Design and receptor interactions of obligate dimeric mutant of chemokine monocyte chemoattractant protein-1 (MCP-1)

Joshua Tan Hoong Yu, Meritxell Canals, Justin P Ludeman, Jamie Wedderburn, Christopher Boston, Stephen J Butler, Ann Marie Carrick, Todd R Parody, Deni Taleski, Arthur Christopoulos, Richard J Payne, Martin J Stone

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Chemokine-receptor interactions regulate leukocyte trafficking during inflammation. CC chemokines exist in equilibrium between monomeric and dimeric forms. Although the monomers can activate chemokine receptors, dimerization is required for leukocyte recruitment in vivo, and it remains controversial whether dimeric CC chemokines can bind and activate their receptors. We have developed an obligate dimeric mutant of the chemokine monocyte chemoattractant protein-1 (MCP-1) by substituting Thr(10) at the dimer interface with Cys. Biophysical analysis showed that MCP-1(T10C) forms a covalent dimer with similar structure to the wild type MCP-1 dimer. Initial cell-based assays indicated that MCP-1(T10C) could activate chemokine receptor CCR2 with potency reduced 1 to 2 orders of magnitude relative to wild type MCP-1. However, analysis of size exclusion chromatography fractions demonstrated that the observed activity was due to a small proportion of MCP-1(T10C) being monomeric and highly potent, whereas the majority dimeric form could neither bind nor activate CCR2 at concentrations up to 1 mum. These observations help to reconcile previous conflicting results and indicate that dimeric CC chemokines do not bind to their receptors with affinities approaching those of the corresponding monomeric chemokines.
Original languageEnglish
Pages (from-to)14692 - 14702
Number of pages11
JournalJournal of Biological Chemistry
Volume287
Issue number18
DOIs
Publication statusPublished - 2012

Cite this

Tan Hoong Yu, Joshua ; Canals, Meritxell ; Ludeman, Justin P ; Wedderburn, Jamie ; Boston, Christopher ; Butler, Stephen J ; Carrick, Ann Marie ; Parody, Todd R ; Taleski, Deni ; Christopoulos, Arthur ; Payne, Richard J ; Stone, Martin J. / Design and receptor interactions of obligate dimeric mutant of chemokine monocyte chemoattractant protein-1 (MCP-1). In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 18. pp. 14692 - 14702.
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title = "Design and receptor interactions of obligate dimeric mutant of chemokine monocyte chemoattractant protein-1 (MCP-1)",
abstract = "Chemokine-receptor interactions regulate leukocyte trafficking during inflammation. CC chemokines exist in equilibrium between monomeric and dimeric forms. Although the monomers can activate chemokine receptors, dimerization is required for leukocyte recruitment in vivo, and it remains controversial whether dimeric CC chemokines can bind and activate their receptors. We have developed an obligate dimeric mutant of the chemokine monocyte chemoattractant protein-1 (MCP-1) by substituting Thr(10) at the dimer interface with Cys. Biophysical analysis showed that MCP-1(T10C) forms a covalent dimer with similar structure to the wild type MCP-1 dimer. Initial cell-based assays indicated that MCP-1(T10C) could activate chemokine receptor CCR2 with potency reduced 1 to 2 orders of magnitude relative to wild type MCP-1. However, analysis of size exclusion chromatography fractions demonstrated that the observed activity was due to a small proportion of MCP-1(T10C) being monomeric and highly potent, whereas the majority dimeric form could neither bind nor activate CCR2 at concentrations up to 1 mum. These observations help to reconcile previous conflicting results and indicate that dimeric CC chemokines do not bind to their receptors with affinities approaching those of the corresponding monomeric chemokines.",
author = "{Tan Hoong Yu}, Joshua and Meritxell Canals and Ludeman, {Justin P} and Jamie Wedderburn and Christopher Boston and Butler, {Stephen J} and Carrick, {Ann Marie} and Parody, {Todd R} and Deni Taleski and Arthur Christopoulos and Payne, {Richard J} and Stone, {Martin J}",
year = "2012",
doi = "10.1074/jbc.M111.334201",
language = "English",
volume = "287",
pages = "14692 -- 14702",
journal = "Journal of Biological Chemistry",
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Tan Hoong Yu, J, Canals, M, Ludeman, JP, Wedderburn, J, Boston, C, Butler, SJ, Carrick, AM, Parody, TR, Taleski, D, Christopoulos, A, Payne, RJ & Stone, MJ 2012, 'Design and receptor interactions of obligate dimeric mutant of chemokine monocyte chemoattractant protein-1 (MCP-1)' Journal of Biological Chemistry, vol. 287, no. 18, pp. 14692 - 14702. https://doi.org/10.1074/jbc.M111.334201

Design and receptor interactions of obligate dimeric mutant of chemokine monocyte chemoattractant protein-1 (MCP-1). / Tan Hoong Yu, Joshua; Canals, Meritxell; Ludeman, Justin P; Wedderburn, Jamie; Boston, Christopher; Butler, Stephen J; Carrick, Ann Marie; Parody, Todd R; Taleski, Deni; Christopoulos, Arthur; Payne, Richard J; Stone, Martin J.

In: Journal of Biological Chemistry, Vol. 287, No. 18, 2012, p. 14692 - 14702.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Design and receptor interactions of obligate dimeric mutant of chemokine monocyte chemoattractant protein-1 (MCP-1)

AU - Tan Hoong Yu, Joshua

AU - Canals, Meritxell

AU - Ludeman, Justin P

AU - Wedderburn, Jamie

AU - Boston, Christopher

AU - Butler, Stephen J

AU - Carrick, Ann Marie

AU - Parody, Todd R

AU - Taleski, Deni

AU - Christopoulos, Arthur

AU - Payne, Richard J

AU - Stone, Martin J

PY - 2012

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N2 - Chemokine-receptor interactions regulate leukocyte trafficking during inflammation. CC chemokines exist in equilibrium between monomeric and dimeric forms. Although the monomers can activate chemokine receptors, dimerization is required for leukocyte recruitment in vivo, and it remains controversial whether dimeric CC chemokines can bind and activate their receptors. We have developed an obligate dimeric mutant of the chemokine monocyte chemoattractant protein-1 (MCP-1) by substituting Thr(10) at the dimer interface with Cys. Biophysical analysis showed that MCP-1(T10C) forms a covalent dimer with similar structure to the wild type MCP-1 dimer. Initial cell-based assays indicated that MCP-1(T10C) could activate chemokine receptor CCR2 with potency reduced 1 to 2 orders of magnitude relative to wild type MCP-1. However, analysis of size exclusion chromatography fractions demonstrated that the observed activity was due to a small proportion of MCP-1(T10C) being monomeric and highly potent, whereas the majority dimeric form could neither bind nor activate CCR2 at concentrations up to 1 mum. These observations help to reconcile previous conflicting results and indicate that dimeric CC chemokines do not bind to their receptors with affinities approaching those of the corresponding monomeric chemokines.

AB - Chemokine-receptor interactions regulate leukocyte trafficking during inflammation. CC chemokines exist in equilibrium between monomeric and dimeric forms. Although the monomers can activate chemokine receptors, dimerization is required for leukocyte recruitment in vivo, and it remains controversial whether dimeric CC chemokines can bind and activate their receptors. We have developed an obligate dimeric mutant of the chemokine monocyte chemoattractant protein-1 (MCP-1) by substituting Thr(10) at the dimer interface with Cys. Biophysical analysis showed that MCP-1(T10C) forms a covalent dimer with similar structure to the wild type MCP-1 dimer. Initial cell-based assays indicated that MCP-1(T10C) could activate chemokine receptor CCR2 with potency reduced 1 to 2 orders of magnitude relative to wild type MCP-1. However, analysis of size exclusion chromatography fractions demonstrated that the observed activity was due to a small proportion of MCP-1(T10C) being monomeric and highly potent, whereas the majority dimeric form could neither bind nor activate CCR2 at concentrations up to 1 mum. These observations help to reconcile previous conflicting results and indicate that dimeric CC chemokines do not bind to their receptors with affinities approaching those of the corresponding monomeric chemokines.

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DO - 10.1074/jbc.M111.334201

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EP - 14702

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 1083-351X

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