Selective Allosteric Modulation of N-Terminally Cleaved, but Not Full Length CCL3 in CCR1

Chemokines undergo post-translational modification such as N-terminal truncations. Here, we describe how N-terminal truncation of full length CCL3^(1-70) affects its activity at CCR1. Truncated CCL3^(5-70) has 10-fold higher potency and enhanced efficacy in β-arrestin recruitment, but less than 2-fold increased potencies in G protein signaling determined by calcium release, cAMP and IP₃ formation. Small positive ago-allosteric ligands modulate the two CCL3 variants differently as the metal ion chelator bipyridine in complex with zinc (ZnBip) enhances the binding of truncated, but not full length CCL3, while a size-increase of the chelator to a chloro-substituted terpyridine (ZnClTerp), eliminates its allosteric, but not agonistic action. By employing a series of receptor mutants and in silico modeling we describe residues of importance for chemokine and small molecule binding. Notably, the chemokine receptor-conserved Glu287^7.39 interacts with the N-terminal amine of truncated CCL3^(5-70) and with Zn²⁺ of ZnBip, thereby bridging their binding sites and enabling the positive allosteric effect. Our study emphasizes that small allosteric molecules may act differently toward chemokine variants and thus selectively modulate interactions of specific chemokine subsets with their cognate receptors.