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.     

驱动蛋白结构与运动机制

驱动蛋白是一类能够利用ATP水解释放的化学能驱动其所携带的“货物”分子沿着微管（microtubule,MT）定向运动的分子马达,在细胞器运输、有丝分裂、轴突运输等方面有着重要的生理作用。随着驱动蛋白结合ADP、ATP和未结合核苷酸（APO）三种特征状态的晶体结构的解析,驱动蛋白构象变化的研究得到了进一步发展,而在力产生机制和运动模型方面仍然存在较大争议。本文以kinesin-1家族为例,分析了驱动蛋白三种特征状态结构的特点、状态结构间的构象转变,论述了驱动蛋白的力产生机制和整个迈步过程。并探讨了驱动蛋白的运动模型,同时采用分子动力学模拟比较了驱动蛋白的两种迈步方式,为深入研究驱动蛋白提供了一定的理论计算。最后,基于本课题组对复杂体系的研究,对驱动蛋白体系的控制机制提出了新的假设,并对未来的研究方向进行了展望。     

The mechanical properties of kinesin-1: a holistic approach

During the last 25?years, a vast amount of research has gone into understanding the mechanochemical cycle of kinesin-1 and similar processive motor proteins. An experimental method that has been widely used to this effect is the in vitro study of kinesin-1 molecules moving along microtubules while pulling a bead, the position of which is monitored optically while trapped in a laser focus. Analysing results from such experiments, in which thermally excited water molecules are violently buffeting the system components, can be quite difficult. At low loads, the effect of the mechanical properties of the entire molecule must be taken into account, as stalk compliance means the bead position recorded is only weakly coupled to the movement of the motor domains, the sites of ATP hydrolysis and microtubule binding. In the present review, findings on the mechanical and functional properties of the various domains of full-length kinesin-1 molecules are summarized and a computer model is presented that uses this information to simulate the motion of a bead carried by a kinesin molecule along a microtubule, with and without a weak optical trap present. A video sequence made from individual steps of the simulation gives a three-dimensional visual insight into these types of experiment at the molecular level.     

Subunits interactions in kinesin motors

Kinesins form a large and diverse superfamily of proteins involved in numerous important cellular processes. The majority of them are molecular motors moving along microtubules. Conversion of chemical energy into mechanical work is accomplished in a sequence of events involving both biochemical and conformational alternation of the motor structure called the mechanochemical cycle. Different members of the kinesin superfamily can either perform their function in large groups or act as single molecules. Conventional kinesin, a member of the kinesin-1 subfamily, exemplifies the second type of motor which requires tight coordination of the mechanochemical cycle in two identical subunits to accomplish processive movement toward the microtubule plus end. Recent results strongly support an asymmetric hand-over-hand model of "walking" for this protein. Conformational strain between two subunits at the stage of the cycle where both heads are attached to the microtubule seems to be a major factor in intersubunit coordination, although molecular and kinetic details of this phenomenon are not yet deciphered. We discuss also current knowledge concerning intersubunit coordination in other kinesin subfamilies. Members of the kinesin-3 class use at least three different mechanisms of movement and can translocate in monomeric or dimeric forms. It is not known to what extent intersubunit coordination takes place in Ncd, a dimeric member of the kinesin-14 subfamily which, unlike conventional kinesin, exercises a power-stroke toward the microtubule minus end. Eg5, a member of the kinesin-5 subfamily is a homotetrameric protein with two kinesin-1-like dimeric halves controlled by their relative orientation on two microtubules. It seems that diversity of subunit organization, quaternary structures and cellular functions in the kinesin superfamily are reflected also by the divergent extent and mechanism of intersubunit coordination during kinesin movement along microtubules.     

Role and regulation of kinesin-8 motors through the cell cycle

Members of the kinesin-8 motor family play a central role in controlling microtubule length throughout the eukaryotic cell cycle. Inactivation of kinesin-8 causes defects in cell polarity during interphase and astral and mitotic spindle length, metaphase chromosome alignment, timing of anaphase onset and accuracy of chromosome segregation. Although the biophysical mechanism by which kinesin-8 molecules influence microtubule dynamics has been studied extensively in a variety of species, a consensus view has yet to emerge. One reason for this might be that some members of the kinesin-8 family can associate to other microtubule-associated proteins, cell cycle regulatory proteins and other kinesin family members. In this review we consider how cell cycle specific modification and its association to other regulatory proteins may modulate the function of kinesin-8 to enable it to function as a master regulator of microtubule dynamics.     

Diffusive Movement of Processive Kinesin-1 on Microtubules

The processive motor kinesin-1 moves unidirectionally toward the plus end of microtubules. This process can be visualized by total internal reflection fluorescence microscopy of kinesin bound to a carboxylated quantum dot (Qdot), which acts both as cargo and label. Surprisingly, when kinesin is bound to an anti-HIS Qdot, it shows diffusive movement on microtubules, which decreased in favor of processive runs with increasing salt concentration. This observation implies that kinesin movement on microtubules is governed by its conformation, as it is well established that kinesin undergoes a salt-dependent transition from a folded (inactive) to an extended (active) molecule. A truncated kinesin lacking the last 75 amino acids (kinesin-ΔC) showed both processive and diffusive movement on microtubules. The extent of each behavior depends on the relative amounts of ADP and ATP, with purely diffusive movement occurring in ADP alone. Taken together, these data imply that folded kinesin.ADP can exist in a state that diffuses along the microtubule lattice without expending energy. This mechanism may facilitate the ability of kinesin to pick up cargo, and/or allow the kinesin/cargo complex to stay bound after encountering obstacles.     

植物驱动蛋白: 从微管阵列到生理活动调控

驱动蛋白(kinesin)是以微管为轨道的分子马达, 其催化ATP水解为ADP, 将贮藏在ATP分子中的化学能高效地转化为机械能, 在细胞形态建成、细胞分裂、细胞运动、胞内物质运输和信号转导等多种生命活动中发挥重要作用。对植物驱动蛋白的研究落后于动物和真菌, 其原因不仅由于植物进化出独有的驱动蛋白家族, 而且其家族成员数量远多于动物驱动蛋白。该文主要总结了驱动蛋白在微管阵列动态组织, 包括周质微管和有丝分裂早前期微管带、纺锤体及成膜体中的角色和功能, 以及其对植物生理活动的调控作用。同时对重要经济作物大豆(Glycine max)中的驱动蛋白进行了系统分析、分类及功能预测, 发现大豆驱动蛋白数量庞大。结合公共数据库中大豆转录组数据, 对部分大豆驱动蛋白进行功能预测, 以期对大豆及其它作物驱动蛋白功能研究提供线索和启示。     

A kinetic dissection of the fast and superprocessive kinesin-3 KIF1A reveals a predominant one-head-bound state during its chemomechanical cycle

The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to WT KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and -2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate-limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate-limiting transition in the KIF1A stepping cycle. Thus, KIF1A''s activity can be explained by a fast rear-head detachment rate, a rate-limiting step of tethered-head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly bound post-hydrolysis state.     

A seesaw model for intermolecular gating in the kinesin motor protein

Recent structural observations of kinesin-1, the founding member of the kinesin group of motor proteins, have led to substantial gains in our understanding of this molecular machine. Kinesin-1, similar to many kinesin family members, assembles to form homodimers that use alternating ATPase cycles of the catalytic motor domains, or “heads”, to proceed unidirectionally along its partner filament (the microtubule) via a hand-over-hand mechanism. Cryo-electron microscopy has now revealed 8-Å resolution, 3D reconstructions of kinesin-1?microtubule complexes for all three of this motor’s principal nucleotide-state intermediates (ADP-bound, no-nucleotide, and ATP analog), the first time filament co-complexes of any cytoskeletal motor have been visualized at this level of detail. These reconstructions comprehensively describe nucleotide-dependent changes in a monomeric head domain at the secondary structure level, and this information has been combined with atomic-resolution crystallography data to synthesize an atomic-level "seesaw" mechanism describing how microtubules activate kinesin’s ATP-sensing machinery. The new structural information revises or replaces key details of earlier models of kinesin’s ATPase cycle that were based principally on crystal structures of free kinesin, and demonstrates that high-resolution characterization of the kinesin–microtubule complex is essential for understanding the structural basis of the cycle. I discuss the broader implications of the seesaw mechanism within the cycle of a fully functional kinesin dimer and show how the seesaw can account for two types of "gating" that keep the ATPase cycles of the two heads out of sync during processive movement.     

Kinesin-2 is a motor for late endosomes and lysosomes

The bidirectional nature of late endosome/lysosome movement suggests involvement of at least two distinct motors, one minus-end directed and one plus-end directed. Previous work has identified dynein as the minus-end-directed motor for late endosome/lysosome localization and dynamics. Conventional kinesin (kinesin-1) has been implicated in plus-end-directed late endosome/lysosome movement, but other kinesin family members may also be involved. Kinesin-2 is known to drive the movement of pigment granules, a type of lysosomally derived organelle, and was recently found to be associated with purified late endosomes. To determine whether kinesin-2 might also power endosome movement in non-pigmented cells, we overexpressed dominant negative forms of the KIF3A motor subunit and KAP3 accessory subunit and knocked down KAP3 levels using RNAi. We found kinesin-2 to be required for the normal steady-state localization of late endosomes/lysosomes but not early endosomes or recycling endosomes. Despite the abnormal subcellular distribution of late endosomes/lysosomes, the uptake and trafficking of molecules through the conventional endocytic pathway appeared to be unaffected. The slow time-course of inhibition suggests that both kinesin-2 itself and its attachment to membranes do not turn over quickly.     

Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters

So far, there has been a discrepancy between the velocities of kinesin-dependent microtubule motility measured in vitro and within cells. By changing ATP, Mg(2+), and kinesin concentrations, pH and ionic strength, we tried to find conditions that favour microtubule gliding across kinesin-covered glass surfaces. For porcine brain kinesin, we found that raising the molar Mg(2+)/ATP ratio can substantially elevate gliding velocity. Gliding became also faster after temperature elevation or lowering the number of kinesin molecules bound to the glass surface. The highest mean gliding velocity (1.8 microm/s+/-0.09 microm/s), approaching velocities measured for anterograde transport in vivo, was achieved by combination of favourable factors (2.5 m m ATP, 12.5 m m Mg(2+), 37 degrees C, 450 kinesin molecules/microm(2)).     

Kinesin’s Neck-Linker Determines its Ability to Navigate Obstacles on the Microtubule Surface

The neck-linker is a structurally conserved region among most members of the kinesin superfamily of molecular motor proteins that is critical for kinesin’s processive transport of intracellular cargo along the microtubule surface. Variation in the neck-linker length has been shown to directly modulate processivity in different kinesin families; for example, kinesin-1, with a shorter neck-linker, is more processive than kinesin-2. Although small differences in processivity are likely obscured in vivo by the coupling of most cargo to multiple motors, longer and more flexible neck-linkers may allow different kinesins to navigate more efficiently around the many obstacles, including microtubule-associated proteins (MAPs), that are found on the microtubule surface within cells. We hypothesize that, due to its longer neck-linker, kinesin-2 can more easily navigate obstacles (e.g., MAPs) on the microtubule surface than kinesin-1. We used total internal reflection fluorescence microscopy to observe single-molecule motility from different kinesin-1 and kinesin-2 neck-linker chimeras stepping along microtubules in the absence or presence of two Tau isoforms, 3RS-Tau and 4RL-Tau, both of which are MAPs that are known to differentially affect kinesin-1 motility. Our results demonstrate that unlike kinesin-1, kinesin-2 is insensitive to the presence of either Tau isoform, and appears to have the ability to switch protofilaments while stepping along the microtubule when challenged by an obstacle, such as Tau. Thus, although kinesin-1 may be more processive, the longer neck-linker length of kinesin-2 allows it to be better optimized to navigate the complex microtubule landscape. These results provide new insight, to our knowledge, into how kinesin-1 and kinesin-2 may work together for the efficient delivery of cargo in cells.     

Kinesin-1 Regulates Microtubule Dynamics via a c-Jun N-terminal Kinase-dependent Mechanism

In the kinesin family, all the molecular motors that have been implicated in the regulation of microtubule dynamics have been shown to stimulate microtubule depolymerization. Here, we report that kinesin-1 (also known as conventional kinesin or KIF5B) stimulates microtubule elongation and rescues. We show that microtubule-associated kinesin-1 carries the c-Jun N-terminal kinase (JNK) to allow its activation and that microtubule elongation requires JNK activity throughout the microtubule life cycle. We also show that kinesin-1 and JNK promoted microtubule rescues to similar extents. Stimulation of microtubule rescues by the kinesin-1/JNK pathway could not be accounted for by the rescue factor CLIP-170. Indeed only a dual inhibition of kinesin-1/JNK and CLIP-170 completely blocked rescues and led to extensive microtubule loss. We propose that the kinesin-1/JNK signaling pathway is a major regulator of microtubule dynamics in living cells and that it is required with the rescue factor CLIP-170 to allow cells to build their interphase microtubule network.     

The axonal transport motor kinesin‐2 navigates microtubule obstacles via protofilament switching

Axonal transport involves kinesin motors trafficking cargo along microtubules that are rich in microtubule‐associated proteins (MAPs). Much attention has focused on the behavior of kinesin‐1 in the presence of MAPs, which has overshadowed understanding the contribution of other kinesins such as kinesin‐2 in axonal transport. We have previously shown that, unlike kinesin‐1, kinesin‐2 in vitro motility is insensitive to the neuronal MAP Tau. However, the mechanism by which kinesin‐2 efficiently navigates Tau on the microtubule surface is unknown. We hypothesized that mammalian kinesin‐2 side‐steps to adjacent protofilaments to maneuver around MAPs. To test this, we used single‐molecule imaging to track the characteristic run length and protofilament switching behavior of kinesin‐1 and kinesin‐2 motors in the absence and presence of 2 different microtubule obstacles. Under all conditions tested, kinesin‐2 switched protofilaments more frequently than kinesin‐1. Using computational modeling that recapitulates run length and switching frequencies in the presence of varying roadblock densities, we conclude that kinesin‐2 switches protofilaments to navigate around microtubule obstacles. Elucidating the kinesin‐2 mechanism of navigation on the crowded microtubule surface provides a refined view of its contribution in facilitating axonal transport.      

Insight into the molecular mechanism of the multitasking kinesin‐8 motor

Members of the kinesin‐8 motor class have the remarkable ability to both walk towards microtubule plus‐ends and depolymerise these ends on arrival, thereby regulating microtubule length. To analyse how kinesin‐8 multitasks, we studied the structure and function of the kinesin‐8 motor domain. We determined the first crystal structure of a kinesin‐8 and used cryo‐electron microscopy to calculate the structure of the microtubule‐bound motor. Microtubule‐bound kinesin‐8 reveals a new conformation compared with the crystal structure, including a bent conformation of the α4 relay helix and ordering of functionally important loops. The kinesin‐8 motor domain does not depolymerise stabilised microtubules with ATP but does form tubulin rings in the presence of a non‐hydrolysable ATP analogue. This shows that, by collaborating, kinesin‐8 motor domain molecules can release tubulin from microtubules, and that they have a similar mechanical effect on microtubule ends as kinesin‐13, which enables depolymerisation. Our data reveal aspects of the molecular mechanism of kinesin‐8 motors that contribute to their unique dual motile and depolymerising functions, which are adapted to control microtubule length.     

Multiple conformations of the nucleotide site of Kinesin family motors in the triphosphate state

Identifying conformational changes in kinesin family motors associated with nucleotide and microtubule (MT) binding is essential to determining an atomic-level model for force production and motion by the motors. Using the mobility of nucleotide analog spin probes bound at the active sites of kinesin family motors to monitor conformational changes, we previously demonstrated that, in the ADP state, the open nucleotide site closes upon MT binding [Naber, N., Minehardt, T. J., Rice, S., Chen, X., Grammer, J., Matuska, M., et al. (2003). Closing of the nucleotide pocket of kinesin family motors upon binding to microtubules. Science, 300, 798-801]. We now extend these studies to kinesin-1 (K) and ncd (nonclaret disjunctional protein) motors in ATP and ATP-analog states. Our results reveal structural differences between several triphosphate and transition-state analogs bound to both kinesin and ncd in solution. The spectra of kinesin/ncd in the presence of SLADP•AlFx/BeFx and kinesin, with the mutation E236A (K-E236A; does not hydrolyze ATP) bound to ATP, show an open conformation of the nucleotide pocket similar to that seen in the kinesin/ncd•ADP states. In contrast, the triphosphate analogs K•SLAMPPNP and K-E236A•SLAMPPNP induce a more immobilized component of the electron paramagnetic resonance spectrum, implying closing of the nucleotide site. The MT-bound states of all of the triphosphate analogs reveal two novel spectral components. The equilibrium between these two components is only weakly dependent on temperature. Both components have more restricted mobility than observed in MT-bound diphosphate states. Thus, the closing of the nucleotide pocket when the diphosphate state binds to MTs is amplified in the triphosphate state, perhaps promoting accelerated ATP hydrolysis. Consistent with this idea, molecular dynamics simulations show a good correlation between our spectroscopic data, X-ray crystallography, and the electron microscopy of MT-bound triphosphate-analog states.     

Diffusion and directed movement: in vitro motile properties of fission yeast kinesin-14 Pkl1

Fission yeast Pkl1 is a kinesin-14A family member that is known to be localized at the cellular spindle and is capable of hydrolyzing ATP. However, its motility has not been detected. Here, we show that Pkl1 is a slow, minus end-directed microtubule motor with a maximum velocity of 33+/-9 nm/s. The Km,MT value of steady-state ATPase activity of Pkl1 was as low as 6.4+/-1.1 nM, which is 20-30 times smaller than that of kinesin-1 and another kinesin-14A family member, Ncd, indicating a high affinity of Pkl1 for microtubules. However, the duty ratio of 0.05 indicates that Pkl1 spends only a small fraction of the ATPase cycle strongly associated with a microtubule. By using total internal reflection fluorescence microscopy, we demonstrated that single molecules of Pkl1 were not highly processive but only exhibited biased one-dimensional diffusion along microtubules, whereas several molecules of Pkl1, probably fewer than 10 molecules, cooperatively moved along microtubules and substantially reduced the diffusive component in the movement. Our results suggest that Pkl1 molecules work in groups to move and generate forces in a cooperative manner for their mitotic functions.     

Controlling kinesin by reversible disulfide cross-linking. Identifying the motility-producing conformational change

Conventional kinesin, a dimeric molecular motor, uses ATP-dependent conformational changes to move unidirectionally along a row of tubulin subunits on a microtubule. Two models have been advanced for the major structural change underlying kinesin motility: the first involves an unzippering/zippering of a small peptide (neck linker) from the motor catalytic core and the second proposes an unwinding/rewinding of the adjacent coiled-coil (neck coiled-coil). Here, we have tested these models using disulfide cross-linking of cysteines engineered into recombinant kinesin motors. When the neck linker motion was prevented by cross-linking, kinesin ceased unidirectional movement and only showed brief one-dimensional diffusion along microtubules. Motility fully recovered upon adding reducing agents to reverse the cross-link. When the neck linker motion was partially restrained, single kinesin motors showed biased diffusion towards the microtubule plus end but could not move effectively against a load imposed by an optical trap. Thus, partial movement of the neck linker suffices for directionality but not for normal processivity or force generation. In contrast, preventing neck coiled-coil unwinding by disulfide cross-linking had relatively little effect on motor activity, although the average run length of single kinesin molecules decreased by 30-50%. These studies indicate that conformational changes in the neck linker, not in the neck coiled-coil, drive processive movement by the kinesin motor.     

Coupling viruses to dynein and kinesin-1

It is now clear that transport on microtubules by dynein and kinesin family motors has an important if not critical role in the replication and spread of many different viruses. Understanding how viruses hijack dynein and kinesin motors using a limited repertoire of proteins offers a great opportunity to determine the molecular basis of motor recruitment. In this review, we discuss the interactions of dynein and kinesin-1 with adenovirus, the α herpes viruses: herpes simplex virus (HSV1) and pseudorabies virus (PrV), human immunodeficiency virus type 1 (HIV-1) and vaccinia virus. We highlight where the molecular links to these opposite polarity motors have been defined and discuss the difficulties associated with identifying viral binding partners where the basis of motor recruitment remains to be established. Ultimately, studying microtubule-based motility of viruses promises to answer fundamental questions as to how the activity and recruitment of the dynein and kinesin-1 motors are coordinated and regulated during bi-directional transport.     

Arabidopsis thaliana protein,ATK1, is a minus-end directed kinesin that exhibits non-processive movement

The microtubule cytoskeleton forms the scaffolding of the meiotic spindle. Kinesins, which bind to microtubules and generate force via ATP hydrolysis, are also thought to play a critical role in spindle assembly, maintenance, and function. The A. thaliana protein, ATK1 (formerly known as KATA), is a member of the kinesin family based on sequence similarity and is implicated in spindle assembly and/or maintenance. Thus, we want to determine if ATK1 behaves as a kinesin in vitro, and if so, determine the directionality of the motor activity and processivity character (the relationship between molecular "steps" and microtubule association). The results show that ATK1 supports microtubule movement in an ATP-dependent manner and has a minus-end directed polarity. Furthermore, ATK1 exhibits non-processive movement along the microtubule and likely requires at least four ATK1 motors bound to the microtubule to support movement. Based on these results and previous data, we conclude that ATK1 is a non-processive, minus-end directed kinesin that likely plays a role in generating forces in the spindle during meiosis.