Importance of basic residues and quaternary structure in the function of MIP-1 beta: CCR5 binding and cell surface sugar interactions

Chemokines direct immune cells toward sites of infection by establishing a gradient across the extracellular matrix of the tissue. This gradient is th ought to be stabilized by ligation of chemokines to sulfated polysaccharide s known as glycosaminoglycans (GAGs) that are found on the surface of endot helial and other cells as well as in the tissue matrix. GAGs interact with chemokines and in some cases cause them to aggregate. The interaction betwe en cell surface GAGs and chemokines has also been postulated to play a role in the anti-HIV activity of some chemokines, including MIP-1 beta. Since m any proteins interact with GAGs by utilizing basic residues, we mutated R18 , K45, R46, and K48 in MIP-1 beta to investigate the role of these residues in GAG binding and CCR5 function. We find that no single amino acid substi tution alone has a dramatic effect on heparin binding, although change at R 46 has a moderate effect. However, binding to heparin is completely abrogat ed in a mutant (K45A/R46A/K48A) in which the entire "40's loop" has been ne utralized. A functional study of these mutants reveals that the charged res idues in this 40's loop, particularly K48 and R46, are critical mediators o f MIP-1 beta binding to its receptor CCRS. However, despite the partially o verlapping function of the residues in the 40's loop in binding to both CCR S and heparin, the presence of cell surface sugars does not appear to be ne cessary for the ability of MIP-1 beta to function on its receptor CCR5, as enzymatic removal of GAGs from cells results in little effect on MIP-1 beta activity. Because the means by which the chemokine gradient transmits info rmation to the recruited cells is not well defined, we also mutated the bas ic residues in MIP(9), a truncated form of MIP-1 beta that is impaired in i ts ability to dimerize, to probe whether the quaternary structure of this c hemokine influences its ability to bind heparin. None of the truncated vari ants bound as well as the full-length proteins containing the same mutation , suggesting that the MIP-IP dimer participates in heparin binding.