CRYSTALLOGRAPHIC STUDIES OF MUTANTS OF THE CHEMOKINE MACROPHAGE INFLAMMATORY PROTEIN-1[[alpha]]. J. Maclean^a, N. W. Isaacs^a, G. J. Graham^b, ^aDept. of Chemistry, Glasgow University, Glasgow, Scotland, ^bBeatson Institute for Cancer Research, Glasgow, Scotland

Macrophage Inflammatory Protein 1, a member of the CC family of chemokines, has been isolated in two forms, namely MIP-l[[alpha]] and MIP-1[[beta]]. Both are low mw proteins (M_r 8kD) which form aggregates of mw up to 100 kD at high ionic strength and neutral pH. MIP-l[[alpha]], a potent inhibitor of haematopoietic stem cell proliferation in vitro, is of potential use in cancer therapy.

Although the NMR structure of MIP-1[[beta]] showed that protein to exist as a discrete dimer at pH values below 3.5¹, the propensity of these proteins to aggregate under physiological conditions had made crystallographic analyses impossible. On the assumption that the interactions giving rise to the aggregation were electrostatic, the self-aggregation potential of MIP- 1[[alpha]] was removed by systematically neutralising acidic residues in the carboxy-terminal region of the protein. Thus a single mutation (66 Glu Gln) gave rise to a stable tetramer (PM1) of mw 32 kD. A double mutation (66 Glu Gln; 64 Asp Asn) gave a stable dimer (PM2) of mw 16 kD. Finally, a stable monomer (PM3) was obtained when three residues were mutated (66 Glu Gln; 64 Asp Asn; 60 Glu Gln).

The structures of all three mutants have been determined by X-ray crystallography. PM3 was solved by SIRAS and the structures of PM2 and PM1 were solved by Molecular Replacement using the refined PM3 coordinates as a search model. The crystal packing is found to be identical for PM2 and PM3, and the dimer thus formed is similar to that of MIP- 1[[beta]]. The structure of the tetramer shows how the predicted interactions could give rise to aggregation of the molecules.

Lodi, P. J. et al, Science, Vol 263, 1994, pl762-1767.