PROTON NMR ASSIGNMENTS AND SOLUTION CONFORMATION OF RANTES, A CHEMOKINE OF THE C-C TYPE

H-1 NMR has been used to investigate the structural properties of RANT ES, a protein from the C-C branch of the chemotactic cytokine family t hat has a strong chemoattractive effect on monocytes, lymphocytes, and eosinophils. Titration of pH from 5.0 to 2.5 indicates that RANTES is extensively aggregated in solution above pH 4.0. At pH 3.7 the protei n is mostly dimeric, although this species does dissociate to the mono mer with a K-d of 35 mu M. NMR data have been acquired and resonance a ssignments made for the dimeric species. Structures of the dimer have been generated by distance geometry and simulated annealing calculatio ns that utilized 1956 intramolecular distance restraints, 120 intermol ecular distance restraints, 164 dihedral angle restraints, and 68 rest raints enforcing 34 hydrogen bonds (17.0 restraints per residue). The structure is well-defined (average root mean square deviation from the average structure of 0.38 +/- 0.06 and 0.53 +/- 0.12 Angstrom for bac kbone heavy atoms of residues 4-66 of the monomer and dimer, respectiv ely). Each monomer consists of a C-terminal alpha-helix packing agains t a three-stranded antiparallel beta-sheet and two short N-terminal be ta-strands; dimerization occurs between the N-terminal regions of each monomer. This quaternary structure is very different from that of the C-X-C chemokines such as interleukin-8 and melanoma growth stimulator y activity but similar to that found for the C-C chemokine macrophage inflammatory factor 1 beta. Distinct structural differences between RA NTES and other chemokines at both the tertiary and quaternary level ar e discussed with regard to the distinct biological functions of the C- C and C-X-C members of this protein family.