Unconventional motoring: an overview of the Kin C and Kin I kinesins

All kinesins share a conserved core motor domain implying a common mechanism for generating force from ATP hydrolysis. How is it then that kinesins exhibit such divergent activities: motility, microtubule cross‐linking and microtubule depolymerization? Although conventional motile kinesins have served as the paradigm for understanding kinesin function, the unconventional kinesins exploit variations on the motile theme to perform unexpected tasks. This review summarizes the biological functions and examines the possible molecular mechanisms of Kin C and Kin I unconventional kinesins. We also discuss the possible differences between the microtubule destabilization models proposed for Kar3 and Kin I kinesins .     

Importance of a flexible hinge near the motor domain in kinesin-driven motility.

Conventional kinesin is a molecular motor consisting of an N-terminal catalytic motor domain, an extended stalk and a small globular C-terminus. Whereas the structure and function of the catalytic motor domain has been investigated, little is known about the function of domains outside the globular head. A short coiled-coil region adjacent to the motor domain, termed the neck, is known to be important for dimerization and may be required for kinesin processivity. We now provide evidence that a helix-disrupting hinge region (hinge 1) that separates the neck from the first extended coiled-coil of the stalk plays an essential role in basic motor activity. A fast fungal kinesin from Syncephalastrum racemosum was used for these studies. Deletion, substitution by a coiled-coil and truncation of the hinge 1 region all reduce motor speed and uncouple ATP turnover from gliding velocity. Insertion of hinge 1 regions from two conventional kinesins, Nkin and DmKHC, fully restores motor activity, whereas insertion of putative flexible linkers of other proteins does not, suggesting that hinge 1 regions of conventional kinesins can functionally replace each other. We suggest that this region is essential for kinesin movement in its promotion of chemo-mechanical coupling of the two heads and therefore the functional motor domain should be redefined to include not only the catalytic head but also the adjacent neck and hinge 1 domains.     

Dissection of Kinesin's Processivity

The protein family of kinesins contains processive motor proteins that move stepwise along microtubules. This mechanism requires the precise coupling of the catalytic steps in the two heads, and their precise mechanical coordination. Here we show that these functionalities can be uncoupled in chimera of processive and non-processive kinesins. A chimera with the motor domain of Kinesin-1 and the dimerization domain of a non-processive Kinesin-3 motor behaves qualitatively as conventional kinesin and moves processively in TIRF and bead motility assays, suggesting that spatial proximity of two Kinein-1 motor domains is sufficient for processive behavior. In the reverse chimera, the non-processive motor domains are unable to step along microtubules, despite the presence of the Kinesin-1 neck coiled coil. Still, ATP-binding to one head of these chimera induces ADP-release from the partner head, a characteristic feature of alternating site catalysis. These results show that processive movement of kinesin dimers requires elements in the motor head that respond to ADP-release and induce stepping, in addition to a proper spacing of the motor heads via the neck coiled coil.     

Metazoan motor models: kinesin superfamily in C. elegans

In eukaryotic cells members of the kinesin family mediate intracellular transport by carrying cellular cargo on microtubule tracks. The nematode Caenorhabditis elegans genome encodes 21 members of the kinesin family, which show significant homology to their mammalian orthologs. Based on motor domain sequence homology and placement of the motor domain in the protein, the C. elegans kinesins have been placed in eight distinct groups; members of which participate in embryonic development, protein transport, synaptic membrane vesicles movement and in the axonal growth. Among 21 kinesins, at least 11 play a central role in spindle movement and chromosomal segregation. Understanding the function of C. elegans kinesins and related proteins may help navigate through the intricacies of intracellular traffic in a simple animal.     

How kinesin motor proteins drive mitotic spindle function: Lessons from molecular assays

Kinesins are enzymes that use the energy of ATP to perform mechanical work. There are approximately 14 families of kinesins within the kinesin superfamily. Family classification is derived primarily from alignments of the sequences of the core motor domain. For this reason, the enzymatic behavior and motility of each motor generally reflects its family. At the cellular level, kinesin motors perform a variety of functions during cell division and within the mitotic spindle to ensure that chromosomes are segregated with the highest fidelity possible. The cellular functions of these motors are intimately related to their mechanical and enzymatic properties at the single molecule level. For this reason, motility studies designed to evaluate the activity of purified molecular motors are a requirement in order to understand, mechanistically, how these motors make the mitotic spindle work and what can cause the spindle to fail. This review will focus on a selection of illustrative kinesins, which have been studied at the molecular level in order to inform our understanding of their function in cells. In addition, the review will endeavor to point out some kinesins that have been studied extensively but which still lack sufficient molecular underpinnings to fully predict their contribution to spindle function.     

A new model for binding of kinesin 13 to curved microtubule protofilaments

Kinesin motor proteins use adenosine triphosphate hydrolysis to do work on microtubules (MTs). Most kinesins walk along the MT, but class 13 kinesins instead uniquely recognize MT ends and depolymerize MT protofilaments. We have used electron microscopy (EM) to understand the molecular interactions by which kinesin 13 performs these tasks. Although a construct of only the motor domain of kinesin 13 binds to every heterodimer of a tubulin ring, a construct containing the neck and the motor domain occupies alternate binding sites. Likewise, EM maps of the dimeric full-length (FL) protein exhibit alternate site binding but reveal density for only one of two motor heads. These results indicate that the second head of dimeric kinesin 13 does not have access to adjacent binding sites on the curved protofilament and suggest that the neck alone is sufficient to obstruct access. Additionally, the FL construct promotes increased stacking of rings compared with other constructs. Together, these data suggest a model for kinesin 13 depolymerization in which increased efficiency is achieved by binding of one kinesin 13 molecule to adjacent protofilaments.     

The kinesin-13 MCAK has an unconventional ATPase cycle adapted for microtubule depolymerization

Unlike other kinesins, members of the kinesin-13 subfamily do not move directionally along microtubules but, instead, depolymerize them. To understand how kinesins with structurally similar motor domains can have such dissimilar functions, we elucidated the ATP turnover cycle of the kinesin-13, MCAK. In contrast to translocating kinesins, ATP cleavage, rather than product release, is the rate-limiting step for ATP turnover by MCAK; unpolymerized tubulin and microtubules accelerate this step. Further, microtubule ends fully activate the ATPase by accelerating the exchange of ADP for ATP. This tuning of the cycle adapts MCAK for its depolymerization activity: lattice-stimulated ATP cleavage drives MCAK into a weakly bound nucleotide state that reaches microtubule ends by diffusion, and end-specific acceleration of nucleotide exchange drives MCAK into a strongly bound state that promotes depolymerization. This altered cycle accounts well for the different mechanical behaviour of this kinesin, which depolymerizes microtubules from their ends, compared to translocating kinesins that walk along microtubules. Thus, the kinesin motor domain is a nucleotide-dependent engine that can be differentially tuned for transport or depolymerization functions.     

One axon,many kinesins: What's the logic?

A large number of membrane-bounded organelles, protein complexes, and mRNAs are transported along microtubules to different locations within the neuronal axon. Axonal transport in the anterograde direction is carried out by members of a superfamily of specialized motor proteins, the kinesins. All kinesins contain a conserved motor domain that hydrolyses ATP to generate movement along microtubules. Regions outside the motor domain are responsible for cargo binding and regulation of motor activity. Present in a soluble, inactive form in the cytoplasm, kinesins are activated upon cargo binding. Selective targeting of different types of kinesin motors to specific cargoes is directed by amino acid sequences situated in their variable tails. Cargo proteins with specific function at their destination, bind directly to specific kinesins for transport. Whereas most kinesins move to microtubule plus-ends, a small number of them move to microtubule minus-ends, and may participate in retrograde axonal transport. Axonal transport by kinesins has a logic: Fully assembled, multisubunit, functional complexes (e.g., ion channel complexes, signaling complexes, RNA-protein complexes) are transported to their destination by kinesin motors that interact transiently (i.e., during transport only) with one of the complexes' subunits.     

Kinesin, 30 years later: Recent insights from structural studies

Motile kinesins are motor proteins that move unidirectionally along microtubules as they hydrolyze ATP. They share a conserved motor domain (head) which harbors both the ATP‐ and microtubule‐binding activities. The kinesin that has been studied most moves toward the microtubule (+)‐end by alternately advancing its two heads along a single protofilament. This kinesin is the subject of this review. Its movement is associated to alternate conformations of a peptide, the neck linker, at the C‐terminal end of the motor domain. Recent progress in the understanding of its structural mechanism has been made possible by high‐resolution studies, by cryo electron microscopy and X‐ray crystallography, of complexes of the motor domain with its track protein, tubulin. These studies clarified the structural changes that occur as ATP binds to a nucleotide‐free microtubule‐bound kinesin, initiating each mechanical step. As ATP binds to a head, it triggers orientation changes in three rigid motor subdomains, leading the neck linker to dock onto the motor core, which directs the other head toward the microtubule (+)‐end. The relationship between neck linker docking and the orientations of the motor subdomains also accounts for kinesin's processivity, which is remarkable as this motor protein only falls off from a microtubule after taking about a hundred steps. As tools are now available to determine high‐resolution structures of motor domains complexed to their track protein, it should become possible to extend these studies to other kinesins and relate their sequence variations to their diverse properties.     

Single fungal kinesin motor molecules move processively along microtubules

Conventional kinesins are two-headed molecular motors that move as single molecules micrometer-long distances on microtubules by using energy derived from ATP hydrolysis. The presence of two heads is a prerequisite for this processive motility, but other interacting domains, like the neck and K-loop, influence the processivity and are implicated in allowing some single-headed kinesins to move processively. Neurospora kinesin (NKin) is a phylogenetically distant, dimeric kinesin from Neurospora crassa with high gliding speed and an unusual neck domain. We quantified the processivity of NKin and compared it to human kinesin, HKin, using gliding and fluorescence-based processivity assays. Our data show that NKin is a processive motor. Single NKin molecules translocated microtubules in gliding assays on average 2.14 micro m (N = 46). When we tracked single, fluorescently labeled NKin motors, they moved on average 1.75 micro m (N = 182) before detaching from the microtubule, whereas HKin motors moved shorter distances (0.83 micro m, N = 229) under identical conditions. NKin is therefore at least twice as processive as HKin. These studies, together with biochemical work, provide a basis for experiments to dissect the molecular mechanisms of processive movement.     

Kinectin-kinesin binding domains and their effects on organelle motility

Intracellular organelle motility involves motor proteins that move along microtubules or actin filaments. One of these motor proteins, kinesin, was proposed to bind to kinectin on membrane organelles during movement. Whether kinectin is the kinesin receptor on organelles with a role in organelle motility has been controversial. We have characterized the sites of interaction between human kinectin and conventional kinesin using in vivo and in vitro assays. The kinectin-binding domain on the kinesin tail partially overlaps its head-binding domain and the myosin-Va binding domain. The kinesin-binding domain on kinectin resides near the COOH terminus and enhances the microtubule-stimulated kinesin-ATPase activity, and the overexpression of the kinectin-kinesin binding domains inhibited kinesin-dependent organelle motility in vivo. These data, when combined with other studies, suggest a role for kinectin in organelle motility.     

Kinesin passing permanent blockages along its protofilament track

During movement along microtubules, kinesin usually follows a track parallel to the axis of a single protofilament. The question arises what happens when kinesin encounters blockages. The present study describes the movement of kinesin labeled by 20-nm gold beads along immobilized microtubules artificially decorated with blocking proteins. To guarantee that exactly the kinesin-binding sites were occupied and to avoid steric effects exerted by large molecules, the KIF5A motor domain was used for blocking. After binding, the blockages were irreversibly cross-linked to the microtubules to make them non-exchangeable. Under such conditions, kinesin movement became a non-continuous one. As a rule, after temporary stopping the kinesin moved on without being released from the microtubule. The results strongly suggest a bypassing mechanism based on the postulation that kinesin changes to and continues movement along a neighbouring protofilament. Bypassing is considered to ensure an efficient long-distance transport of cellular cargoes by kinesins.     

These motors were made for walking

Kinesins are a diverse group of adenosine triphosphate (ATP)‐dependent motor proteins that transport cargos along microtubules (MTs) and change the organization of MT networks. Shared among all kinesins is a ~40 kDa motor domain that has evolved an impressive assortment of motility and MT remodeling mechanisms as a result of subtle tweaks and edits within its sequence. Several elegant studies of different kinesin isoforms have exposed the purpose of structural changes in the motor domain as it engages and leaves the MT. However, few studies have compared the sequences and MT contacts of these kinesins systematically. Along with clever strategies to trap kinesin–tubulin complexes for X‐ray crystallography, new advancements in cryo‐electron microscopy have produced a burst of high‐resolution structures that show kinesin–MT interfaces more precisely than ever. This review considers the MT interactions of kinesin subfamilies that exhibit significant differences in speed, processivity, and MT remodeling activity. We show how their sequence variations relate to their tubulin footprint and, in turn, how this explains the molecular activities of previously characterized mutants. As more high‐resolution structures become available, this type of assessment will quicken the pace toward establishing each kinesin's design–function relationship.     

Universal and unique features of kinesin motors: insights from a comparison of fungal and animal conventional kinesins.

Kinesins are microtubule motors that use the energy derived from the hydrolysis of ATP to move unidirectionally along microtubules. The founding member of this still growing superfamily is conventional kinesin, a dimeric motor that moves processively towards the plus end of microtubules. Within the family of conventional kinesins, two groups can be distinguished to date, one derived from animal species, and one originating from filamentous fungi. So far no conventional kinesin has been reported from plant cells. Fungal and animal conventional kinesins differ in several respects, both in terms of their primary sequence and their physiological properties. Thus all fungal conventional kinesins move at velocities that are 4-5 times higher than those of animal conventional kinesins, and all of them appear to lack associated light chains. Both groups of motors are characterized by a number of group-specific sequence features which are considered here with respect to their functional importance. Animal and fungal conventional kinesins also share a number of sequence characteristics which point to common principles of motor function. The overall domain organization is remarkably similar. A C-terminal sequence motif common to all kinesins, which constitutes the only region of high homology outside the motor domain, suggests common principles of cargo association in both groups of motors. Consideration of the differences of, and similarities between, fungal and animal kinesins offers novel possibilities for experimentation (e. g., by constructing chimeras) that can be expected to contribute to our understanding of motor function.     

Processivity of single-headed kinesin motors

The processive movement of single-headed kinesins is studied by using a ratchet model of non-Markov process, which is built on the experimental evidence that the strong binding of kinesin to microtubule in rigor state induces a large apparent change in the local microtubule conformation. In the model, the microtubule plays a crucial active role in the kinesin movement, in contrast to the previous belief that the microtubule only acts as a passive track for the kinesin motility. The unidirectional movement of single-headed kinesin is resulted from the asymmetric periodic potential between kinesin and microtubule while its processivity is determined by its binding affinity for microtubule in the weak ADP state. Using the model, various experimental results for monomeric kinesin KIF1A, such as the mean step size, the step-size distribution, the long run length and the mean velocity versus load, can be well explained quantitatively. This local conformational change of the microtubule may also play important roles in the processive movement of conventional two-headed kinesins. An experiment to verify the model is suggested.     

Functional anatomy of the kinesin molecule in vivo.

We have developed an assay that allows the functional efficiency of mutant kinesins to be probed in vivo. We show here that the growth rate of the filamentous fungus Neurospora crassa can be used as a sensitive reporter for the ability of mutant kinesins to suppress the phenotype of the kinesin null mutant of Neurospora. Truncation mutants, internal deletion mutants and chimeras, in which homologous domains were exchanged between different fungal kinesins, were generated and transformed into the kinesin-deficient strain. None of the mutations affect motor velocity in vitro, but even minor alterations in the tail domain severely compromise kinesin's performance in vivo. The analysis of these mutants has identified subdomains in the stalk and tail likely to be involved in cargo binding and/or regulation of motor activity. The phenotypes of several mutants strongly suggest that kinesin requires a folded conformation to achieve full functionality in vivo. Folding critically depends on two flexible domains in the stalk that allow an interaction of the tail with the neck/hinge region near the catalytic motor domain. The assay has proven to be a valuable tool in the analysis of kinesin function in vivo and should help to characterize the sites involved in intra- and intermolecular interactions.     

Processivity of single-headed kinesin motors

The processive movement of single-headed kinesins is studied by using a ratchet model of non-Markov process, which is built on the experimental evidence that the strong binding of kinesin to microtubule in rigor state induces a large apparent change in the local microtubule conformation. In the model, the microtubule plays a crucial active role in the kinesin movement, in contrast to the previous belief that the microtubule only acts as a passive track for the kinesin motility. The unidirectional movement of single-headed kinesin is resulted from the asymmetric periodic potential between kinesin and microtubule while its processivity is determined by its binding affinity for microtubule in the weak ADP state. Using the model, various experimental results for monomeric kinesin KIF1A, such as the mean step size, the step-size distribution, the long run length and the mean velocity versus load, can be well explained quantitatively. This local conformational change of the microtubule may also play important roles in the processive movement of conventional two-headed kinesins. An experiment to verify the model is suggested.     

Reversing a ‘backwards’ motor

Ncd, a kinesin-related microtubule motor protein that moves the ‘wrong’ way on microtubules, towards the minus ends, has now been made to move like kinesin, towards plus ends, by fusing regions from outside the kinesin motor domain to the Ncd motor.^1,2 Since it is the kinesin motor domain that binds to and moves on the microtubule, the finding that regions outside the motor domain can confer directionality of Ncd movement is remarkable—it implies a structural basis for motor polarity. Here we consider this finding from a structural point of view and discuss the implications for motor function and evolution. BioEssays 20:108–112, 1998. © 1998 John Wiley & Sons, Inc.     

Motoring down the highways of the cell

All eukaryotic cells contain large numbers of motor proteins (kinesins, dyneins and myosins), each of which appears to carry out a specialized force-generating function within the cell. They are known to have roles in muscle contraction, ciliary movement, organelle and vesicle transport, mitosis and cytokinesis. These motor proteins operate on different cytoskeletal filaments; myosins move along actin filaments, and kinesins and dyneins along microtubules. Recently published crystal structures of the motor domains of two members of the kinesin superfamily reveal that they share the same overall fold that is also found at the core of the larger myosin motor. This suggests that they may share a common mechanism as well as a common ancestry.     

Structural dynamics of the microtubule binding and regulatory elements in the kinesin-like calmodulin binding protein

Kinesins are molecular motors that power cell division and transport of various proteins and organelles. Their motor activity is driven by ATP hydrolysis and depends on interactions with microtubule tracks. Essential steps in kinesin movement rely on controlled alternate binding to and detaching from the microtubules. The conformational changes in the kinesin motors induced by nucleotide and microtubule binding are coordinated by structural elements within their motor domains. Loop L11 of the kinesin motor domain interacts with the microtubule and is implicated in both microtubule binding and sensing nucleotide bound to the active site of kinesin. Consistent with its proposed role as a microtubule sensor, loop L11 is rarely seen in crystal structures of unattached kinesins. Here, we report four structures of a regulated plant kinesin, the kinesin-like calmodulin binding protein (KCBP), determined by X-ray crystallography. Although all structures reveal the kinesin motor in the ATP-like conformation, its loop L11 is observed in different conformational states, both ordered and disordered. When structured, loop L11 adds three additional helical turns to the N-terminal part of the following helix α4. Although interactions with protein neighbors in the crystal support the ordering of loop L11, its observed conformation suggests the conformation for loop L11 in the microtubule-bound kinesin. Variations in the positions of other features of these kinesins were observed. A critical regulatory element of this kinesin, the calmodulin binding helix positioned at the C-terminus of the motor domain, is thought to confer negative regulation of KCBP. Calmodulin binds to this helix and inserts itself between the motor and the microtubule. Comparison of five independent structures of KCBP shows that the positioning of the calmodulin binding helix is not decided by crystal packing forces but is determined by the conformational state of the motor. The observed variations in the position of the calmodulin binding helix fit the regulatory mechanism previously proposed for this kinesin motor.