CC chemokine MIP-1 beta can function as a monomer and depends on Phe13 forreceptor binding

The reported structures of many CC chemokines show a conserved dimer interf ace along their N-terminal region, raising the possibility that the quatern ary arrangement of these small immune proteins might influence their functi on. We have produced and analyzed several mutants of MIP-1 beta having a ra nge of dimer K-d values in order to determine the significance of dimerizat ion in receptor binding and cellular activation, NMR and analytical ultrace ntrifugation were used to analyze the oligomeric state of the mutants. Func tional relevance was determined by receptor binding affinity and the abilit y to invoke intracellular calcium release from CHO cells transfected with t he MIP-1 beta receptor CCR5, The monomeric N-terminally truncated mutant MI P(9) was able to bind the CCR5 receptor with a K-i of 600 pM but displayed weak agonistic properties, while the monomeric mutant P8A still retained th e ability to tightly bind (K-i = 480 pM) and to activate (EC50 = 12 nM) the receptor. These data suggest that the MIP-1 beta dimer is not required for CCR5 binding or activation. In addition, we identified Phe13, the residue immediately following the conserved CC motif in MIP-1 beta, as a key determ inant for binding to CCR5, Replacement of Phe13 by Tyr, Leu, Lys, and Ala s howed the aromatic side chain to be important fur both binding to CCR5 and chemokine dimerization.