THE HUMAN CC CHEMOKINE MIP-1b DIMER IS NOT COMPETENT TO BIND TO THE CCR5 RECEPTOR

Hongjun Jin, Xiaohong Shen, Brandi Renee Baggett, Xiangming Kong and Patricia J. LiWang

From the Department of Biochemistry & Biophysics, Texas A&M University, College Station, TX 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, while the dimer has been implicated in binding cell surface glycosaminoglycans (GAGs). However, while 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 MIP-1b by placing a disulfide bond at the center of its dimer interface through a single amino acid substitution (MIP-1b-A10C). This variant was shown to be a non-dissociating dimer by SDS-PAGE and analytical ultracentrifugation. NMR reveals a structure largely the same as the wild type protein. In studies of GAG binding, MIP-1b-A10C binds to a heparin sepharose column as tightly as the wild type protein and more tightly than monomeric variants. However, MIP-1b-A10C neither binds nor activates the MIP-1b receptor CCR5. It was found that the ability to activate CCR5 was recovered upon reduction of the intermolecular disulfide crosslinking by incubation with 1 mM DTT. This work provides the first definitive evidence that the CC chemokine MIP-1b dimer is not able to bind or activate its receptor, and implicates the CC chemokine monomer as the sole receptor-interacting unit.