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

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

Eyepath Lab

8 Citationer (Scopus)

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-283^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 Trp-248^VI: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-116^III: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-37^I:07/1.39, Trp-86^II:20/2.60, and Phe-109^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.

Originalsprog	Engelsk
Tidsskrift	Journal of Biological Chemistry
Vol/bind	291
Udgave nummer	52
Sider (fra-til)	26860-26874
ISSN	0021-9258
DOI	https://doi.org/10.1074/jbc.M116.740183
Status	Udgivet - 23 dec. 2016

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10.1074/jbc.M116.740183Licens: Ikke-specificeret

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@article{28cc11319a3b43afb98582e124cf742f,

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

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.",

author = "Stefanie Karlsh{\o}j and Amarandi, {Roxana Maria} and Olav Larsen and Viktorija Daugvilaite and Anne Steen and Matja{\v z} Brvar and Aurel Pui and Frimurer, {Thomas Michael} and Trond Ulven and Rosenkilde, {Mette Marie}",

note = "Copyright {\textcopyright} 2016, The American Society for Biochemistry and Molecular Biology.",

year = "2016",

month = dec,

day = "23",

doi = "10.1074/jbc.M116.740183",

language = "English",

volume = "291",

pages = "26860--26874",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology, Inc.",

number = "52",

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TY - JOUR

T1 - Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5)

AU - Karlshøj, Stefanie

AU - Amarandi, Roxana Maria

AU - Larsen, Olav

AU - Daugvilaite, Viktorija

AU - Steen, Anne

AU - Brvar, Matjaž

AU - Pui, Aurel

AU - Frimurer, Thomas Michael

AU - Ulven, Trond

AU - Rosenkilde, Mette Marie

PY - 2016/12/23

Y1 - 2016/12/23

N2 - 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.

AB - 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.

U2 - 10.1074/jbc.M116.740183

DO - 10.1074/jbc.M116.740183

M3 - Journal article

C2 - 27834679

SN - 0021-9258

VL - 291

SP - 26860

EP - 26874

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 52

ER -

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

Abstract

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