微管解聚型驱动蛋白的结构与功能

Kin-I 驱动蛋白(Kin-I kinesins)是一类重要的微管调节蛋白,具有依赖ATP的微管解聚活性.这类驱动蛋白在神经元的发育、纺锤体的组装和染色体的分离过程中起着重要的作用.自被发现以来的十几年里,人们对Kin-I驱动蛋白做了大量的研究工作.现对Kin-I驱动蛋白的结构、微管解聚活性及生理功能等方面进行简要综述.     

鼠脑驱动蛋白ATP水解机制的晶体学分析

鼠脑驱动蛋白是一类利用ATP水解释放的能量在微管系统上高连续性运动的常规驱动蛋白。了解ATP水解的化学能如何转化为机械动能是驱动蛋白研究中的重大课题。为此,鼠脑驱动蛋白单体(rK354)的晶体通过浸泡的方式引入ATP的结构类似物AMPPNP。rK354-AMPPNP复合物和rK354-ADP复合物结构的比较,揭示了开关区域Ⅱ的Glu237起连接ATP的γ-磷酸和驱动蛋白微管结合区的枢纽作用。     

驱动蛋白在一种新的微管动力中可能的生物学作用

细胞器沿着微管的转运能够用分离出的成份重新建立,从而对这一复杂的运动过程得以进行生物化学的剖析.这一技术途径己导致一个新颖的、普遍存在的、以微管为基础产生动力的蛋白质的提纯,称之为驱动蛋白(Kinesin,源于希腊语"运动").驱动蛋白的体外运动性质和免疫细胞化学定位提示,它可能作为细胞器转运和有丝分裂中微管依赖性运动的原动力而起作用.     

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。     

驱动蛋白颈链对接机制的研究进展

驱动蛋白能够携带"货物"沿微管高速连续行走,在行走过程中,将ATP结合与水解释放的化学能转化为机械能。驱动蛋白的由十几个氨基酸组成的颈链周期性地与驱动蛋白头部对接和分离是其行走的关键步骤,也是驱动蛋白发力做功的关键环节。现结合本课题组最新的研究结果,对驱动蛋白颈链3个部分不同的对接机制的研究进展进行综述。驱动蛋白颈链对接机制的阐明,加深了人们对于驱动蛋白沿微管行走机制的理解,同时也为其他分子马达工作机理的研究提供了参考。     

驱动蛋白的结构与功能

驱动蛋白是一类蛋白质超家族的总称,其中驱动蛋白-1(以下简称驱动蛋白)是目前已知的有机体内最小的马达蛋白.驱动蛋白能够催化三磷酸腺苷(adenosine triphosphate,ATP)分子的水解反应,将贮藏在ATP中的化学能转变为自身机械运动所需的机械能.驱动蛋白能够沿着微管连续定向运动,在细胞的有丝分裂和胞内物质运输中发挥重要作用.在真核细胞中,驱动蛋白主要以二聚体的形式存在,其结构主要包括4个部分,即马达头部、茎部、连接头部与茎部的颈链以及与"货物"相结合的尾部.驱动蛋白二聚体独特的结构特征以及各个组成部分协调的构象变化,保证了其沿微管的连续行走.目前,驱动蛋白的结构与功能之间的关系的研究取得了重要的进展.随着实验和计算水平的不断提高,彻底了解驱动蛋白的运动机理已经为期不远了.     

微管蛋白亚型及其功能

微管是真核细胞构成细胞骨架的主要成分,由α/β微管蛋白组装而成。微管在细胞多种活动中发挥着重要的作用,其功能主要受微管结合蛋白、微管蛋白的翻译后修饰以及微管蛋白亚型的调控。已有研究发现,α/β微管蛋白存在多种亚型,微管蛋白亚型在不同组织以及发育过程中的表达模式差异较大。多种微管蛋白亚型基因的突变可以引起神经系统疾病。该文综述了微管蛋白亚型的研究进展,尤其在微管功能调控、神经系统发育及其相关疾病中的作用。     

秋水仙碱处理为何能得到多倍体植物?

答在细胞有丝分裂的中期会发生染色体向两极移动的现象,而染色体的运动又有赖于微管的参与作用。微管的组成成分是微管蛋白。在活的细胞中,微管蛋白既可以聚合成微管,有时微管又可以解聚成微管蛋白。另外,微管蛋白能与秋水仙碱等植物碱特异结合,而且一旦结合即能阻止微管蛋白聚合成微管。正在分     

性激素对成年小鼠脑微管蛋白合成的影响

以3H-秋水仙碱为探针,测定小鼠脑微管蛋白含量．结果表明雌性激素具有显著的促进成年鼠脑微管蛋白合成的作用．与雌性激素相比,雄性激素促进脑微管蛋白合成的作用较弱．特别值得指出的是雌性激素促进脑微管蛋白合成的作用发生在脑发育的临界期之外,而此时甲状腺激素早已丧失了促进脑微管蛋白合成的作用．因此雌性激素在维护成年脑结构和功能的完整完善方面起着重要作用,而且这种作用可能会获得新的应用．     

靶向微管抗肿瘤药物研究进展

微管由微管蛋白组成,在细胞分裂、细胞内物质运输、信号传递、维持细胞形态等过程中起着重要作用.一些干扰微管功能的化合物可使细胞停滞在有丝分裂期而抑制细胞增殖.相对于正常细胞,肿瘤细胞有丝分裂异常频繁,以微管作为抗肿瘤的靶点已成为研究热点.作用于微管的微管蛋白抑制剂通过抑制微管蛋白的聚合促进微管解聚或者抑制微管解聚促进微管蛋白聚合来破坏微管动态平衡、干扰肿瘤细胞纺锤体形成、阻断细胞分裂、抑制肿瘤增殖,现就微管蛋白抑制剂的研究进展作一综述.     

Nucleotide-dependent movements of the kinesin motor domain predicted by simulated annealing.

The structure of an ATP-bound kinesin motor domain is predicted and conformational differences relative to the known ADP-bound form of the protein are identified. The differences should be attributed to force-producing ATP hydrolysis. Candidate ATP-kinesin structures were obtained by simulated annealing, by placement of the ATP gamma-phosphate in the crystal structure of ADP-kinesin, and by interatomic distance constraints. The choice of such constraints was based on mutagenesis experiments, which identified Gly-234 as one of the gamma-phosphate sensing residues, as well as on structural comparison of kinesin with the homologous nonclaret disjunctional (ncd) motor and with G-proteins. The prediction of nucleotide-dependent conformational differences reveals an allosteric coupling between the nucleotide pocket and the microtubule binding site of kinesin. Interactions of ATP with Gly-234 and Ser-202 trigger structural changes in the motor domain, the nucleotide acting as an allosteric modifier of kinesin's microtubule-binding state. We suggest that in the presence of ATP kinesin's putative microtubule binding regions L8, L12, L11, alpha4, alpha5, and alpha6 form a face complementary in shape to the microtubule surface; in the presence of ADP, the microtubule binding face adopts a more convex shape relative to the ATP-bound form, reducing kinesin's affinity to the microtubule.     

All-atom structural investigation of kinesin-microtubule complex constrained by high-quality cryo-electron-microscopy maps

In this study, we have performed a comprehensive structural investigation of three major biochemical states of a kinesin complexed with microtubule under the constraint of high-quality cryo-electron-microscopy (EM) maps. In addition to the ADP and ATP state which were captured by X-ray crystallography, we have also modeled the nucleotide-free or APO state for which no crystal structure is available. We have combined flexible fitting of EM maps with regular molecular dynamics simulations, hydrogen-bond analysis, and free energy calculation. Our APO-state models feature a subdomain rotation involving loop L2 and α6 helix of kinesin, and local structural changes in active site similar to a related motor protein, myosin. We have identified a list of hydrogen bonds involving key residues in the active site and the binding interface between kinesin and microtubule. Some of these hydrogen bonds may play an important role in coupling microtubule binding to ATPase activities in kinesin. We have validated our models by calculating the binding free energy between kinesin and microtubule, which quantitatively accounts for the observation of strong binding in the APO and ATP state and weak binding in the ADP state. This study will offer promising targets for future mutational and functional studies to investigate the mechanism of kinesin motors.     

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.     

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.     

A simulation model of the conventional kinesin based on the Driven-by-Detachment mechanism

Kinesins are molecular motors that unidirectionally move along microtubules using the chemical energy of ATP. Although the core structure of kinesins is similar to that of myosins, the lever-arm hypothesis, which is widely accepted as a plausible mechanism to explain the behaviors of myosins, cannot be directly applied to kinesins. Masuda has proposed a mechanochemical process called the ‘Driven-by-Detachment (DbD)’ mechanism to explain the characteristic behaviors of myosins, including the backward movement of myosin VI and the loose coupling phenomenon of myosin II. The DbD mechanism assumes that the energy of ATP is mainly used to detach a myosin head from an actin filament by temporarily reducing the affinity of the myosin against the actin. After the affinity is recovered, the detached head has potential energy originating from the attractive force between the myosin and the actin. During the docking process, the potential energy is converted into elastic energy within the myosin molecule, and the intramolecular elastic energy is finally used to produce the power strokes. In the present paper, the DbD mechanism was used to explain the hand-over-hand motion of the conventional kinesin. The neck linker of the kinesin is known to determine the directionality of the motility but, in this paper, it was assumed that the neck linker was not directly engaged in the power strokes, which were driven by the attractive force between the kinesin head and the microtubule. Based on this assumption, simple mechanical simulations showed that the model of a kinesin dimer processively moved along a microtubule protofilament, if the affinity of the kinesin against the microtubule is appropriately controlled. Moreover, if an external force was applied to the center of the kinesin dimer, the dimer moved backward along a microtubule, as observed in experimental motility assays.     

Chemomechanical coupling of the forward and backward steps of single kinesin molecules

The molecular motor kinesin travels processively along a microtubule in a stepwise manner. Here we have studied the chemomechanical coupling of the hydrolysis of ATP to the mechanical work of kinesin by analysing the individual stepwise movements according to the directionality of the movements. Kinesin molecules move primarily in the forward direction and only occasionally in the backward direction. The hydrolysis of a single ATP molecule is coupled to either the forward or the backward movement. This bidirectional movement is well described by a model of Brownian motion assuming an asymmetric potential of activation energy. Thus, the stepwise movement along the microtubule is most probably due to Brownian motion that is biased towards the forward direction by chemical energy stored in ATP molecules.     

The role of microtubules in processive kinesin movement

Kinesins are microtubule-based motors that are important for various intracellular transport processes. To understand the mechanism of kinesin movement, X-ray crystallography has been used to study the atomic structures of kinesin. However, as crystal structures of kinesin alone accumulate, it is becoming clear that kinesin structures should also be investigated with the microtubule to understand the contribution of the microtubule track to the nucleotide-induced conformational changes of kinesin. Recently, several high-resolution structures of kinesin with microtubules were obtained using cryo-electron microscopy. Comparison with X-ray crystallographic structures revealed the importance of the microtubule in determining the conformation of kinesin. Together with recent biophysical data, we describe different structural models of processive kinesin movement and provide a framework for future experiments.     

Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport

In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end- directed movement. The latter was selectively blocked in the rigor- mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo.     

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.     

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.