HETERONUCLEAR (H-1, C-13, N-15) NMR ASSIGNMENTS AND SOLUTION STRUCTURE OF THE MONOCYTE CHEMOATTRACTANT PROTEIN-1 (MCP-1) DIMER

A full high-resolution three-dimensional solution structure of the mon ocyte chemoattractant protein-1 (MCP-1 or MCAF) homodimer has been det ermined by heteronuclear multidimensional NMR. MCP-1 is a member of a family of small proteins which play a crucial role in immune surveilla nce by orchestrating the recruitment of specific leukocytes to areas o f immune challenge. The protein was uniformly isotopically enriched wi th C-13 and N-15 by expression in Escherichia coli, and complete seque nce-specific resonance assignments were obtained by a combination of h eteronuclear double- and triple-resonance experiments. The secondary s tructure was deduced from characteristic patterns of NOEs, C-13(alpha/ beta) chemical shifts, measurements of (3)J(HNH alpha) scalar coupling s, and patterns of slowly exchanging amide protons. Because MCP-1 form s symmetrical homodimers, additional experiments were carried out to u nambiguously establish the quaternary contacts. NOEs from these novel experiments were merged with more traditional heteronuclear separated NOE measurements in an iterative strategy to partition the restraints between explicit inter/intrasubunit contacts and a class wherein both were retained as ambiguous. With more than 30 restraints per residue, the three-dimensional structure is well-defined with a backbone rmsd o f 0.37 Angstrom to the mean over residues 5-69 of the dimer. We compar e the structure with those recently reported for the related chemokine s MIP-1 beta and RANTES and highlight the differences in terms of rece ptor specificity and function as well as interpret the known biologica l activity data of MCP-1 mutants.