Step-by-step: defining the catalytic properties of heterodimeric kinesin-2 motors

Abstract

Kinesin is a class of MT-based molecular motors that is involved in vesicle transport, signal transduction, microtubule cytoskeletal remodeling, and cell division for proper organismal physiology and development. The kinesin-2 family, for example, is well-known for its transport roles because it is highly processive, meaning it can take multiple 8-nm steps along the microtubule track before it detaches. The expression of four genes, namely KIF3A, KIF3B, KIF3C, and KIF17 can result in mammalian kinesin-2s heterodimeric KIF3AB and KIF3AC as well as homodimeric KIF17. KIF3AB, which is associated with a cargo adaptor protein called kinesin accessory protein (KAP), is essential for intraflagellar transport for ciliary assembly and can act as a scaffold for hedgehog signaling. Much more is known about KIF3AB/KAP than KIF3AC, mainly because there is no KIF3C orthologue in other model organisms. Unlike KIF3AB/KAP, heterodimeric KIF3AC is primarily expressed in neurons. One of the longstanding questions in the field has been why mammalian kinesin-2 is preferentially expressed as a heterodimer. One role for heterodimerization may be to specify adaptor and cargo binding. However, we hypothesize that heterodimeric kinesins may have also evolved to tune the catalytic properties of the heterodimer for its transport roles. KIF3AC serves as an ideal kinesin for testing this hypothesis because the intrinsic properties of KIF3A within engineered KIF3AA are significantly faster than KIF3C within engineered KIF3CC. For example, the single-molecule velocity of KIF3AA is 240 nm/s whereas the velocity of KIF3CC is 7.5 nm/s. However, KIF3AC achieves a velocity of 186 nm/s which is intermediate of KIF3AA and KIF3CC. This leads to the question of how the catalytic properties of KIF3A and KIF3C within KIF3AC differ from their intrinsic properties within homodimeric KIF3AA and KIF3CC. We addressed this question using stopped-flow presteady-state ADP release kinetics experiments and computational modeling of the KIF3AC stepping cycle. The modeling predicted that KIF3A and KIF3C collide with the microtubule with similar rates. However, once KIF3AC is on the microtubule, KIF3A and KIF3C retain their relative intrinsic catalytic properties. To better understand the mechanism of KIF3AC, we also modeled the stepping cycle of KIF3AB. The modeling predicted that heterodimerization alters the microtubule association properties of KIF3A and KIF3B but once on the microtubule, each head steps with equivalent fast rates of ~40 s-1. To confirm these results, both presteady-state phosphate release and dissociation kinetics experiments were conducted. Mathematical modeling of the data from the phosphate release kinetics experiments, which capture the steps from ATP association through coupled phosphate release and dissociation, is currently in progress. However, the experiment results demonstrate that KIF3A likely dominates the fast initial exponential rate of phosphate release and dissociation of KIF3AC. Together, these results suggest that heterodimerization serves as a mechanism for regulating the motility of heterodimeric KIF3AC.