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 trimer interface. The mutants were substitutions at residue Pro-100 (P100S and P100G) and deletions of 1-5 residues at the C terminus. Single mutants yielded proteins that kept both activity and hexameric structure. However, they were severely affected in their stability toward temperature 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 protein was performed at 2.6 A resolution. The x-ray structure showed no direct alteration of intersubunits contacts at residue 100, but 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. Consequences of the mutation are discussed in view of the structural and biochemical properties observed in the mutant Dictyostelium protein.
Karlsson
A
A
Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 1149, Institut Pasteur, 75724, Paris Cedex 15, France.
Mesnildrey
S
S
Xu
Y
Y
Moréra
S
S
Janin
J
J
Véron
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000737:chemistry; D004023:Dictyostelium / Q000201:enzymology; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D011487:Protein Conformation; D011489:Protein Denaturation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014461:Ultracentrifugation; D014508:Urea / Q000737:chemistry
D017931:DNA Primers; D046911:Macromolecular Substances; D014508:Urea; D009701:Nucleoside-Diphosphate Kinase