Comprehensive structural model of the mechanochemical cycle of a mitotic motor highlights molecular adaptations in the kinesin family

Abstract

Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and essential cellular activities are poorly understood. It has been assumed that the first identified kinesin-the transport motor kinesin-1-is the mechanistic paradigm for the entire superfamily, but accumulating evidence suggests otherwise. To address the deficits in our understanding of the molecular basis of functional divergence within the kinesin superfamily, we studied kinesin-5s, which are essential mitotic motors whose inhibition blocks cell division. Using cryo-electron microscopy and determination of structure at subnanometer resolution, we have visualized conformations of microtubule-bound human kinesin-5 motor domain at successive steps in its ATPase cycle. After ATP hydrolysis, nucleotide-dependent conformational changes in the active site are allosterically propagated into rotations of the motor domain and uncurling of the drug-binding loop L5. In addition, the mechanical neck-linker element that is crucial for motor stepping undergoes discrete, ordered displacements. We also observed large reorientations of the motor N terminus that indicate its importance for kinesin-5 function through control of neck-linker conformation. A kinesin-5 mutant lacking this N terminus is enzymatically active, and ATP-dependent neck-linker movement and motility are defective, although not ablated. All these aspects of kinesin-5 mechanochemistry are distinct from kinesin-1. Our findings directly demonstrate the regulatory role of the kinesin-5 N terminus in collaboration with the motor's structured neck-linker and highlight the multiple adaptations within kinesin motor domains that tune their mechanochemistries according to distinct functional requirements. molecular motors | macromolecular assemblies | mitosis | cancer N ucleotide triphosphates are the fuel that powers the cell's machinery. Conversion of this fuel into mechanical work, i.e., mechanochemistry, depends on individual machines and the functional context in which they have evolved. Indeed, elucidation of the mechanochemistry of a particular machine provides critical insight into both its functions and modes of regulation. Kinesins are a superfamily of motors that use ATP to undertake microtubule (MT)-based work. Kinesins operate throughout the cell cycle in many contexts and can generate force toward the MT plus or minus end and also depolymerize MTs (1). The kinesin mechanochemical engine-the motor domain (MD)-is highly conserved, and conformational changes in the active site during the motor's ATPase cycle are transmitted to other parts of the MD to generate force (2). Most of our current knowledge about kinesin mechanochemistry comes from studies of the superfamily founding member, the transport motor kinesin-1 (K1) (2). However, accumulating evidence suggests that small modifications within kinesin MDs have profound effects on their cellular function. The molecul...

Kinesin processivity is gated by phosphate release

“…For example, does the partial NL docking implied by phosphate sensitivity represent a distinct structural state-and therefore a mechanical substepor a shift in some rapid equilibrium between fully docked and undocked conformations? Cryoelectron microscope structural studies of Eg5, a mitotic kinesin-5, have found hydrolysis-induced shifts in the NL conformation (36,37), and similar shifts may take place in kinesin-1. Moreover, previous work characterizing the motility of Eg5 has shown that its processivity can be also be enhanced by the addition of P i (38), suggesting that the P i release gating mechanism proposed here may not be limited to kinesin-1 motors.…”

The structural kinetics of switch-1 and the neck linker explain the functions of kinesin-1 and Eg5

“…1B for Eg5. These labeling sites were selected based on prior structural studies (2,6,7,(9)(10)(11)(12)(17)(18)(19) to detect changes in the distance between the NL or switch-1 and relatively fixed locations in β1 and β7 by using time-resolved FRET between a fluorescent donor (AEDANS) and a nonfluorescent acceptor (DDPM) (20,21).…”

“…The NL has been proposed to isomerize between two conformations: one that is flexible and termed undocked, and the other that is ordered and termed docked, where it interacts with a cleft in the motor domain formed by the twisted β-sheet and is oriented along the MT axis (5-7). NL isomerization (5,8) is hypothesized to be the force-generating transition in kinesin motors (6,7,(9)(10)(11), and its position has also been proposed to coordinate the ATPase cycles of processive kinesin dimers by regulating nucleotide binding and hydrolysis (11).…”

“…MT binding accelerates ADP dissociation, thereby allowing ATP to bind, the NL to dock, and mechanical work to be performed. ATP hydrolysis and phosphate release are then followed by dissociation from the MT to complete the cycle (5,(7)(8)(9)(10)14). This model also argues that: (i) NL docking of the MT-attached motor domain moves the tethered, trailing head into a forward position, where it undergoes a biased diffusional search to attach to the next MT-binding site (11,14); (ii) switch-1, which coordinates the γ-phosphate of ATP, alternates between two conformations, referred to as "open" and "closed," and the NL alternates between docked and undocked (5,6,10,(13)(14)(15); and (iii) coordination between the conformations of switch-1 and the NL regulates the timing of the ATPase cycles of the two motor domains in processive kinesin dimers (11).…”

The Kinesin-5 Chemomechanical Cycle Is Dominated by a Two-heads-bound State

“…Eg5 is roughly 10-fold slower than kinesin-1, although how this speed relates to the motor's processivity and force-generating ability is unclear. From CryoEM reconstructions, the Eg5 neck linker domain adopts a rearwardfacing orientation in the ADP state, in contrast to the more disordered kinesin-1 (9,10), although recent structural kinetics data identifies discrete neck linker positions in both motor classes (11). Eg5 has an extended Loop 5, which has been shown to affect nucleotide binding and serve as a latch for structural coupling within the Switch I domain (12,13) and as a binding target of several potential drugs (14 -16).…”