The human CC chemokine MIP-1beta dimer is not competent to bind to the CCR5 receptor.
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-1beta (MIP-1beta) by placing a disulfide bond at the center of its dimer interface through a single amino acid substitution (MIP-1beta-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-1beta-A10C binds to a heparin-Sepharose column as tightly as the wild type protein and more tightly than monomeric variants. However, MIP-1beta-A10C neither binds nor activates the MIP-1beta 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-1beta dimer is not able to bind or activate its receptor and implicates the CC chemokine monomer as the sole receptor-interacting unit.
Jin
Hongjun
H
Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77843, USA.
Shen
Xiaohong
X
Baggett
Brandi Renee
BR
Kong
Xiangming
X
LiWang
Patricia J
PJ
D000818:Animals; D016466:CHO Cells; D054407:Chemokine CCL4; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004229:Dithiothreitol / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D019402:Macrophage Inflammatory Proteins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry
D054407:Chemokine CCL4; D003432:Cross-Linking Reagents; D019402:Macrophage Inflammatory Proteins; D019713:Receptors, CCR5; D012685:Sepharose; D004229:Dithiothreitol