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

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37 Citations (Scopus)

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
JournalThe Journal of Biological Chemistry
Volume287
Issue number18
DOIs
Publication statusPublished - 2012

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