Glycosaminoglycan disaccharide alters the dimer dissociation constant of the chemokine MIP-1 beta.
Chemokines are immune system proteins that recruit and activate leukocytes to sites of infection. This recruitment is believed to involve the establishment of a chemokine concentration gradient by the binding of chemokines to glycosaminoglycans (GAGs). In previous studies, we elucidated the GAG binding site of the chemokine MIP-1beta and implicated the involvement of the chemokine dimer in GAG binding through residues across the dimer interface. In the present studies, nuclear magnetic resonance spectroscopy was used to investigate the effect of GAG binding on MIP-1beta dimerization. Using several dimerization-impaired variants of MIP-1beta (F13Y, F13L, L34W, and L34K), these studies indicate that the addition of disaccharide to the mutants increases their dimerization affinities. For MIP-1beta F13Y, the presence of the disaccharide increases the chemokine dimerization affinity about 9-fold as evidenced by a decrease in the dimer dissociation constant from 610 to 66 microM. Even more dramatically, the dimerization affinity of MIP-1beta L34W also increases upon addition of disaccharide, with the dimer dissociation constant decreasing from 97 to 6.5 microM. After this effect for the mutants of MIP-1beta was shown, similar experiments were conducted with the CC chemokine RANTES, and it was demonstrated that the presence of disaccharide increases its dimerization affinity by almost 7-fold. These findings provide further evidence of the importance of the dimer in chemokine function and provide the first quantitative investigation of the role of GAGs in the manipulation of the MIP-1beta quaternary structure.
McCornack
Melissa A
MA
Department of Biochemistry and Biophysics, Texas A&M University, TAMU 2128, College Station, Texas 77843-2128, USA.
Boren
Danielle M
DM
LiWang
Patricia J
PJ
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D054407:Chemokine CCL4; D018946:Chemokine CCL5 / Q000378:metabolism; D019281:Dimerization; D004187:Disaccharides / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000235:genetics; D008253:Macaca mulatta; D019402:Macrophage Inflammatory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010649:Phenylalanine / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D019713:Receptors, CCR5 / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics; D014461:Ultracentrifugation
D054407:Chemokine CCL4; D018946:Chemokine CCL5; D004187:Disaccharides; D019402:Macrophage Inflammatory Proteins; D019713:Receptors, CCR5; C464536:heparin disaccharide; D014443:Tyrosine; D010649:Phenylalanine; D006493:Heparin; D007930:Leucine