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.
The target residence time (RT) for a given ligand is one of the important parameters that have to be optimized during drug design. It is well established that shielding the receptor−ligand hydrogen bond (H-bond) interactions from water has been one of the factors in increasing ligand RT. Building on this foundation, here we report that shielding an intra-protein H-bond, which confers rigidity to the binding pocket and which is not directly involved in drug−receptor interactions, can strongly influence RT for CCR2 antagonists. Based on our recently solved CCR2 structure with MK-0812 and molecular dynamics (MD) simulations, we show that the RT for this and structurally related ligands is directly dependent on the shielding of the Tyr120-Glu291 H-bond from the water. If solvated this H-bond is often broken, making the binding pocket flexible and leading to shorter RT.
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