| In humans, the kinesin-related protein Kif2b, a potent regulator of microtubule dynamics, plays an important role in high fidelity chromosome segregation during mitotic progression. As with some other members of the kinesin 13 subfamily, Kif2b is unusual in that its activity is regulated by phosphorylation, and phosphorylation at threonine 125 and serine 204 can regulate the activity and function of Kif2b in microtubule dynamics. Due to the lack of detailed structural information, the molecular basis for regulation by phosphorylation, as well as the mechanism of microtubule depolymerization catalyzed by Kif2b, is unknown. In order to investigate these activities, the primary goal of this project was to determine the high-resolution crystal structure of the Kif2b catalytic domain, as well as the phosphomimetic mutants Kif2b T125D, Kif2bS204D and Kif2bT125D, S204D. As bacterial expression and protein purification protocols for of Kif2b have not been described previously, we successfully developed a purification of the Kif2b motor domain involving a three-step protocol including ion-exchange chromatography, affinity chromatography, and size-exclusion chromatography. Characterization using circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC) illustrated a well-folded Kif2b motor domain with denaturation temperatures between 31 and 34°C and a robust ATPase activity was confirmed using EnzChek phosphate assay. Although preliminary crystals were obtained for the wild-type construct, diffraction was not yet observed. Finally, to identity the structural consequences of phosphorylation, the phosphomimetic mutants Kif2bT125D, Kif2bS204D and Kif2b T125D, S204D were generated and purified following a similar procedure to the wild-type motor domain. |
