Kinesin-2 Heterodimerization Alters Entry into a Processive Run along the Microtubule but not Stepping Within the Run

Heterodimeric KIF3AC and KIF3AB are two members of the mammalian kinesin-2 family that generate force for microtubule plus-end directed cargo transport. However, the advantage of heterodimeric kinesins over homodimeric ones is not well understood. While there is high sequence and structural similarity in the catalytic motor domains of KIF3A, KIF3B, and KIF3C, it is well recognized that small changes in sequence within the motor domain can encode differences in motile properties that are critical for cellular function. We showed previously that microtubule association for entry into a processive run was similar in rate for KIF3AC and KIF3AB at 6.6 µM^-1s^-1 and 7.0 µM^-1s^-1, respectively. Yet for engineered homodimers of KIF3AA and KIF3BB, this constant was significantly faster at 11 µM^-1s^-1 and much slower for KIF3CC at 2.1 µM^-1s^-1. These results led us to hypothesize that heterodimerization of KIF3A with KIF3C and KIF3A with KIF3B altered the intrinsic catalytic properties of each motor domain. Here, we test this hypothesis directly by a combination of presteady-state stopped-flow kinetics approaches and mathematical modeling. Surprisingly, the modeling revealed that the catalytic properties of KIF3A and KIF3B/C were altered upon microtubule binding, yet each motor domain retained its intrinsic kinetics for ADP release and subsequent ATP binding and the nucleotide-promoted transitions thereafter. The results are consistent with the interpretation that for KIF3AB, each motor head is catalytically similar and therefore each step is approximately equivalent. In contrast, for KIF3AC the results predict that the processive steps will alternate between a fast step for KIF3A followed by a slow step for KIF3C resulting in asymmetric stepping during a processive run. This study reveals the impact of heterodimerization of the motor domains for microtubule association to start the processive run and the fundamental differences in the motile properties of KIF3C in comparison to KIF3A and KIF3B.