驱动蛋白的能量转换过程——从ATP分子结合到颈链对接

驱动蛋白是一种分子马达,同时也是一种核苷酸酶,它能够将ATP分子所携带的化学能转化为其沿微管蛋白行走的机械能,每消耗一个ATP分子行走一步。对于驱动蛋白如何将ATP的化学能转化为构象变化的机械能的研究一直是生物物理学研究的热点问题。本文从3个方面对此问题的研究进展进行了综述:ATP分子与驱动蛋白结合; ATP结合引起驱动蛋白头部产生转动;驱动蛋白头部转动引起驱动蛋白颈链向头部的对接。将这三个方面的内容合并起来就构成了驱动蛋白的能量传递路径。     

Ser203在大鼠脑驱动蛋白水解ATP中重要作用的晶体学分析

鼠脑驱动蛋白（rat brain kinesin）是一种利用水解ATP所释放的能量在微管束上高速并且连续性运动的常规驱动蛋白. 它在神经突触的物质运输中起着重要作用. 研究驱动蛋白是如何将ATP中储藏的化学能转化为机械动能是理解其运动机能的重要课题. 本课题获得了鼠脑驱动蛋白单体与ATP结构类似物AMPPCP形成的复合物晶体结构. 将这个晶体结构与鼠脑驱动蛋白单体-另一种ATP结构类似物AMPPNP形成的复合物晶体结构以及鼠脑驱动蛋白单体-ATP水解产物ADP形成的复合物晶体结构进行相互比较,揭示了活性中心的开关区域I中丝氨酸203可能作为质子的供体,加速了ATP中gamma-磷酸和beta-磷酸的断裂,从而导致ATP的水解.     

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。     

ABC转运蛋白的结构与转运机制

腺苷三磷酸结合盒转运蛋白（ATP-binding cassette transponer,ABC转运蛋白）超家族是一组跨膜蛋白,具有ATP结合区域的单向底物转运泵,以主动转运方式完成多种分子的跨膜转运.ABC转运蛋白的一个亚家族与多药抗性（multidrug resistance,MDR）有关,而多药抗性是临床肿瘤化疗中需要解决的主要问题,所以其结构与转运机制一直是研究的热点.最近几年获得了一些高分辨率的ABC转运蛋白的晶体结构,该文将根据ABC转运蛋白的结构的研究进展对其可能的转运机制进行讨论.     

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.     

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。     

ClpP蛋白酶研究进展：从细菌到人线粒体

Caseinolytic protease（ClpP）是一种包含丝氨酸蛋白酶催化三联体结构域的ATP依赖的蛋白水解酶,广泛存在于原核生物以及真核生物的线粒体和叶绿体中。它通常与AAA＋家族的分子伴4gClpX结合形成ClpXV蛋白酶复合物,AAA＋家族成员能利用水解ATP提供的能量将蛋白底物去折叠,随后将底物分子转移至ClpP蛋白酶的水解腔体进行降解。ClpP蛋白酶对细胞内蛋白质量控制及维持体内稳态起到至关重要的作用。该文综述了近年来有关ClpP蛋白酶在结构、功能以及与细菌毒力的关系和有关药物开发等方面的研究。     

分子马达KIF1A的研究进展

驱动蛋白家族成员1A(kinesin family member 1A,KIF1A)属于向微管正向端移动的驱动蛋白第三家族,它能够利用三磷酸腺苷(adenosine triphosphate,ATP)水解释放的能量实现沿微管定向运动。KIF1A是轴突末端的突触囊泡体沿微管输运的重要载体,其马达结构域的突变将导致多种与神经有关的疾病和缺陷。文中主要综述了近年来KIF1A有关的生命过程和疾病的研究进展,介绍了KIF1A催化ATP水解反应的各中间态结构,同时基于这些结构信息,阐述了KIF1A的运动形式、核苷酸轮换机制和运动机理,并对今后的研究前景进行了展望,旨为KIF1A相关研究提供思路。     

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。     

光谱及分子模拟法对HSA和BSA与酪氨酸激酶抑制剂Imatinib相互作用的比较分析

研究一种酪氨酸激酶抑制剂（tyrosine kinase inhibitor, TKI）伊马替尼（imatinib, IMA）与人血清清蛋白（HSA）及牛血清清蛋白（BSA）的相互作用,比较分析HSA和BSA与IMA相互作用机制的差异. 模拟生理条件下,计算机模拟技术结合荧光光谱和紫外光谱法,研究IMA与蛋白质的作用机制. 分子模建IMA与血清清蛋白的结合模型,表明伊马替尼与蛋白质的相互作用力为疏水作用力,兼有氢键作用. 光谱结果表明,IMA与HSA和BSA的相互作用表现为静态结合过程,结合强度较强,IMA与HSA和BSA分子的结合距离r值较小,说明发生了能量转移现象. IMA对HSA和BSA的结构域微区构象产生影响,使结合位域的疏水性发生改变. 荧光相图技术解析出IMA与HSA和BSA反应构象型态的变迁为“二态”模型. HSA与IMA相互作用的热力学参数表明,IMA与HSA之间是以疏水作用为主的分子间作用,而IMA与BSA之间的作用力为氢键和范德华力,兼有少量的疏水作用力. 光谱实验与计算机模拟结果基本一致,可为研究IMA与HSA和BSA相互作用本质提供一定参考.     

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.     

Understanding a molecular motor walking along a microtubule: an asymmetric Brownian motor driven by bubble formation with a focus on binding affinity

In recent years, many studies on a molecular motor have been conducted in the fields of biorheology and nanoengineering. The molecular motor is a molecule that converts the chemical energy obtained by ATP hydrolysis into mechanical energy. Explaining this mechanism is important for nanoengineering. A kinesin, which is a type of molecular motor, has the characteristics to move on a microtubule with hand-over-hand steps. The kinesin walking behaviour is explained by the ‘asymmetric Brownian ratchet model’. Previously, we had suggested that the walking mechanism was achieved by the bubble formation in a nanosized channel surrounded by hydrophobic atoms with the transition between the two states – bubble state and liquid state. However, the walking behaviour of the model motor was different from that of a single molecule measurement of a kinesin. In this study, we constructed a new motor system focused on the asymmetric binding affinity of a motor protein and performed a model simulation using the dissipative particle dynamics method. As a result, it was observed that hand-over-hand walking depends on the transition position ratio and the transition frequency coefficient. Moreover, the efficiency of the new motor system is higher than that of the previous motor systems. The new motor model can provide a simulation guide for the design of biomimetic nanomachines.     

Autoinhibition of the kinesin-2 motor KIF17 via dual intramolecular mechanisms

Long-distance transport in cells is driven by kinesin and dynein motors that move along microtubule tracks. These motors must be tightly regulated to ensure the spatial and temporal fidelity of their transport events. Transport motors of the kinesin-1 and kinesin-3 families are regulated by autoinhibition, but little is known about the mechanisms that regulate kinesin-2 motors. We show that the homodimeric kinesin-2 motor KIF17 is kept in an inactive state in the absence of cargo. Autoinhibition is caused by a folded conformation that enables nonmotor regions to directly contact and inhibit the enzymatic activity of the motor domain. We define two molecular mechanisms that contribute to autoinhibition of KIF17. First, the C-terminal tail interferes with microtubule binding; and second, a coiled-coil segment blocks processive motility. The latter is a new mechanism for regulation of kinesin motors. This work supports the model that autoinhibition is a general mechanism for regulation of kinesin motors involved in intracellular trafficking events.     

Are Coiled-Coils of Dimeric Kinesins Unwound during Their Walking on Microtubule?

Dimeric kinesin motor proteins such as homodimeric kinesin-1, homodimeric Ncd and heterodimeric Kar3/Vik1are composed of two head domains which are connected together by a rod-shaped, coiled-coil stalk. Despite the extensive and intensive studies on structures, kinetics, dynamics and walking mechanism of the dimers, whether their coiled-coils are unwound or not during their walking on the microtubule is still an unclear issue. Here, we try to clarify this issue by using molecular dynamics simulations. Our simulation results showed that, for Ncd, a large change in potential of mean force is required to unwind the coiled-coil by only several pairs of residues. For both Ncd and kinesin-1, the force required to initiate the coiled-coil unwinding is larger than that required for unfolding of the single [Formula: see text]-helix that forms the coiled-coil or is larger than that required to unwind the DNA duplex, which is higher than the unbinding force of the kinesin head from the microtubule in strong microtubule-binding states. Based on these results and the comparison of the sequence between the coiled-coil of Kar3/Vik1 and those of Ncd and kinesin-1, it was deduced that the coiled-coil of the Kar3/Vik1 should also be very stable. Thus, we concluded that the coiled-coils of kinesin-1, Ncd and Kar3/Vik1 are almost impossible to unwind during their walking on the microtubule.     

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.     

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.     

Neck-linker docking coordinates the kinetics of kinesin's heads

Conventional kinesin is a two-headed homodimeric motor protein, which is able to walk along microtubules processively by hydrolyzing ATP. Its neck linkers, which connect the two motor domains and can undergo a docking/undocking transition, are widely believed to play the key role in the coordination of the chemical cycles of the two motor domains and, consequently, in force production and directional stepping. Although many experiments, often complemented with partial kinetic modeling of specific pathways, support this idea, the ultimate test of the viability of this hypothesis requires the construction of a complete kinetic model. Considering the two neck linkers as entropic springs that are allowed to dock to their head domains, and incorporating only the few most relevant kinetic and structural properties of the individual heads, we develop here the first, to our knowledge, detailed, thermodynamically consistent model of kinesin that can 1), explain the cooperation of the heads (including their gating mechanisms) during walking, and 2), reproduce much of the available experimental data (speed, dwell-time distribution, randomness, processivity, hydrolysis rate, etc.) under a wide range of conditions (nucleotide concentrations, loading force, neck-linker length and composition, etc.). Besides revealing the mechanism by which kinesin operates, our model also makes it possible to look into the experimentally inaccessible details of the mechanochemical cycle and predict how certain changes in the protein affect its motion.     

The Origin of Minus-end Directionality and Mechanochemistry of Ncd Motors

Adaptation of molecular structure to the ligand chemistry and interaction with the cytoskeletal filament are key to understanding the mechanochemistry of molecular motors. Despite the striking structural similarity with kinesin-1, which moves towards plus-end, Ncd motors exhibit minus-end directionality on microtubules (MTs). Here, by employing a structure-based model of protein folding, we show that a simple repositioning of the neck-helix makes the dynamics of Ncd non-processive and minus-end directed as opposed to kinesin-1. Our computational model shows that Ncd in solution can have both symmetric and asymmetric conformations with disparate ADP binding affinity, also revealing that there is a strong correlation between distortion of motor head and decrease in ADP binding affinity in the asymmetric state. The nucleotide (NT) free-ADP (φ-ADP) state bound to MTs favors the symmetric conformation whose coiled-coil stalk points to the plus-end. Upon ATP binding, an enhanced flexibility near the head-neck junction region, which we have identified as the important structural element for directional motility, leads to reorienting the coiled-coil stalk towards the minus-end by stabilizing the asymmetric conformation. The minus-end directionality of the Ncd motor is a remarkable example that demonstrates how motor proteins in the kinesin superfamily diversify their functions by simply rearranging the structural elements peripheral to the catalytic motor head domain.     

Kinesin‐2 motors adapt their stepping behavior for processive transport on axonemes and microtubules

Two structurally distinct filamentous tracks, namely singlet microtubules in the cytoplasm and axonemes in the cilium, serve as railroads for long‐range transport processes in vivo. In all organisms studied so far, the kinesin‐2 family is essential for long‐range transport on axonemes. Intriguingly, in higher eukaryotes, kinesin‐2 has been adapted to work on microtubules in the cytoplasm as well. Here, we show that heterodimeric kinesin‐2 motors distinguish between axonemes and microtubules. Unlike canonical kinesin‐1, kinesin‐2 takes directional, off‐axis steps on microtubules, but it resumes a straight path when walking on the axonemes. The inherent ability of kinesin‐2 to side‐track on the microtubule lattice restricts the motor to one side of the doublet microtubule in axonemes. The mechanistic features revealed here provide a molecular explanation for the previously observed partitioning of oppositely moving intraflagellar transport trains to the A‐ and B‐tubules of the same doublet microtubule. Our results offer first mechanistic insights into why nature may have co‐evolved the heterodimeric kinesin‐2 with the ciliary machinery to work on the specialized axonemal surface for two‐way traffic.     

Modulation of the Kinesin ATPase Cycle by Neck Linker Docking and Microtubule Binding

Kinesin motor proteins use an ATP hydrolysis cycle to perform various functions in eukaryotic cells. Many questions remain about how the kinesin mechanochemical ATPase cycle is fine-tuned for specific work outputs. In this study, we use isothermal titration calorimetry and stopped-flow fluorometry to determine and analyze the thermodynamics of the human kinesin-5 (Eg5/KSP) ATPase cycle. In the absence of microtubules, the binding interactions of kinesin-5 with both ADP product and ATP substrate involve significant enthalpic gains coupled to smaller entropic penalties. However, when the wild-type enzyme is titrated with a non-hydrolyzable ATP analog or the enzyme is mutated such that it is able to bind but not hydrolyze ATP, substrate binding is 10-fold weaker than ADP binding because of a greater entropic penalty due to the structural rearrangements of switch 1, switch 2, and loop L5 on ATP binding. We propose that these rearrangements are reversed upon ATP hydrolysis and phosphate release. In addition, experiments on a truncated kinesin-5 construct reveal that upon nucleotide binding, both the N-terminal cover strand and the neck linker interact to modulate kinesin-5 nucleotide affinity. Moreover, interactions with microtubules significantly weaken the affinity of kinesin-5 for ADP without altering the affinity of the enzyme for ATP in the absence of ATP hydrolysis. Together, these results define the energy landscape of a kinesin ATPase cycle in the absence and presence of microtubules and shed light on the role of molecular motor mechanochemistry in cellular microtubule dynamics.