor CCR2A in complex with the orthosteric antagonist MK-0812. Full-length CCR2A, stabilized by rubredoxin and a series of five mutations were resolved at 3.3 Å . An N-and C-terminally truncated CCR2A construct was crystallized in an alternate crystal form, which yielded a 2.7 Å resolution structure using serial synchrotron crystallography. Our structures provide a clear structural explanation for the observed key role of residue E291 7.39 in highaffinity binding of several orthosteric CCR2 antagonists. By combining all the structural information collected, we generated models of co-structures for the structurally diverse pyrimidine amide class of CCR2 antagonists. Even though the representative Ex15 overlays well with MK-0812, it also interacts with the non-conserved H121 3.33 , resulting in a significant selectivity over CCR5. Insights derived from this work will facilitate drug discovery efforts directed toward highly selective CCR2 antagonists with potentially superior efficacy.

In situ crystallography as an emerging method for structure solution of membrane proteins: the case of CCR2A

“…The application of the IMISX method bypassed this mechanical stress and preserved the inherent diffraction properties of the crystals (Figure S3A‐C by Apel et al . ). After cryo‐freezing of the IMISX plate, mini‐datasets were collected for each crystal using the conventional rotation imaging method.…”

“…Three distinct CCR2 crystal structures corresponding to both CCR2A and CCR2B isoforms have recently been determined (PDB http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5T1A, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6GPS, and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6GPX). Zheng et al .…”

“…The final structure is a ternary complex with ligands CCR2‐RA‐[R] and BMS‐681, which bind to the allosteric and orthosteric sites, respectively. The two crystal structures (6GPS and 6GPX) of the chemokine receptor CCR2A splice variant were determined in complex with the orthosteric antagonist MK‐0812 . The 3.3 Å resolution structure (PDB http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6GPS) from Apel et al was obtained from a full‐length CCR2A construct (CCR2A‐fl) containing amino acid residues 2–374, rubredoxin inserted between residues 231 and 235, and five point mutations N14Q, C70Y, G175N, A241D, and K311E.…”

“…The two crystal structures (6GPS and 6GPX) of the chemokine receptor CCR2A splice variant were determined in complex with the orthosteric antagonist MK‐0812 . The 3.3 Å resolution structure (PDB http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6GPS) from Apel et al was obtained from a full‐length CCR2A construct (CCR2A‐fl) containing amino acid residues 2–374, rubredoxin inserted between residues 231 and 235, and five point mutations N14Q, C70Y, G175N, A241D, and K311E. As we identified proteolysis of the N‐ and/or C‐termini of the CCR2A‐fl construct to be a prerequisite for crystallization, we designed a truncated construct CCR2A‐ΔN.C (−) containing residues 29–321, including the ICL3 rubredoxin fusion and three point mutations C70Y, G175N, and A241D (Fig.…”

“…Here, we review the current development status of in meso in situ serial X‐ray crystallization (IMISX), a specific example of a solid support crystal delivery and its recent application to the structure determination of the chemokine receptor 2A (CCR2A) at 2.7 Å resolution . CCL2/CCR2 signaling has been implicated in the pathogenesis of several neurological disorders and autoimmune diseases like multiple sclerosis, obesity, and atherosclerosis, suggesting that CCR2 is an attractive target for potential therapeutic intervention of these diseases .…”