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

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.Nucleotide 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 molecular basis of such adaptations is largely unknown.Our interests focus on kinesin-5s (K5s), which cross-link MTs and are essential for the formation and maintenance of bipolar mitotic spindles in most eukaryotes (3). Like K1s, K5s have an N-terminal MD followed by a neck-linker (NL) that connects the MD to the rest of the motor. However, K1s are dimeric, whereas K5 tetramerization is mediated by the coiled-coil that follows the K5 NL and gives rise to a dumbbell-shaped molecule with pairs of MDs at either end (3, 4). However, it is not simply the K5 tetrameric structure, with each MD pair taking ATP-driven steps toward the MT plus-ends (5), that is required for its mitotic functions: K5 MDs have specific mechanochemical properties that are matched to their spindle functions (6). Typically, K5s are slow motors thought to work in teams when cross-linking and sliding MTs (5). The MT-stimulated ATPase turnover of monomeric K5 is slower than that of K1 (5–9/s vs. ∼50/s) (7–9), with phosphate release being the rate-limiting step (6–8/s) (10). K5 dimer stepping is concomitantly slower than that of K1 (∼100 nm/s vs. ∼650 nm/s unloaded velocity) and has a different response to external load (11, 12).A complete molecular explanation for these divergent properties remains elusive. Although it is well established that ATP binding induces NL docking along the MD toward the MT plus end in plus-end kinesins, different physical properties of the NL and variations in nucleotide-dependent conformational changes affect modes of force generation (3). Recently, the collaborative role of the K1 MD N terminus and, in particular, its proximal portion, called the “cover strand,” has emerged. The cover strand forms a short β-sheet, the cover-neck bundle (CNB) with the proximal section of the NL to assist docking and thus force production (13, 14). The K5 N terminus is longer than the N terminus of K1s (19 vs. 9 residues), but we recently described K5 CNB formation on ATP (AMPPNP) binding (15), and a role for the K5 CNB in force generation was inferred indirectly using a K1/K5 chimera (16).Fundamental questions remain concerning the fate of the CNB throughout the kinesin ATPase cycle and more generally about how information is conveyed from the MD catalytic center to the site of force generation at the CNB. Deciphering the K5-specific molecular mechanism also is of great interest because vertebrate K5-specific inhibitors bind to the highly variable loop L5 that regulates the K5 ATPase cycle and NL movement (17–19). These inhibitors allosterically block ATPase activity and directional movement (20, 21), arrest mitosis (22), and currently are being investigated in cancer clinical trials (3). Using cryo-electron microscopy (cryo-EM), we have addressed deficiencies in our understanding of kinesin mechanochemistry by directly visualizing MT-bound K5 in its ATP hydrolysis transition and ADP-bound states. Combined with our previous characterization of the ATP-binding step of MT-bound K5 (15), we provide a comprehensive picture of the molecular mechanism of K5 throughout its ATPase cycle and highlight critical molecular adaptations in comparison with other kinesins, particularly K1s. Unexpectedly, we demonstrate a role for the N terminus beyond force generation in controlling the NL conformation of K5s throughout their ATPase cycle. We also identify the role of the short helix α0 proximal to the active site in conveying conformational changes to the N terminus. Using biophysical methods, we directly demonstrate the kinetic role of the N terminus in assisting NL movement and hence in supporting efficient K5 activity. We also highlight intrinsic differences in the NL of K5, as compared with K1, that contribute to the very different mechanochemistries of these functionally divergent motors.