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J. Biol. Chem., Vol. 282, Issue 38, 27976-27983, September 21, 2007

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The Human CC Chemokine MIP-1 Dimer Is Not Competent to Bind to the CCR5 Receptor^*

From the Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77843

Chemokine dimerization has been the subject of much interest in recent years as evidence has accumulated that different quaternary states of chemokines play different biological roles; the monomer is believed to be the receptor-binding unit, whereas the dimer has been implicated in binding cell surface glycosaminoglycans. However, although several studies have provided evidence for this paradigm by making monomeric chemokine variants or dimer-impaired chemokines, few have provided direct evidence of the receptor function of a chemokine dimer. We have produced a covalent dimer of the CC chemokine macrophage inflammatory protein-1 (MIP-1) by placing a disulfide bond at the center of its dimer interface through a single amino acid substitution (MIP-1-A10C). This variant was shown to be a nondissociating dimer by SDS-PAGE and analytical ultracentrifugation. NMR reveals a structure largely the same as the wild type protein. In studies of glycosaminoglycan binding, MIP-1-A10C binds to a heparin-Sepharose column as tightly as the wild type protein and more tightly than monomeric variants. However, MIP-1-A10C neither binds nor activates the MIP-1 receptor CCR5. It was found that the ability to activate CCR5 was recovered upon reduction of the intermolecular disulfide cross-link by incubation with 1 mM dithiothreitol. This work provides the first definitive evidence that the CC chemokine MIP-1 dimer is not able to bind or activate its receptor and implicates the CC chemokine monomer as the sole receptor-interacting unit.

Received for publication, March 28, 2007 , and in revised form, June 25, 2007.

^* This work was supported by National Institutes of Health Grant AI47832, National Science Foundation Grant MCB-0212700, and by the Robert Welch Foundation Grant A1472. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Table S1.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, Texas A & M University, TAMU 2128, College Station, TX 77843-2128. Tel.: 979-845-5616; Fax: 979-845-9274; E-mail: pliwang{at}tamu.edu.

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