The Experts below are selected from a list of 29136 Experts worldwide ranked by ideXlab platform
Marvin Bentley - One of the best experts on this subject based on the ideXlab platform.
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a novel strategy to visualize vesicle bound Kinesins reveals the diversity of Kinesin mediated transport
Traffic, 2019Co-Authors: Gary Banker, Rui Yang, Zoe Bostick, Alex Garbouchian, Julie Luisi, Marvin BentleyAbstract:In mammals, 15 to 20 Kinesins are thought to mediate vesicle transport. Little is known about the identity of vesicles moved by each Kinesin or the functional significance of such diversity. To characterize the transport mediated by different Kinesins, we developed a novel strategy to visualize vesicle-bound Kinesins in living cells. We applied this method to cultured neurons and systematically determined the localization and transport parameters of vesicles labeled by different members of the Kinesin-1, -2, and -3 families. We observed vesicle labeling with nearly all Kinesins. Only six Kinesins bound vesicles that undergo long-range transport in neurons. Of these, three had an axonal bias (KIF5B, KIF5C and KIF13B), two were unbiased (KIF1A and KIF1Bβ), and one transported only in dendrites (KIF13A). Overall, the trafficking of vesicle-bound Kinesins to axons or dendrites did not correspond to their motor domain preference, suggesting that on-vesicle regulation is crucial for Kinesin targeting. Surprisingly, several Kinesins were associated with populations of somatodendritic vesicles that underwent little long-range transport. This assay should be broadly applicable for investigating Kinesin function in many cell types.
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A novel strategy to visualize vesicle‐bound Kinesins reveals the diversity of Kinesin‐mediated transport
Traffic, 2019Co-Authors: Rui Yang, Gary Banker, Zoe Bostick, Alex Garbouchian, Julie Luisi, Marvin BentleyAbstract:In mammals, 15 to 20 Kinesins are thought to mediate vesicle transport. Little is known about the identity of vesicles moved by each Kinesin or the functional significance of such diversity. To characterize the transport mediated by different Kinesins, we developed a novel strategy to visualize vesicle-bound Kinesins in living cells. We applied this method to cultured neurons and systematically determined the localization and transport parameters of vesicles labeled by different members of the Kinesin-1, -2, and -3 families. We observed vesicle labeling with nearly all Kinesins. Only six Kinesins bound vesicles that undergo long-range transport in neurons. Of these, three had an axonal bias (KIF5B, KIF5C and KIF13B), two were unbiased (KIF1A and KIF1Bβ), and one transported only in dendrites (KIF13A). Overall, the trafficking of vesicle-bound Kinesins to axons or dendrites did not correspond to their motor domain preference, suggesting that on-vesicle regulation is crucial for Kinesin targeting. Surprisingly, several Kinesins were associated with populations of somatodendritic vesicles that underwent little long-range transport. This assay should be broadly applicable for investigating Kinesin function in many cell types.
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a novel split Kinesin assay identifies motor proteins that interact with distinct vesicle populations
Journal of Cell Biology, 2012Co-Authors: Brian Jenkins, Marvin Bentley, Julie Luisi, Helena Decker, Gary BankerAbstract:Identifying the Kinesin motors that interact with different vesicle populations is a longstanding and challenging problem with implications for many aspects of cell biology. Here we introduce a new live-cell assay to assess Kinesin–vesicle interactions and use it to identify Kinesins that bind to vesicles undergoing dendrite-selective transport in cultured hippocampal neurons. We prepared a library of “split Kinesins,” comprising an axon-selective Kinesin motor domain and a series of Kinesin tail domains that can attach to their native vesicles; when the split Kinesins were assembled by chemical dimerization, bound vesicles were misdirected into the axon. This method provided highly specific results, showing that three Kinesin-3 family members—KIF1A, KIF13A, and KIF13B—interacted with dendritic vesicle populations. This experimental paradigm allows a systematic approach to evaluate motor–vesicle interactions in living cells.
William O. Hancock - One of the best experts on this subject based on the ideXlab platform.
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Motor Reattachment Kinetics Play a Dominant Role in Multimotor-Driven Cargo Transport.
Biophysical Journal, 2018Co-Authors: Qingzhou Feng, Geng-yuan Chen, Keith J Mickolajczyk, William O. HancockAbstract:Abstract Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including Kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple Kinesins coordinate their activity during cargo transport are poorly understood. To understand these multimotor coordination mechanisms, defined pairs of Kinesin-1 and Kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than Kinesin-1 at the single-molecule level, addition of Kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the Kinesin-2 microtubule reattachment rate was shown to be fourfold faster than that of Kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of a gold-nanoparticle-labeled motor with 1 ms and 2 nm precision revealed that Kinesin-2 motors detach and rebind to the microtubule much more frequently than does Kinesin-1. Finally, compared to cargo transported by two Kinesin-1, cargo transported by two Kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multimotor transport and suggest a coordinated transport model in which Kinesin-1 motors step effectively against loads whereas Kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by Kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.
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Motor reattachment kinetics play a dominant role in multimotor-driven cargo transport
bioRxiv, 2017Co-Authors: Qingzhou Feng, Geng-yuan Chen, Keith J Mickolajczyk, William O. HancockAbstract:Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including Kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple Kinesins coordinate their activity during cargo transport are poorly understood. To understand these multi-motor coordination mechanisms, defined pairs of Kinesin-1 and Kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than Kinesin-1 at the single-molecule level, addition of Kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the Kinesin-2 microtubule reattachment rate was shown to be 4-fold faster than that of Kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of gold-nanoparticle-labeled cargo with 1 ms and 2 nm precision revealed that Kinesin-2 motors detach and rebind to the microtubule much more frequently than do Kinesin-1. Finally, cargo transported by Kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multi-motor transport and suggest a coordinated transport model in which Kinesin-1 motors step effectively against loads while Kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by Kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.
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the axonal transport motor Kinesin 2 navigates microtubule obstacles via protofilament switching
Traffic, 2017Co-Authors: Gregory J Hoeprich, William O. Hancock, Keith J Mickolajczyk, Shane R Nelson, Christopher L BergerAbstract:: 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.
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engineered Kinesin motor proteins amenable to small molecule inhibition
Nature Communications, 2016Co-Authors: Martin F Engelke, Shankar Shastry, Michael Winding, Yang Yue, Federico Teloni, Sanjay Reddy, Lynne T Blasius, Pushpanjali Soppina, William O. HancockAbstract:The human genome encodes 45 Kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each Kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer Kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using Kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable Kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific Kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest.
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engineered Kinesin motor proteins amenable to small molecule inhibition
bioRxiv, 2016Co-Authors: Martin F Engelke, William O. Hancock, Shankar Shastry, Michael Winding, Yang Yue, Federico Teloni, Sanjay Reddy, Lynne T Blasius, Pushpanjali Soppina, Vladimir I GelfandAbstract:The human genome encodes 45 Kinesins that drive cell division, cell motility, intracellular trafficking, and ciliary function. Determining the cellular function of each Kinesin would be greatly facilitated by specific small molecule inhibitors, but screens have yielded inhibitors that are specific to only a small number of Kinesins, likely due to the high conservation of the Kinesin motor domain across the superfamily. Here we present a chemical-genetic approach to engineer Kinesin motors that retain microtubule-dependent motility in the absence of inhibitor yet can be efficiently inhibited by small, cell-permeable molecules. Using Kinesin-1 as a prototype, we tested two independent strategies to design inhibitable motors. First, we inserted the six amino acid tetracysteine tag into surface loops of the motor domain such that binding of biarsenic dyes allosterically inhibits processive motility. Second, we fused DmrB dimerization domains to the motor heads such that addition of B/B homodimerizer cross-links the two motor domains and inhibits motor stepping. We show, using cellular assays that the engineered Kinesin-1 motors are able to transport artificial and natural Kinesin-1 cargoes, but are efficiently inhibited by the addition of the relevant small molecule. Single-molecule imaging in vitro revealed that inhibitor addition reduces the number of processively moving motors on the microtubule, with minor effects on motor run length and velocity. It is likely that these inhibition strategies can be successfully applied to other members of the Kinesin superfamily due to the high conservation of the Kinesin motor domain. The described engineered motors will be of great utility to dynamically and specifically study Kinesin function in cells and animals.
Nobutaka Hirokawa - One of the best experts on this subject based on the ideXlab platform.
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Kinesin superfamily motor proteins and intracellular transport
Nature Reviews Molecular Cell Biology, 2009Co-Authors: Nobutaka Hirokawa, Yosuke Tanaka, Yasuko Noda, Shinsuke NiwaAbstract:Forty-five genes that encode Kinesin superfamily proteins (also known as KIFs) have been discovered in the mouse and human genomes. KIFs are molecular motors that directionally transport various cargos, including membranous organelles, protein complexes and mRNAs, along the microtubule system. The mechanisms by which different Kinesins recognize, bind and unload specific cargo have been identified. The spatiotemporal delivery of cargos by KIF-based transport can be regulated by phosphorylation, G proteins and Ca^2+ levels. It is now recognized that Kinesins have unexpected roles in the regulation of physiological processes, such as higher brain function, tumour suppression and developmental patterning. Kinesins are molecular motors that directionally transport various cargos, including membranous organelles, protein complexes and mRNAs. The mechanisms by which Kinesins recognize, bind and unload cargo, and also regulate processes such as higher brain function, tumour suppression and developmental patterning, are becoming clear. Intracellular transport is fundamental for cellular function, survival and morphogenesis. Kinesin superfamily proteins (also known as KIFs) are important molecular motors that directionally transport various cargos, including membranous organelles, protein complexes and mRNAs. The mechanisms by which different Kinesins recognize and bind to specific cargos, as well as how Kinesins unload cargo and determine the direction of transport, have now been identified. Furthermore, recent molecular genetic experiments have uncovered important and unexpected roles for Kinesins in the regulation of such physiological processes as higher brain function, tumour suppression and developmental patterning. These findings open exciting new areas of Kinesin research.
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Kinesin superfamily motor proteins and intracellular transport
Nature Reviews Molecular Cell Biology, 2009Co-Authors: Nobutaka Hirokawa, Yosuke Tanaka, Yasuko Noda, Shinsuke NiwaAbstract:Intracellular transport is fundamental for cellular function, survival and morphogenesis. Kinesin superfamily proteins (also known as KIFs) are important molecular motors that directionally transport various cargos, including membranous organelles, protein complexes and mRNAs. The mechanisms by which different Kinesins recognize and bind to specific cargos, as well as how Kinesins unload cargo and determine the direction of transport, have now been identified. Furthermore, recent molecular genetic experiments have uncovered important and unexpected roles for Kinesins in the regulation of such physiological processes as higher brain function, tumour suppression and developmental patterning. These findings open exciting new areas of Kinesin research.
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analysis of the Kinesin superfamily insights into structure and function
Trends in Cell Biology, 2005Co-Authors: Harukata Miki, Yasushi Okada, Nobutaka HirokawaAbstract:Kinesin superfamily proteins (KIFs) are key players or ‘hub' proteins in the intracellular transport system, which is essential for cellular function and morphology. The KIF superfamily is also the first large protein family in mammals whose constituents have been completely identified and confirmed both in silico and in vivo . Numerous studies have revealed the structures and functions of individual family members; however, the relationships between members or a perspective of the whole superfamily structure until recently remained elusive. Here, we present a comprehensive summary based on a large, systematic phylogenetic analysis of the Kinesin superfamily. All available sequences in public databases, including genomic information from all model organisms, were analyzed to yield the most complete phylogenetic Kinesin tree thus far, comprising 14 families. This comprehensive classification builds on the recently proposed standardized nomenclature for Kinesins and allows systematic analysis of the structural and functional relationships within the Kinesin superfamily.
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Molecular motors and mechanisms of directional transport in neurons
Nature Reviews Neuroscience, 2005Co-Authors: Nobutaka Hirokawa, Reiko TakemuraAbstract:Intracellular transport is fundamental for neuronal morphogenesis, function and survival. Many proteins are selectively transported to either axons or dendrites. In addition, some specific mRNAs are transported to dendrites for local translation. Proteins of the Kinesin superfamily participate in selective transport by using adaptor or scaffolding proteins to recognize and bind cargoes. The molecular components of RNA-transporting granules have been identified, and it is becoming clear how cargoes are directed to axons and dendrites by Kinesin superfamily proteins. Here we discuss the molecular mechanisms of directional axonal and dendritic transport with specific emphasis on the role of motor proteins and their mechanisms of cargo recognition. In neurons, most proteins that are needed in the axon are synthesized in the cell body and selectively transported to the axon. Most dendritic proteins are also selectively transported from the cell body, but several specific mRNAs are transported into dendrites to support local protein synthesis. For transport of membranous organelles, macromolecular complexes and mRNAs in the axons and dendrites, microtubules serve as rails and Kinesin superfamily proteins (KIFs) function as anterograde motors. Microtubules have polarity, with a plus end and a minus end. Forty-five KIF genes have been identified in mice and humans. All Kinesins have a motor domain that shows high degrees of homology. However, regions outside the motor domains are unique, and these regions allow various cargoes to be recognized and differentially transported. Recently, unified family names for classification of Kinesins in all phylogenies (Kinesin 1 to Kinesin 14) have been defined, but individual motor names will remain the same. The KIF5 family corresponds to conventional Kinesin. Most Kinesins move towards the plus end of microtubules, which takes them from the cell body towards the nerve terminal. The KIF2 family is unique in having both plus-end-directed motor activity and microtubule-depolymerizing activity, which is used to control axon collateral extension at the growth cone. Adaptor/scaffolding proteins tend to be used for the binding of Kinesins to cargoes. Examples are the use of the adaptor protein 1 (AP1) adaptor complex, scaffolding proteins, including proteins of the JNK signalling pathway called JIPs and glutamate receptor-interacting protein 1 (GRIP1), and a tripartite scaffolding protein complex containing LIN10, LIN2 and LIN7 for transporting selective membrane cargoes. The molecular interactions of Kinesins, adaptor/scaffolding proteins and cargoes have been elucidated in detail in these examples. KIF5 transports various cargoes to both axons and dendrites. Both the carboxy-terminal tail of KIF5 and the associated light chain can serve as binding sites for cargoes, and they might be differentially used for selective transport. Cargoes bound to Kinesin light chain tend to be transported to axons, whereas those bound to the KIF5 tail are transported to dendrites. mRNAs are transported in dendrites as a large multisubunit complex of 42 proteins that binds to the tail of KIF5. The microtubule bundle at the initial segment, which shows characteristically strong binding to EB1 (a microtubule-associated protein), serves as a cue for KIF5 to enter axons. Many mechanisms might be used to achieve selective transport of various cargoes. However, a basic understanding of the transport process from the viewpoint of motors and their association with cargoes will clarify the common principles of selective transport.
Rui Yang - One of the best experts on this subject based on the ideXlab platform.
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a novel strategy to visualize vesicle bound Kinesins reveals the diversity of Kinesin mediated transport
Traffic, 2019Co-Authors: Gary Banker, Rui Yang, Zoe Bostick, Alex Garbouchian, Julie Luisi, Marvin BentleyAbstract:In mammals, 15 to 20 Kinesins are thought to mediate vesicle transport. Little is known about the identity of vesicles moved by each Kinesin or the functional significance of such diversity. To characterize the transport mediated by different Kinesins, we developed a novel strategy to visualize vesicle-bound Kinesins in living cells. We applied this method to cultured neurons and systematically determined the localization and transport parameters of vesicles labeled by different members of the Kinesin-1, -2, and -3 families. We observed vesicle labeling with nearly all Kinesins. Only six Kinesins bound vesicles that undergo long-range transport in neurons. Of these, three had an axonal bias (KIF5B, KIF5C and KIF13B), two were unbiased (KIF1A and KIF1Bβ), and one transported only in dendrites (KIF13A). Overall, the trafficking of vesicle-bound Kinesins to axons or dendrites did not correspond to their motor domain preference, suggesting that on-vesicle regulation is crucial for Kinesin targeting. Surprisingly, several Kinesins were associated with populations of somatodendritic vesicles that underwent little long-range transport. This assay should be broadly applicable for investigating Kinesin function in many cell types.
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A novel strategy to visualize vesicle‐bound Kinesins reveals the diversity of Kinesin‐mediated transport
Traffic, 2019Co-Authors: Rui Yang, Gary Banker, Zoe Bostick, Alex Garbouchian, Julie Luisi, Marvin BentleyAbstract:In mammals, 15 to 20 Kinesins are thought to mediate vesicle transport. Little is known about the identity of vesicles moved by each Kinesin or the functional significance of such diversity. To characterize the transport mediated by different Kinesins, we developed a novel strategy to visualize vesicle-bound Kinesins in living cells. We applied this method to cultured neurons and systematically determined the localization and transport parameters of vesicles labeled by different members of the Kinesin-1, -2, and -3 families. We observed vesicle labeling with nearly all Kinesins. Only six Kinesins bound vesicles that undergo long-range transport in neurons. Of these, three had an axonal bias (KIF5B, KIF5C and KIF13B), two were unbiased (KIF1A and KIF1Bβ), and one transported only in dendrites (KIF13A). Overall, the trafficking of vesicle-bound Kinesins to axons or dendrites did not correspond to their motor domain preference, suggesting that on-vesicle regulation is crucial for Kinesin targeting. Surprisingly, several Kinesins were associated with populations of somatodendritic vesicles that underwent little long-range transport. This assay should be broadly applicable for investigating Kinesin function in many cell types.
Benoît Gigant - One of the best experts on this subject based on the ideXlab platform.
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New Insights into the Coupling between Microtubule Depolymerization and ATP Hydrolysis by Kinesin-13 Protein Kif2C.
Journal of Biological Chemistry, 2015Co-Authors: Weiyi Wang, Benoît Gigant, Marcel Knossow, Ting Shen, Raphaël Guérois, Fuming Zhang, Hureshitanmu Kuerban, Chunguang WangAbstract:Kinesin-13 proteins depolymerize microtubules in an ATP hydrolysis-dependent manner. The coupling between these two activities remains unclear. Here, we first studied the role of the Kinesin-13 subfamily-specific loop 2 and of the KVD motif at the tip of this loop. Shortening the loop, the lysine/glutamate interchange and the additional Val to Ser substitution all led to Kif2C mutants with decreased microtubule-stimulated ATPase and impaired depolymerization capability. We rationalized these results based on a structural model of the Kif2C-ATP-tubulin complex derived from the recently determined structures of Kinesin-1 bound to tubulin. In this model, upon microtubule binding Kif2C undergoes a conformational change governed in part by the interaction of the KVD motif with the tubulin interdimer interface. Second, we mutated to an alanine the conserved glutamate residue of the switch 2 nucleotide binding motif. This mutation blocks motile Kinesins in a post-conformational change state and inhibits ATP hydrolysis. This Kif2C mutant still depolymerized microtubules and yielded complexes of one Kif2C with two tubulin heterodimers. These results demonstrate that the structural change of Kif2C-ATP upon binding to microtubule ends is sufficient for tubulin release, whereas ATP hydrolysis is not required. Overall, our data suggest that the conformation reached by Kinesin-13s upon tubulin binding is similar to that of tubulin-bound, ATP-bound, motile Kinesins but that this conformation is adapted to microtubule depolymerization.
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The structure of apo-Kinesin bound to tubulin links the nucleotide cycle to movement
Nature Communications, 2014Co-Authors: Luyan Cao, Weiyi Wang, Qiyang Jiang, Chunguang Wang, Marcel Knossow, Benoît GigantAbstract:Kinesin-1 is a dimeric ATP-dependent motor protein that moves towards microtubules (+) ends. This movement is driven by two conformations (docked and undocked) of the two motor domains carboxy-terminal peptides (named neck linkers), in correlation with the nucleotide bound to each motor domain. Despite extensive data on Kinesin-1, the structural connection between its nucleotide cycle and movement has remained elusive, mostly because the structure of the critical tubulin-bound apo-Kinesin state was unknown. Here we report the 2.2 Å structure of this complex. From its comparison with detached Kinesin-ADP and tubulin-bound Kinesin-ATP, we identify three Kinesin motor subdomains that move rigidly along the nucleotide cycle. Our data reveal how these subdomains reorient on binding to tubulin and when ATP binds, leading respectively to ADP release and to neck linker docking. These results establish a framework for understanding the transformation of chemical energy into mechanical work by (+) end-directed Kinesins.
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Structure of a Kinesin–tubulin complex and implications for Kinesin motility
Nature Structural and Molecular Biology, 2013Co-Authors: Benoît Gigant, Weiyi Wang, Birgit Dreier, Qiyang Jiang, Ludovic Pecqueur, Andreas Plückthun, Chunguang Wang, Marcel KnossowAbstract:The typical function of Kinesins is to transport cargo along microtubules. Binding of ATP to microtubule-attached motile Kinesins leads to cargo displacement. To better understand the nature of the conformational changes that lead to the power stroke that moves a Kinesin's load along a microtubule, we determined the X-ray structure of human Kinesin-1 bound to αβ-tubulin. The structure defines the mechanism of microtubule-stimulated ATP hydrolysis, which releases the Kinesin motor domain from microtubules. It also reveals the structural linkages that connect the ATP nucleotide to the Kinesin neck linker, a 15-amino acid segment C terminal to the catalytic core of the motor domain, to result in the power stroke. ATP binding to the microtubule-bound Kinesin favors neck-linker docking. This biases the attachment of Kinesin's second head in the direction of the movement, thus initiating each of the steps taken.
