The Kinesin Family Member BimC Contains a Second Microtubule Binding Region Attached to the N terminus of the Motor Domain

Abstract

The kinesin family member BimC has a highly positively charged domain of ϳ70 amino acids at the N terminus of the motor domain. Motor domain constructs of BimC were prepared with and without this extra domain to determine its influence. The level of microtubules needed for half saturation of the ATPase of BimC motor domain constructs is reduced by ϳ7000-fold at low ionic strength upon addition of this extra N-terminal extension. Although the change in microtubule affinity is less at higher salt, addition of the N-terminal domain still produces a 20-fold increase in affinity for microtubules in 200 mM potassium acetate. A fusion protein of the N-terminal domain and thioredoxin binds tightly to MTs at low salt, consistent with the increased affinity of motor domain constructs (which contain the N-terminal domain) being due to the additional binding of the N-terminal domain to the microtubule. Hydrodynamic analysis indicates that the N-terminal extension is in a highly extended conformation, suggesting that it may be intrinsically disordered. Fusion of the N-terminal extension of BimC onto the motor domain of conventional kinesin produces a similar large increase in microtubule affinity without significant reduction in k cat or velocity in an in vitro motility assay, suggesting that the N-terminal extension can act in a modular manner to increase the microtubule affinity of kinesin motor domains without a decrease in velocity.

The EB1 Homolog Mal3 Stimulates the ATPase of the Kinesin Tea2 by Recruiting It to the Microtubule

“…A. Doudna). General procedures for expression and lysis were as described previously (6,21). All solutions containing Tea2 constructs were supplemented with Ն0.1 mM ATP.…”

“…Sedimentation and diffusion coefficients were determined by velocity centrifugation in a sucrose gradient and gel filtration, essentially as described previously (6) in A25 buffer supplemented with 25 mM KCl and 200 mM NaCl for Mal3 and 400 mM NaCl and 0.1 mM MgATP for Tea2 constructs. Gel filtration was not performed with the Tea2 constructs because they exhibited partial adsorption during passage down the column.…”

“…nesin (25) and BC62M of BimC (6). T2NM contains the motor domain plus the Nte (corresponding to monomeric BC1M), and T2NMC1 also includes the first predicted coiled coil region (corresponding to dimeric DKH405).…”

The tethered motor domain of a kinesin-microtubule complex catalyzes reversible synthesis of bound ATP

“…This difficulty can be overcome by the use of BKinM (9). This construct consists of the full-length monomer motor domain of DKH346 fused to the N-terminal extension of BimC, which loosely tethers the motor domain to the MT with greatly increased net MT affinity but without perturbation of the ATPase rate or sliding velocity (9). The exchange reaction occurs slowly, but its rate is approxi- mately linear, as indicated in Fig.…”

“…Analysis of the fully MT-activated process is difficult to achieve with monomer motor domains, because their net affinity for MTs is weak in the presence of saturating ADP levels. This difficulty can be overcome by the use of BKinM (9). This construct consists of the full-length monomer motor domain of DKH346 fused to the N-terminal extension of BimC, which loosely tethers the motor domain to the MT with greatly increased net MT affinity but without perturbation of the ATPase rate or sliding velocity (9).…”