Abstract
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.
Highlights
Most cytoskeletal motor proteins comprise two globular domains, called “heads,” joined by a long superhelical segment
Many lines of experimental evidence support the view that Ncd is a nonprocessive motor, it was reported recently that full-length Ncd may exhibit some characteristics of a processive protein [13]
Several studies using electron microscopy clearly indicated that only one of the Ncd motor domains binds to the microtubule [15,16,17]
Summary
Expression Constructs—We developed a two-plasmid expression system for Ncd; one of the plasmids (a derivative of pET28a (Novagen)) directed expression of a subunit with an N-terminal His tag (NH2-MGSSHHHHHHSSGLVPRGSHM-); the other (a derivative of pBIOEx) directed expression of a subunit with a short amino acid sequence (NH2-MAGGLNDIFEAQKIEWHEDTGGSS-) that in Escherichia coli is biotinylated in vivo at the underlined lysine [25]. After 30 min, the microtubule polymers were stabilized and diluted 100-fold at room temperature in BRB80 buffer containing 40 M paclitaxel. After removing unbound Ncd, tetramethylrhodamine-labeled MTs were introduced in buffer N and supplemented with anti-fades (20 mM D-glucose, 0.02 mg/ml glucose oxidase, 0.008 mg/ml catalase, 1 mM dithiothreitol), 1 mM ATP, and 10 M paclitaxel. Fb ϭ (F0 Ϫ Fc)/F0, where F0 and Fc denote the values of fluorescence intensities of 1,5-IAEDANS-Ncd in the supernatant at the total tubulin concentration equal to 0 and MT0, respectively. Ncd homodimers were labeled with 1,5-IAEDANS and incubated with MT for 15 min at room temperature and centrifuged to sediment the complex. Using Equation 2, one can compute the fraction of Ncd molecules bound through Head 1 (WT head) as a function of microtubule concentration
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