The Processivity of Kinesin-2 Motors Suggests Diminished Front-Head Gating

Abstract

Summary Kinesin-2 motors, which are involved in intraflagellar transport and cargo transport along cytoplasmic microtubules, differ from motors in the canonical Kinesin-1 family in having a heterodimeric rather than homodimeric structure and in possessing a three amino acid insertion in their neck linker domain. To determine how these structural features alter the chemomechanical coupling in Kinesin-2, we used single-molecule bead experiments to measure the processivity and velocity of mouse Kinesin-2 heterodimer (KIF3A/B) and the engineered homodimers KIF3A/A and KIF3B/B, and compared their behavior to Drosophila Kinesin-1 heavy chain (KHC). Single motor run lengths of Kinesin-2 were four-fold shorter than Kinesin-1. Extending the Kinesin-1 neck linker by three amino acids led to a similar reduction in processivity. Furthermore, Kinesin-2 processivity varied inversely with ATP concentration. Stochastic simulations of the Kinesin-1 and Kinesin-2 hydrolysis cycles suggest that “front head gating”, in which rearward tension prevents ATP binding to the front head when both heads are bound to the microtubule, is diminished in Kinesin-2. Because the mechanical tension that underlies front head gating must be transmitted through the neck linker domains, we propose that the diminished coordination in Kinesin-2 is a result of its longer and hence more compliant neck linker element.

Regulation of a heterodimeric kinesin-2 through an unprocessive motor domain that is turned processive by its partner

“…S4). This contrasts with equivalent constructs of mouse kinesin-2 where the homodimers are reported to be processive (18). Importantly, also the constitutively active KLP11 EE ∕11 EE chimera did not show signs of processive movement ruling out the possibility that KLP11/11 is simply autoinhibited by its wild-type tail (Fig.…”

“…To probe for the presence of such an autoregulatory mechanism in KLP11/KLP20, we introduced mutations in the tail domains of the KLP11 and the KLP20 polypeptide chains (20). Moreover, we designed chimeras with splice sites between the head and the tail domains (18), resulting in heterodimeric constructs with identical head domains, or constructs whose head domains are swapped compared to the wild type. These chimeras allowed us to dissect the significance of KLP11 and KLP20 for autoinhibition and processivity in C. elegans kinesin-2.…”

“…However, the evolutionary advantage of combining two different motor proteins is not yet fully understood. Moreover, depending on their origin, heteromeric kinesins seem to differ dramatically in their kinetic behavior; e.g., the ciliary sea urchin kinesin-2 is an unprocessive motor, whereas in the mouse kinesin-2 (a motor that is no longer confined only to cilia but is also found in the cytoplasm) both head domains are processive as shown with homodimeric chimeras (18,19).…”

Molecular crowding creates traffic jams of kinesin motors on microtubules

“…Thus we could describe these properties of kinesin-1 (and other transport motors such as kinesin-2, ref. 27, and kinesin-3, refs. 28 and 29, which also have comparatively low processivities compared to the kinesin-8s such as Kip3 studied here or Kif18A, ref.…”

Microtubule shuttles on kinesin-coated glass micro-wire tracks

“…Collective operation of the motor domains holding one microtubule results in its translocation, allowing its use as a potential transporter (Brunner et al, 2007). This cooperative mode has a great advantage in that the distance of travel of microtubules is several orders of magnitude longer compared to the run lengths of an individual kinesin (Agarwal and Hess, 2010;Muthukrishnan et al, 2009). However, for a practical application of the biomotile system randomness in the initial gliding direction of microtubules still needs to be overcome.…”