Mechanochemical Properties of the Kinesin-2 Motor, KIF3A/B, Studied by Optical Trapping

Abstract

The kinesin-2 motor KIF3A/B is a processive transport motor that incorporates two different motor domains, coded by separate KIF3A and KIF3B polypeptides. In intraflagellar transport, kinesin-2 motors transport cargo towards the tips of cilia, and dynein motors attached to the cargo are responsible for transport back towards the cell body. While it is known that these opposing motors are responsible for bidirectional transport, little is known about the performance of kinesin-2 motors under load. Here, we used a feedback-controlled optical trap to probe the nanomechanical properties of full-length mouse KIF3A/B under various load regimes and nucleotide concentrations. In addition, each motor domain was characterized by studying mutants consisting of two identical motor domains. Compared to conventional kinesin-1, kinesin-2 velocities were less dependent on load. Moreover, motor processivity, as measured by the run length, depended strongly upon the external load. In a tug-of-war with dynein, such characteristics are expected to enhance the dynamics of directional switching during transport, compared with kinesin-1 which slows under load but remains processive. Experiments using chimeric motors indicate that the load-dependent properties of kinesin-2 are attributable to their motor domains and not, for example, to the lengths of the neck linkers, nor to the properties of the coiled-coil stalks. The velocity data can be modeled in terms of twin alternating three-state cycles, one for each type of motor domain, where ATP binding is followed by a load-dependent transition, presumably neck-linker docking, before hydrolysis. Modeling also suggests that neck-linker docking represents a key mechanochemical step with a shorter characteristic distance for kinesin-2 than kinesin-1. A reduced characteristic distance may facilitate hydrolysis under load and reduce the probability that the tethered motor domain reaches the next microtubule binding site, leading to diminished processivity.