NUCLEOSIDE DIPHOSPHATE KINASE - INVESTIGATION OF THE INTERSUBUNIT CONTACTS BY SITE-DIRECTED MUTAGENESIS AND CRYSTALLOGRAPHY

NDP kinase from Dictyostelium was mutated by site-directed mutagenesis at positions indicated by structural data to be involved in the trime r interface. The mutants were substitutions at residue Pro-100 (P100S and P100G) and deletions of 1-5 residues at the C terminus. Single mut ants yielded proteins that kept both activity and hexameric structure. However, they were severely affected in their stability toward temper ature and urea denaturation. When the P100S mutation was combined with any of the C-terminal deletions, the enzyme lost most of its activity and dissociated into dimers. Crystallographic analysis of the P100S p rotein was performed at 2.6 Angstrom resolution. The x-ray structure s howed no direct alteration of intersubunits contacts at residue 100, b ut an induced disruption of the interaction between Asp-115 and the C terminus of another subunit. The substitution of proline 100 to serine corresponds to the Killer-of-prune mutation in Drosophila. Consequenc es of the mutation are discussed in view of the structural and biochem ical properties observed in the mutant Dictyostelium protein.