Cover-neck Bundle Research Articles

Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition

Plasmodium parasites cause malaria and are responsible annually for hundreds of thousands of deaths. Kinesins are a superfamily of microtubule-dependent ATPases that play important roles in the parasite replicative machinery, which is a potential target for antiparasite drugs. Kinesin-5, a molecular motor that cross-links microtubules, is an established antimitotic target in other disease contexts, but its mechanism in Plasmodium falciparum is unclear. Here, we characterized P. falciparum kinesin-5 (PfK5) using cryo-EM to determine the motor's nucleotide-dependent microtubule-bound structure and introduced 3D classification of individual motors into our microtubule image processing pipeline to maximize our structural insights. Despite sequence divergence in PfK5, the motor exhibits classical kinesin mechanochemistry, including ATP-induced subdomain rearrangement and cover neck bundle formation, consistent with its plus-ended directed motility. We also observed that an insertion in loop5 of the PfK5 motor domain creates a different environment in the well-characterized human kinesin-5 drug-binding site. Our data reveal the possibility for selective inhibition of PfK5 and can be used to inform future exploration of Plasmodium kinesins as antiparasite targets.

Neck linker docking is critical for Kinesin-1 force generation in cells but at a cost to motor speed and processivity.

Kinesin force generation involves ATP-induced docking of the neck linker (NL) along the motor core. However, the roles of the proposed steps of NL docking, cover-neck bundle (CNB) and asparagine latch (N-latch) formation, during force generation are unclear. Furthermore, the necessity of NL docking for transport of membrane-bound cargo in cells has not been tested. We generated kinesin-1 motors impaired in CNB and/or N-latch formation based on molecular dynamics simulations. The mutant motors displayed reduced force output and inability to stall in optical trap assays but exhibited increased speeds, run lengths, and landing rates under unloaded conditions. NL docking thus enhances force production but at a cost to speed and processivity. In cells, teams of mutant motors were hindered in their ability to drive transport of Golgi elements (high-load cargo) but not peroxisomes (low-load cargo). These results demonstrate that the NL serves as a mechanical element for kinesin-1 transport under physiological conditions.

Cryo-EM Structure (4.5-Å) of Yeast Kinesin-5–Microtubule Complex Reveals a Distinct Binding Footprint and Mechanism of Drug Resistance

Kinesin-5s are microtubule-dependent motors that drive spindle pole separation during mitosis. We used cryo-electron microscopy to determine the 4.5-Å resolution structure of the motor domain of the fission yeast kinesin-5 Cut7 bound to fission yeast microtubules and explored the topology of the motor–microtubule interface and the susceptibility of the complex to drug binding. Despite their non-canonical architecture and mechanochemistry, Schizosaccharomyces pombe microtubules were stabilized by epothilone at the taxane binding pocket. The overall Cut7 footprint on the S. pombe microtubule surface is altered compared to mammalian tubulin microtubules because of their different polymer architectures. However, the core motor–microtubule interaction is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation including cover neck bundle formation. However, the Cut7 ATPase is not blocked by a mammalian-specific kinesin-5 inhibitor, consistent with the non-conserved sequence and structure of its loop5 insertion.

The Role of the Cover-Neck Bundle in Multi-Motor Transport against Load in Cells

Kinesin motor proteins share a highly-conserved catalytic motor domain responsible for coupling the chemical energy of ATP hydrolysis to force generation and microtubule-directed functions. Previous studies of kinesin-1 suggested that ATP binding to the motor domain induces the formation of the cover-neck bundle (CNB), a beta-sheet between β0 at the N-terminus (the coverstrand) and β9 in the first half of the necklinker. The CNB was shown to contribute to force generation by single kinesin-1 motors under purified conditions and has been predicted to function in force generation across the kinesin superfamily. However, differences in coverstrand and necklinker sequences make the role of the CNB across different kinesin families unclear. We adapted artificial cargo systems to delineate key features of the CNB during multi-motor transport against low and high loads in cells. Our results indicate that in teams, kinesin-1 motors can robustly transport cargo against high- and low-load in cells. Molecular modeling was used to predict mutations in the coverstrand and necklinker that disrupt and shorten beta-sheet formation. Experimental testing of these mutations shows that impaired beta-sheet formation compromised the capacity for teams of kinesin-1 motors to transport high-load cargo but not low-load cargo to the periphery of cells. Surprisingly, truncation of the region N-terminal to the coverstrand, predicted to have a minimal role in CNB formation, severely crippled the capacity for teams of kinesin-1 motors to transport both low- and high-load cargoes in cells. We plan to extend these studies to additional kinesin families to identify distinct motility dependencies on formation of the CNB.

A Structural Model of the Mitotic Kinesin-6 Mechanochemical Cycle

MKLP2 is a member of the kinesin-6 family, with critical roles during the metaphase-anaphase transition and in cytokinesis. The MKLP2 motor domain contains the conserved nucleotide and microtubule binding sites characteristic of plus-end mitotic kinesins. However, it is ∼60% larger than most other kinesins due to large inserts, the precise role(s) of which are unknown.Our biochemical studies show that the MKLP2 motor domain has nucleotide-insensitive sub-micromolar affinity for microtubules, except in the presence of the ATP analogue ADP.AlFx, which induces apparent nanomolar affinity. Consistently, we find that ATP binding kinetics appear to be uncoupled from microtubule binding. Using cryo-electron microscopy and structure determination (to ∼5A resolution), we have visualized the microtubule-bound MKLP2 motor domain at successive steps in its ATPase cycle. Its orientation with respect to the microtubule surface is conserved compared to other kinesins. ADP release induces small perturbations of the P-loop and loop11, described for transport kinesins and consistent with high levels of conservation in these regions. However, sequence divergence within the motor manifests itself at larger scales. Strikingly, ADP release does not induce major conformational changes around the nucleotide-binding site. However, in the presence of the ADP.AlFx, loop11 and loop9 are ordered, becoming compact around the bound nucleotide, forming a hydrolysis-competent conformation. The divergent loop2 also forms a contact with the microtubule surface, as do regions of the highly extended loop6. Along with these changes, the elongated kinesin-6 neck-linker is directed towards the microtubule plus-end but does not dock along the motor domain. The N-terminus of the motor is also disordered in all states and a cover neck bundle is not formed.Our data contribute to the mechanochemical dissection of this divergent mitotic kinesin and emphasize the critical mechanistic contribution of the motor interface with the microtubule.

Initial conformation of kinesin's neck linker

How ATP binding initiates the docking process of kinesin's neck linker is a key question in understanding kinesin mechanisms. By exploiting a molecular dynamics method, we investigate the initial conformation of kinesin's neck linker in its docking process. We find that, in the initial conformation, the neck linker has interactions with β0 and forms a ‘cover-neck bundle’ structure with β0. From this initial structure, the formation of extra turns and the docking of the cover-neck bundle structure can be achieved. The motor head provides a forward force on the initial cover-neck bundle structure through ATP-induced rotation. This force, together with the hydrophobic interaction of ILE327 with the hydrophobic pocket on the motor head, drives the formation of the extra turn and initiates the neck linker docking process. Based on these findings, a pathway from ATP binding-induced motor head rotation to neck linker docking is proposed.

Simulation Study of Interactions Between Kinesin’s Neck Linker and Motor Domain

The docking movement of kinesin’s neck linker to the motor domain is the force generation step of kinesin. This docking movement is achieved by various non-bonding interactions between neck linker and motor domain. However, the relative strength and the structural basis for these interactions are still unclear. To elucidate these problems, we unbind the docked neck linker from motor domain using steered molecular dynamics and mutation simulations. We find that the surprisingly high strength of a structure called the “ asparagine latch” arises from exquisite cooperation between hydrogen bonds and hydrophobic interactions. A β-sheet called the “ cover-neck bundle” behaves like an elastic beam in its unbinding process. The hydrophobic interaction between ILE327 (amino acid sequence in 2KIN) and the hydrophobic pocket on the motor domain is strong and provides a fixed support for elastic bending of the cover-neck bundle.

Modular Aspects of Kinesin Force Generation Machinery

The motor head of kinesin carries out microtubule binding, ATP hydrolysis, and force generation. Despite a high level of sequence and structural conservation, subtle variations in subdomains of the motor head determine family-specific properties. In particular, both Kinesin-1 (Kin-1) and Kinesin-5 (Kin-5) walk processively to the microtubule plus-end, yet show distinct motility characteristics suitable for their functions. We studied chimeric Kin-1/Kin-5 constructs with a combination of single molecule motility assays and molecular dynamics simulations to demonstrate that Kin-5 possesses a force-generating element similar to Kin-1, i.e., the cover-neck bundle. Furthermore, the Kin-5 neck linker makes additional contacts with the core of the motor head via loop L13, which putatively compensates for the shorter cover-neck bundle of Kin-5. Our results indicate that Kin-1 is mechanically optimized for individual cargo transport, whereas Kin-5 does not necessarily maximize its mechanical performance. Its biochemical rates and enhanced force sensitivity may instead be beneficial for operation in a group of motors. Such variations in subdomains would be a strategy for achieving diversity in motility with the conserved motor head.

Chimeric Kinesin I/Eg5 Constructs Reveal Important Elements to Motor Activity

Kinesin I (KHC) and kinesin V (Eg5) have very similar structure, yet very different roles. Eg5 is generally though of as having a low stall force (∼1.5pN), slow (unloaded velocity ∼100nm s−1), and having limited processivity, while KHC has a stall force of 5-7pN, a velocity of 500-700nm s−1, and having run lengths in the micron range. We have recently shown that the formation of the cover neck bundle (CNB), which is the formation of a beta sheet between B0 (the coverstrand) and B9 (the first half of the necklinker), is necessary for the motor to generate significant amounts of force. CNB formation along with docking of the necklinker to the motor head ( B7) creates the kinesin's power stroke. Loop 13 (L13), which has previously been shown to affect motor velocity with the mutation of highly conserved glycines to alanine, also forms contacts with B9, and has been shown to make a triple beta sheet structure consisting of B0, B9, and L13. To investigate the relative roles of the coverstrand, B9, and L13 in motor behavior we have created chimeric KHC/Eg5 constructs that incorporate the sequences for these elements from Eg5 into the KHC motorhead. We have found that stall force and unloaded run length are greatly affected by the substitution of Eg5 structural elements into KHC. These results suggest that the motors operate best with a matched CNB and that L13 strongly affects the mechanical strength of the motor. While a match CNB appears to make the relative motor function more robust, B9 has a larger impact on motor function than B0. Furthermore, these structural elements cause the motor to stall at lower forces, be slower, and less processive, but they alone do not turn KHC into Eg5.

Free Energy Changes During Kinesin's Force-Generating Substep

We have previously suggested that Kinesin-1 generates force by transient folding between the N-terminal cover strand and the C-terminal neck linker domains into a beta-sheet, the so-called cover-neck bundle (CNB). Once formed, the CNB has a conformational bias sufficient to move the neck linker forward. Replica exchange molecular dynamics simulations have been performed to elucidate the energetics of CNB formation with and without load. Without load, the CNB state is weakly favorable compared to non-CNB states by 0.85 kcal/mol at 300 K, which is in agreement with a previous experimental value based on electron paramagnetic resonance, 0.72 kcal/mol (Rice et al, Biophys. J. 84:1844 (2003)), although the identity of the states involved is not certain. In non-CNB states the mobile neck linker points mostly forward in the ATP-like conformation of the motor head, so there is relatively little conformational difference with the CNB-state. By contrast, when a 10-pN rearward load is applied at the end of the beta9 part of the neck linker, a new local energy minimum appears for a rearward-pointing state. Compared to the CNB state, the free energy of the rearward-pointing state is higher by 2.96 kcal/mol at 300 K. This indicates that the CNB readily forms under applied load and thus is able to move the neck linker forward. The significance of these results for the mechanism by which kinesin-1 walks on microtubules will be discussed.

Kinesin's cover-neck bundle folds forward to generate force

Each step of the kinesin motor involves a force-generating molecular rearrangement. Although significant progress has been made in elucidating the broad features of the kinesin mechanochemical cycle, molecular details of the force generation mechanism remain a mystery. Recent molecular dynamics simulations have suggested a mechanism in which the forward drive is produced when the N-terminal cover strand forms a beta-sheet with the neck linker to yield the cover-neck bundle. We tested this proposal by comparing optical trapping motility measurements of cover strand mutants with the wild-type. Motility data, as well as kinetic analyses, revealed impairment of the force-generating capacity accompanied by a greater load dependence in the mechanochemical cycle. In particular, a mutant with the cover strand deleted functioned only marginally, despite the fact that the cover strand, the N-terminal "dangling end," unlike the neck linker and nucleotide-binding pocket, is not involved with any previously considered energy transduction pathway. Furthermore, a constant assisting load, likely in lieu of a power stroke, was shown to rescue forward motility in the cover strand deletion mutant. Our results support a stepping mechanism driven by dynamic cover-neck bundle formation. They also suggest a strategy to generate motors with altered mechanical characteristics by targeting the force-generating element.

Force Generation in Kinesin Hinges on Cover-Neck Bundle Formation

Force Generation in Kinesin Hinges on Cover-Neck Bundle Formation

Kinesin's parts pull together

![][1] Force analysis shows the formation of the cover neck bundle (yellow) is at the heart of kinesin's power stroke. HWANG/ELSEVIER Kinesin creates a new domain to generate its power, according to Wonmuk Hwang (Texas A&M University, College Station, TX), Matthew Lang (

Force Generation in Kinesin Hinges on Cover-Neck Bundle Formation

In kinesin motors, a fundamental question concerns the mechanism by which ATP binding generates the force required for walking. Analysis of available structures combined with molecular dynamics simulations demonstrates that the conformational change of the neck linker involves the nine-residue-long N-terminal region, the cover strand, as an element that is essential for force generation. Upon ATP binding, it forms a beta sheet with the neck linker, the cover-neck bundle, which induces the forward motion of the neck linker, followed by a latch-type binding to the motor head. The estimated stall force and anisotropic response to external loads calculated from the model agree with force-clamp measurements. The proposed mechanism for force generation by the cover-neck bundle formation appears to apply to several kinesin families. It also elucidates the design principle of kinesin as the smallest known processive motor.