Abstract
Kinesin-2 refers to the family of motor proteins represented by conserved, heterotrimeric kinesin-II and homodimeric Osm3/Kif17 class of motors.Kinesin-II, a microtubule-based anterograde motor, is composed of three different conserved subunits, named KLP64D, KLP68D and DmKAP in Drosophila. Although previous reports indicated that coiled coil interaction between the middle segments of two dissimilar motor subunits established the heterodimer, the molecular basis of the association is still unknown.Here, we present a detailed heterodimeric association model of the KLP64D/68D stalk supported by extensive experimental analysis and molecular dynamic simulations. We find that KLP64D stalk is unstable, but forms a weak coiled coil heteroduplex with the KLP68D stalk when coexpressed in bacteria. Local instabilities, relative affinities between the C-terminal stalk segments, and dynamic long-range interactions along the stalks specify the heterodimerization. Thermal unfolding studies and independent simulations further suggest that interactions between the C-terminal stalk fragments are comparatively stable, whereas the N-terminal stalk reversibly unfolds at ambient temperature.Results obtained in this study suggest that coiled coil interaction between the C-terminal stalks of kinesin-II motor subunits is held together through a few hydrophobic and charged interactions. The N-terminal stalk segments are flexible and could uncoil reversibly during a motor walk. This supports the requirement for a flexible coiled coil association between the motor subunits, and its role in motor function needs to be elucidated.
