Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5)

Stefanie Karlshøj,Roxana Maria Amarandi,Olav Larsen,Viktorija Daugvilaite,Anne Steen,Matjaž Brvar,Aurel Pui,Thomas Michael Frimurer,Trond Ulven,Mette Marie Rosenkilde

doi:10.1074/jbc.m116.740183

Abstract

The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn2+ (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and 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 Zn2+ is anchored to the chemokine receptor-conserved Glu-283VII: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 Trp-248VI:13/6.48 microswitch, contributing to its 8-fold higher potency. The impact of Trp-248 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 Trp-248 required for receptor activation, thereby explaining its lack of activity. Importantly, ZnClTerp becomes agonist to the same extent as ZnTerp upon Ala mutation of Ile-116III:16/3.40, a residue that constrains the Trp-248 microswitch in its inactive conformation. Binding studies with 125I-CCL3 revealed an allosteric interface between the chemokine and the small molecule binding site, including residues Tyr-37I:07/1.39, Trp-86II:20/2.60, and Phe-109III: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.

Highlights

The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn2؉ (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and competitors for CCL5 binding
We describe the structural basis for CCR5 activation by small molecule agonists, the metal ion chelator complexes ZnBip, ZnTerp, and ZnClTerp
The allosteric enhancement of CCL3 by all three ligands arises from a positive modulation of an allosteric interface between the chelator and chemokine binding sites comprising Phe-109 in TM-3 and aromatic residues in the minor binding pocket

Summary

The abbreviations used are

Allosteric Modulation and Agonism in CCR5 transmembrane receptor area. This agrees with the suggested “pseudo”-two-step model that roughly separates the chemokine-receptor interaction into an affinity-providing step 1 (chemokine core-extracellular receptor domains) and an activation-inducing step 2 (chemokine N terminus-transmembrane receptor domain) [9, 10]. There, the binding of CCL3 was enhanced by the small molecule agonists metal ion chelator complexes, highlighting an allosteric binding mode, whereas another chemokine, CCL5, was displaced with equimolar affinities [16]. CCL5 and the metal ion chelator complexes were affected by the same transmembrane receptor mutations, pointing to an overlap in binding sites that results in the observed competitive binding pattern for CCL5 and the small molecules. It has been discussed whether an allosteric binding mode should be pursued in the development of chemokine receptor antagonists. We thereby describe the molecular mechanism for small molecule-mediated activation and allosteric modulation in CCR5

Results

Discussion

Experimental Procedures