Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5).
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:06/7.39Both 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:13/6.48microswitch, 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 with125I-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:09/3.33The 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.
From the Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark.
Stefanie
http://orcid.org/0000-0001-6505-0883
S
Karlshøj
From the Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark.|the Faculty of Chemistry, Alexandru Ioan Cuza University of Iaşi, Bd. Carol I No. 11, RO-700506 Iaşi, Romania.
Roxana Maria
RM
Amarandi
From the Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark.
Olav
http://orcid.org/0000-0001-9054-4690
O
Larsen
From the Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark.
Viktorija
http://orcid.org/0000-0001-8786-348X
V
Daugvilaite
From the Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark.
Anne
A
Steen
the Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark.
Matjaž
M
Brvar
the Faculty of Chemistry, Alexandru Ioan Cuza University of Iaşi, Bd. Carol I No. 11, RO-700506 Iaşi, Romania.
Aurel
A
Pui
the Novo Nordisk Foundation Center for Basic Metabolic Research, Section for Metabolic Receptology and Enteroendocrinology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark, and.
Thomas Michael
TM
Frimurer
the Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark.
Trond
T
Ulven
From the Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark, rosenkilde@sund.ku.dk.
Mette Marie
http://orcid.org/0000-0001-9600-3254
MM
Rosenkilde
D000494:Allosteric Regulation / Q000187:drug effects*; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002614:Chelating Agents / Q000494:pharmacology*; D054405:Chemokine CCL3 / Q000378:metabolism*; D002522:Chlorocebus aethiops; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology*; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000378:metabolism*
C515429:CCL3 protein, human; C584933:CCR5 protein, human; D002614:Chelating Agents; D054405:Chemokine CCL3; D011725:Pyridines; D019713:Receptors, CCR5; C023666:pyridine
7-transmembrane helix receptor; C-C chemokine receptor type 5 (CCR5); G protein-coupled receptor (GPCR); activation mechanism; allosteric regulation; computer modeling; small molecule