Interactions between subunits in heterodimeric Ncd molecules.
The nonprocessive minus-end-directed kinesin-14 Ncd is involved in the organization of the microtubule (MT) network during mitosis. Only one of the two motor domains is involved in the interaction with the MT. The other head is tethered to the bound one. Here we prepared, purified, and characterized mutated Ncd molecules carrying point mutations in one of the heads, thus producing heterodimeric motors. The mutations tested included substitutions in Switch I and II: R552A, E585A, and E585D; the decoupling mutant N600K; and a deletion in the motor domain in one of the subunits resulting in a single-headed molecule (NcN). These proteins were isolated by two sequential affinity chromatography steps, followed by measurements of their affinities to MT, enzymatic properties, and the velocity of the microtubule gliding test in vitro. A striking observation is a low affinity of the single-headed NcN for MT both without nucleotides and in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate, implying that the tethered head has a profound effect on the structure of the Ncd-MT complex. Mutated homodimers had no MT-activated ATPase and no motility, whereas NcN had motility comparable with that of the wild type Ncd. Although the heterodimers had one fully active and one inactive head, the ATPase and motility of Ncd heterodimers varied dramatically, clearly demonstrating that interactions between motor domains exist in Ncd. We also show that the bulk property of dimeric proteins that interact with the filament with only one of its heads depends also on the distribution of the filament-interacting subunits.
Kocik
Elzbieta
E
Motor Proteins Laboratory, Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw.
Skowronek
Krzysztof J
KJ
Kasprzak
Andrzej A
AA
D019943:Amino Acid Substitution; D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D020836:Protein Structure, Quaternary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics
D029721:Drosophila Proteins; C066696:ncd protein, Drosophila; D016547:Kinesins