The small molecule metal-ion chelators bipyridine and terpyridine complexed with Zn(2+) (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3-binding to CCR5, weak modulators of CCL4-binding, and as competitors for CCL5-binding. Here we describe their binding site using computational modeling, binding and functional studies on WT and mutated CCR5. The metal-ion Zn(2+) is anchored to the chemokine receptor-conserved E283(VII:06/7.39) Both chelators interact with aromatic residues in the transmembrane receptor domain. The additional pyridine ring of ZnTerp binds deeply in the major binding pocket and, in contrast to ZnBip, interacts directly with the W248(VI:13/6.48) micro-switch, contributing to its 8-fold higher potency. The impact of W248 was further confirmed by ZnClTerp, a chloro-substituted version of ZnTerp that showed no inherent agonism, but maintained positive allosteric modulation of CCL3-binding. Despite a similar overall binding mode of all three metal-ion chelator complexes, the pyridine-ring of ZnClTerp blocks the conformational switch of W248 required for receptor activation, explaining its lack of activity. Importantly, ZnClTerp becomes agonist to the same extent as ZnTerp upon Ala-mutation of I116(III:16/3.40) – a residue that constrains the W248 micro-switch in its inactive conformation. Binding studies with 125I-CCL3 revealed an allosteric interface between the chemokine and the small molecule binding site including residues Y37(I:07/1.39), W86(II:20/2.60) and F109(III:09/3.33) The small molecules and CCL3 approach this interface from opposite directions with some residues being mutually exploited. This study provides new insight into the molecular mechanism of CCR5 activation and paves the way for future allosteric drugs for chemokine receptors.
Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5).