Kinesin Motor Domain Research Articles

Anchor Effect of Interactions Between Kinesin's Nucleotide-Binding Pocket and Microtubule.

Microtubule not only provides the track for kinesin but also modulates kinesin's mechanochemical cycle. Microtubule binding greatly increases the rates of two chemical steps occurring inside the nucleotide-binding pocket (NBP) of kinesin, i.e., ATP hydrolysis and ADP release. Kinesin neck linker docking (the key force-generation step) is initiated by the motor head rotation induced by ATP binding which needs an anchor provided by microtubule. These functions of microtubule can only be accomplished through interactions with kinesin. Based on the newly obtained crystal structures of kinesin-microtubule complexes, we investigate the interactions between kinesin's NBP and microtubule using molecular dynamics simulations. We find that the N-3 motif of NBP has direct interactions with a group of negatively charged residues on α-tubulin through Ser235 and Lys237. These specific long-range interactions induce binding of NBP to microtubule at the right position and assist the formation of the indirect interaction between NBP and microtubule. These interactions between N-3 and microtubule have an important anchor effect for kinesin's motor domain during its rotation with Ser235 as the rotation center, and also play a crucial role in stabilizing the ATP-hydrolysis environment.

Sequence and Functional Diversity Across the Kinesin Superfamily

Members of the kinesin superfamily are defined by the presence a kinesin motor domain containing signature sequences for nucleotide and microtubule binding. A variety of biochemical and biophysical studies of the core kinesin-1 motor domain have illuminated how nucleotide binding and hydrolysis lead to force generation and motility along microtubules. Alternating catalysis by the two motor domains enables processive motility along the microtubule surface. It has been assumed that this is the mechanistic paradigm for all kinesin motors. However, recent work indicates that sequence divergence within the kinesin motor domain endows different kinesin motors with distinct microtubule-based properties. We examined how evolutionary tuning of the core motor domain endowed kinesin-3 motors with a high microtubule on-rate, high velocity, and superprocessivity. We also examined sequence divergence within the kinesin-4 family of motors such that some members of this family lost the ability to undergo processive motility.

Interrogating Emergent Transport Properties for Molecular Motor Ensembles: A Semi-analytical Approach.

Intracellular transport is an essential function in eucaryotic cells, facilitated by motor proteins—proteins converting chemical energy into kinetic energy. It is understood that motor proteins work in teams enabling unidirectional and bidirectional transport of intracellular cargo over long distances. Disruptions of the underlying transport mechanisms, often caused by mutations that alter single motor characteristics, are known to cause neurodegenerative diseases. For example, phosphorylation of kinesin motor domain at the serine residue is implicated in Huntington’s disease, with a recent study of phosphorylated and phosphomimetic serine residues indicating lowered single motor stalling forces. In this article we report the effects of mutations of this nature on transport properties of cargo carried by multiple wild-type and mutant motors. Results indicate that mutants with altered stall forces might determine the average velocity and run-length even when they are outnumbered by wild type motors in the ensemble. It is shown that mutants gain a competitive advantage and lead to an increase in the expected run-length when the load on the cargo is in the vicinity of the mutant’s stalling force or a multiple of its stalling force. A separate contribution of this article is the development of a semi-analytic method to analyze transport of cargo by multiple motors of multiple types. The technique determines transition rates between various relative configurations of motors carrying the cargo using the transition rates between various absolute configurations. This enables a computation of biologically relevant quantities like average velocity and run-length without resorting to Monte Carlo simulations. It can also be used to introduce alterations of various single motor parameters to model a mutation and to deduce effects of such alterations on the transport of a common cargo by multiple motors. Our method is easily implementable and we provide a software package for general use.

Nucleotide-free kinesin motor domains reversibly convert to an inactive conformation with characteristics of a molten globule

Nucleotide-free kinesin motor domains from several kinesin families convert reversibly to a refractory conformation that cannot rapidly rebind ADP. In the absence of glycerol, the refractory conformation of Drosophila kinesin motor domains is favored by 50-fold with conversion of the active to the refractory species at ∼0.052 s−1 and reactivating in the presence of ADP at ∼0.001 s−1. This reactivation by ADP is due to conformational selection rather than induced fit because ADP is not bound to the refractory species at concentrations of ADP that are sufficient to saturate the rate of reactivation. Glycerol stabilizes the active conformation by reducing the rate of inactivation, while having little effect on the reactivation rate. Circular dichroism indicates a large conformational change occurs on formation of the refractory species. The refractory conformation binds ANS (8-anilino-1-napthalenesulfonic acid) with a large increase in fluorescence, indicating that it has molten globule character. High ANS binding is also observed with the refractory forms of Eg5 (a kinesin-5) and Ncd (a kinesin-14), indicating that a refractory conformation with molten globule characteristics may be a common feature of nucleotide-free kinesin motor domains.

Review: Mechanochemistry of the kinesin-1 ATPase.

ABSTRACTKinesins are P‐loop NTPases that can do mechanical work. Like small G‐proteins, to which they are related, kinesins execute a program of active site conformational changes that cleaves the terminal phosphate from an NTP substrate. But unlike small G‐proteins, kinesins can amplify and harness these conformational changes in order to exert force. In this short review I summarize current ideas about how the kinesin active site works and outline how the active site chemistry is coupled to the larger‐scale structural cycle of the kinesin motor domain. Focusing largely on kinesin‐1, the best‐studied kinesin, I discuss how the active site switch machinery of kinesin cycles between three distinct states, how docking of the neck linker stabilizes two of these states, and how tension‐sensitive and position‐sensitive neck linker docking may modulate both the hydrolysis step of ATP turnover and the trapping of product ADP in the active site. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 476–482, 2016.

Multiscale method for modeling binding phenomena involving large objects: application to kinesin motor domains motion along microtubules.

Many biological phenomena involve the binding of proteins to a large object. Because the electrostatic forces that guide binding act over large distances, truncating the size of the system to facilitate computational modeling frequently yields inaccurate results. Our multiscale approach implements a computational focusing method that permits computation of large systems without truncating the electrostatic potential and achieves the high resolution required for modeling macromolecular interactions, all while keeping the computational time reasonable. We tested our approach on the motility of various kinesin motor domains. We found that electrostatics help guide kinesins as they walk: N-kinesins towards the plus-end, and C-kinesins towards the minus-end of microtubules. Our methodology enables computation in similar, large systems including protein binding to DNA, viruses, and membranes.

Mapping the Processivity Determinants of the Kinesin-3 Motor Domain

Kinesin superfamily members play important roles in many diverse cellular processes including cell motility, cell division, intracellular transport and regulation of the microtubule cytoskeleton. How the properties of the family-defining motor domain of distinct kinesins are tailored to their differing cellular roles remains largely unknown. Here we employ molecular dynamics simulations coupled with energetic calculations to infer the family-specific interactions of kinesin-1 and kinesin-3 motor domains with microtubules in different nucleotide states. Experimental mutagenesis and single molecule motility assays are then used to further assess the predicted residue-wise determinants of distinct kinesin-microtubule binding properties. Collectively, our results identify residues in the L8, L11 and alpha6 regions that contribute to family-specific microtubule interactions and whose mutation affects motor-microtubule complex stability and processive motility (the ability of an individual motor to take multiple steps along its microtubule filament). In particular, substitutions of prominent kinesin-3 residues with those found in kinesin-1, namely R167S/H171D, K266D, and R346M, were found to decrease kinesin-3 processivity 10-fold and thus approach kinesin-1 levels.

Mechanism of Kinsein-2 Navigation Around Obstacles on the Microubule Surface

Intracellular cargo transport is a complex and highly regulated process. Molecular motors are responsible for navigating these cargos along complex microtubule paths containing microtubule-associated proteins. Many cargos contain more than one directionally similar motors i.e., kinesin-1 and kinesin-2, and it is not well understood why directionally similar teams of motors work together. We have previously shown, in vitro, that kinesin-2's motility is insensitive to a neuronal MAP called Tau, unlike kinesin-1 which is strongly inhibited by Tau. Kinesin-2's longer neck-linker was demonstrated to be essential to successfully navigate Tau obstacles when compared to kinesin-1's shorter neck-linker. However, the mechanism by which kinesin-2 efficiently navigates Tau is unknown. Based on our previous work, we hypothesize that mammalian kinesin-2 side-steps to adjacent protofilaments to maneuver around MAPs. To test this, we use single-molecule imaging to track the side-stepping behavior of kinesin-1 and kinesin-2 in the absence and presence of both static (i.e., monomeric rigor kinesin motor domains) and dynamic (i.e., Tau) obstacles on the microtubule surface. Furthermore, we also examine the rotation of microtubules sparsely decorated with quantum dots in the gliding filament assay to observe torque generation (i.e., side-stepping behavior) in the absence and presence of Tau. Defining kinesin-2's mechanism of navigation on the crowded microtubule surface provides a previously unappreciated and unique contribution to understanding how teams of directionally-similar motors facilitate long range intracellular cargo transport.

N-Terminal Coiled-Coils in the Human Kinesin-5 and Kinesin-1 Stalk are Sufficient for Tetrameric Organization

Oligomerization is a key biological control mechanism in protein function. Homo-dimerization versus homo-tetramerization transforms cellular function for different members of a protein superfamily. Case in point, the homotetrameric organization of Kinesin-5 proteins, via a ∼500 amino acid coiled-coil stalk, underlies their ability to organize the mitotic spindle. Little is known about the rules that govern homo-oligomerization of Kinesin-5 proteins. Our goal is to uncover the trigger sequence, or the element indispensable for tetramer formation, in Kinesin-5. Herein, sequence profiling tools show that the first 150 - 200 residues of the stalk is the only region, common to Kinesin-5 and Kinesin-1 proteins, that is calculated to form coiled-coils; the remaining ∼350 residues in Kinesin-5 exhibit variability in expected coiled-coil formation and do not parallel predictions for the dimeric Kinesin-1 proteins. In two protein constructs containing the first 141 residues of the stalk, in vitro tetramerization of the coiled-coil peptide and the truncated form of the human Kinesin-5 protein was observable by size-exclusion chromatography, static light scattering, and macro-ion mobility spectrometry. We also determined that the human Kinesin-5 and a chimeric protein consisting of the Kinesin-5 motor domain and Kinesin-1 stalk can crosslink and move more than one microtubule in situ. Our data support that, for more than one kinesin family member, the N-terminal portion of the stalk permits tetramerization. Regulation of the dimer-to-tetramer transition may require the sequence of flanking necklinker and C-terminal stalk domains.

Mapping the Processivity Determinants of the Kinesin-3 Motor Domain

Kinesin superfamily members play important roles in many diverse cellular processes, including cell motility, cell division, intracellular transport, and regulation of the microtubule cytoskeleton. How the properties of the family-defining motor domain of distinct kinesins are tailored to their different cellular roles remains largely unknown. Here, we employed molecular-dynamics simulations coupled with energetic calculations to infer the family-specific interactions of kinesin-1 and kinesin-3 motor domains with microtubules in different nucleotide states. We then used experimental mutagenesis and single-molecule motility assays to further assess the predicted residue-wise determinants of distinct kinesin-microtubule binding properties. Collectively, our results identify residues in the L8, L11, and α6 regions that contribute to family-specific microtubule interactions and whose mutation affects motor-microtubule complex stability and processive motility (the ability of an individual motor to take multiple steps along its microtubule filament). In particular, substitutions of prominent kinesin-3 residues with those found in kinesin-1, namely, R167S/H171D, K266D, and R346M, were found to decrease kinesin-3 processivity 10-fold and thus approach kinesin-1 levels.

A new protocol to accurately determine microtubule lattice seam location.

Microtubules (MTs) are cylindrical polymers of αβ-tubulin that display pseudo-helical symmetry due to the presence of a lattice seam of heterologous lateral contacts. The structural similarity between α- and β-tubulin makes it difficult to computationally distinguish them in the noisy cryo-EM images, unless a marker protein for the tubulin dimer, such as kinesin motor domain, is present. We have developed a new data processing protocol that can accurately determine αβ-tubulin register and seam location for MT segments. Our strategy can deal with difficult situations, where the marker protein is relatively small or the decoration of marker protein is sparse. Using this new seam-search protocol, combined with movie processing for data from a direct electron detection camera, we were able to determine the cryo-EM structures of MT at 3.5 Å resolution in different functional states. The successful distinction of α- and β-tubulin allowed us to visualize the nucleotide state at the E-site and the configuration of lateral contacts at the seam.

Identification of two novel KIF5A mutations in hereditary spastic paraplegia associated with mild peripheral neuropathy

Spastic paraplegia type 10 (SPG10) is a rare form of autosomal dominant hereditary spastic paraplegia (AD-HSP) due to mutations in KIF5A, a gene encoding the neuronal kinesin heavy-chain involved in axonal transport. KIF5A mutations have been associated with a wide clinical spectrum, ranging from pure HSP to isolated peripheral nerve involvement or complicated HSP phenotypes. Most KIF5A mutations are clustered in the motor domain of the protein that is necessary for microtubule interaction. Here we describe two Spanish families with an adult onset complicated AD-HSP in which neurological studies revealed a mild sensory neuropathy. Intention tremor was also present in both families. Molecular genetic analysis identified two novel mutations c.773 C>T and c.833 C>T in the KIF5A gene resulting in the P258L and P278L substitutions respectively. Both were located in the highly conserved kinesin motor domain of the protein which has previously been identified as a hot spot for KIF5A mutations. This study adds to the evidence associating the known occurrence of mild peripheral neuropathy in the adult onset SPG10 type of AD-HSP.

Analyzing kinesin motor domain translocation in cultured hippocampal neurons.

Neuronal microtubules are subject to extensive posttranslational modifications and are bound by MAPs, tip-binding proteins, and other accessory proteins. All of these features, which are difficult to replicate in vitro, are likely to influence the translocation of kinesin motors. Here we describe assays for evaluating the translocation of a population of fluorescently labeled kinesin motor domains, based on their accumulation in regions of the cell enriched in microtubule plus ends. Neurons lend themselves to these experiments because of their microtubule organization. In axons, microtubules are oriented with their plus ends out; dendrites contain a mixed population of microtubules, but those near the tips are also plus end out. The assays involve the expression of constitutively active kinesins that can walk processively, but that lack the autoinhibitory domain in the tail that normally prevents their binding to microtubules until they attach to vesicles. The degree to which such motor domains accumulate at neurite tips serves as a measure of the efficiency of their translocation. Although these assays cannot provide the kind of quantitative kinetic information obtained from in vitro assays, they offer a simple way to examine kinesin translocation in living neurons. They can be used to compare the translocation efficiency of different kinesin motors and to evaluate how mutations or posttranslational modifications within the motor domain influence kinesin translocation. Changes to motor domain accumulation in these assays can also serve as readout for changes in the microtubule cytoskeleton that affect kinesin translocation.

Mechanistic Origin of Microtubule Dynamic Instability and Its Modulation by EB Proteins

Microtubule (MT) dynamic instability is driven by GTP hydrolysis and regulated by microtubule-associated proteins, including the plus-end tracking end-binding protein (EB) family. We report six cryo-electron microscopy (cryo-EM) structures of MTs, at 3.5 Å or better resolution, bound to GMPCPP, GTPγS, or GDP, either decorated with kinesin motor domain after polymerization or copolymerized with EB3. Subtle changes around the E-site nucleotide during hydrolysis trigger conformational changes in α-tubulin around an "anchor point," leading to global lattice rearrangements and strain generation. Unlike the extended lattice of the GMPCPP-MT, the EB3-bound GTPγS-MT has a compacted lattice that differs in lattice twist from that of the also compacted GDP-MT. These results and the observation that EB3 promotes rapid hydrolysis of GMPCPP suggest that EB proteins modulate structural transitions at growing MT ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. Our findings explain both EBs end-tracking behavior and their effect on microtubule dynamics.

Fission yeast mitochondria are distributed by dynamic microtubules in a motor-independent manner.

The cytoskeleton plays a critical role in regulating mitochondria distribution. Similar to axonal mitochondria, the fission yeast mitochondria are distributed by the microtubule cytoskeleton, but this is regulated by a motor-independent mechanism depending on the microtubule associated protein mmb1p as the absence of mmb1p causes mitochondria aggregation. In this study, using a series of chimeric proteins to control the subcellular localization and motility of mitochondria, we show that a chimeric molecule containing a microtubule binding domain and the mitochondria outer membrane protein tom22p can restore the normal interconnected mitochondria network in mmb1-deletion (mmb1∆) cells. In contrast, increasing the motility of mitochondria by using a chimeric molecule containing a kinesin motor domain and tom22p cannot rescue mitochondria aggregation defects in mmb1∆ cells. Intriguingly a chimeric molecule carrying an actin binding domain and tom22p results in mitochondria associated with actin filaments at the actomyosin ring during mitosis, leading to cytokinesis defects. These findings suggest that the passive motor-independent microtubule-based mechanism is the major contributor to mitochondria distribution in wild type fission yeast cells. Hence, we establish that attachment to microtubules, but not kinesin-dependent movement and the actin cytoskeleton, is required and crucial for proper mitochondria distribution in fission yeast.

De novo mutations in KIF1A cause progressive encephalopathy and brain atrophy.

ObjectiveTo determine the cause and course of a novel syndrome with progressive encephalopathy and brain atrophy in children.MethodsClinical whole-exome sequencing was performed for global developmental delay and intellectual disability; some patients also had spastic paraparesis and evidence of clinical regression. Six patients were identified with de novo missense mutations in the kinesin gene KIF1A. The predicted functional disruption of these mutations was assessed in silico to compare the calculated conformational flexibility and estimated efficiency of ATP binding to kinesin motor domains of wild-type (WT) versus mutant alleles. Additionally, an in vitro microtubule gliding assay was performed to assess the effects of de novo dominant, inherited recessive, and polymorphic variants on KIF1A motor function.ResultsAll six subjects had severe developmental delay, hypotonia, and varying degrees of hyperreflexia and spastic paraparesis. Microcephaly, cortical visual impairment, optic neuropathy, peripheral neuropathy, ataxia, epilepsy, and movement disorders were also observed. All six patients had a degenerative neurologic course with progressive cerebral and cerebellar atrophy seen on sequential magnetic resonance imaging scans. Computational modeling of mutant protein structures when compared to WT kinesin showed substantial differences in conformational flexibility and ATP-binding efficiency. The de novo KIF1A mutants were nonmotile in the microtubule gliding assay.InterpretationDe novo mutations in KIF1A cause a degenerative neurologic syndrome with brain atrophy. Computational and in vitro assays differentiate the severity of dominant de novo heterozygous versus inherited recessive KIF1A mutations. The profound effect de novo mutations have on axonal transport is likely related to the cause of progressive neurologic impairment in these patients.

Aluminum-induced kinesin inactivation as potential molecular cause of impairment of neuronal transport processes.

It is commonly accepted that aluminum ions may initiate the development of diverse diseases, including neurological disorders. So far, our knowledge of the molecular mechanisms of the interaction of aluminum with defined cellular structures has been still fragmentary. As functional key tasks of neuronal cells essentially depend on the activity of kinesin, we wanted to find out whether this motor protein represents a molecular target for aluminum. We demonstrate that aluminum ions inhibit (IC50 ∼50 μM) the ATPase of the neuron-specific kinesin KIF5A. The ATPase-active center itself, which is located in the kinesin motor domain, does not seem to be directly affected by aluminum. Our results suggest that inhibition is preferentially caused by aluminum binding to some sequence within the kinesin stalk leading to a conformational state of the kinesin molecule, similar to those described in cases of kinesin autoinhibition caused by motor domain-tail binding. Because of the relative high sequence conservation of mammalian kinesin-1 (to which KIF5A belongs), we assume that also in non-neuronal cells the intracellular transport can be affected by aluminum ions.

Rab6 regulation of the kinesin family KIF1C motor domain contributes to Golgi tethering.

Most kinesins transport cargoes bound to their C-termini and use N-terminal motor domains to move along microtubules. We report here a novel function for KIF1C: it transports Rab6A-vesicles and can influence Golgi complex organization. These activities correlate with KIF1C's capacity to bind the Golgi protein Rab6A directly, both via its motor domain and C-terminus. Rab6A binding to the motor domain inhibits microtubule interaction in vitro and in cells, decreasing the amount of motile KIF1C. KIF1C depletion slows protein delivery to the cell surface, interferes with vesicle motility, and triggers Golgi fragmentation. KIF1C can protect Golgi membranes from fragmentation in cells lacking an intact microtubule network. Rescue of fragmentation requires sequences that enable KIF1C to bind Rab6A at both ends, but not KIF1C motor function. Rab6A binding to KIF1C's motor domain represents an entirely new mode of regulation for a kinesin motor, and likely has important consequences for KIF1C's cellular functions.

Characterizing the Allosteric Effect of Nucleotide and Inhibitor Binding to the Kinesin Motor Domain

Kinesin motor domains couple cycles of ATP hydrolysis to cycles of microtubule binding and conformational changes that result in directional force and movement on microtubules. General principles of this mechanochemical coupling have now been established. However, fundamental atomistic details of the underlying allosteric mechanisms remain unknown. This lack of knowledge hampers the development of new inhibitors and limits our understanding of how disease-associated mutations in distal sites can interfere with the fidelity of motor domain function. Here we combine unbiased molecular dynamics simulations, bioinformatics analysis and in-silico mutational studies to elucidate the structural dynamic effects of nucleotide turnover and allosteric inhibition. Multiple replica simulations of kinesin-1 and kinesin-5 bound to ATP-, ADP- and kinesin-5 specific inhibitors were used to create residue-based networks that characterize the internal dynamic coordination of functional regions. This analysis predicts the intervening residues involved in the dynamic coupling of nucleotide, microtubule, neck-linker and inhibitor binding sites. Regions identified include the nucleotide binding switch regions, loop5, loop7, α4-α5-loop13, α1 and β4-β6-β7. Also evident were nucleotide and inhibitor dependent shifts in the dynamic coupling paths linking functional sites. In particular, inhibitor binding to the loop5 region affects β-sheet residues and α1 leading to a dynamic decoupling of nucleotide, microtubule and neck-linker binding sites. Additional analysis of point mutations in kinesin-5, including P131 (loop5), Q78/I79 (α1), E166 (loop7), K272/I273 (β7) G325/G326 (loop13), support their predicted role in mediating the dynamic coupling of distal functional surfaces. Collectively, our results and approach provide a framework for explaining how binding events and point mutations can alter dynamic couplings critical for kinesin motor domain function.

Kinesin-5 Acts as a Microtubule Stabilizer, Polymerase and Plus-Tip Tracker

Kinesin-5 slides antiparallel microtubules apart during mitosis and is necessary for bipolar spindle formation. Besides its unique homotetrameric configuration, determined by the coiled-coil domain, kinesin-5 motor domains also possess specific properties optimal for their spindle organizing function. To study properties intrinsic to the kinesin-5 motor domain, we generated functional kinesin-5 dimers by fusing the kinesin-5 head and 18-residue neck linker to the coiled-coil rod of kinesin-1. In ATP this kinesin-5 dimer decorated plus-ends of taxol-stabilized microtubules and slowed depolymerization of GMPCPP microtubules. On dynamic microtubules, kinesin-5 dimer increased the microtubule growth rate by more than a factor of two and reduced the catastrophe frequency three-fold. These findings are consistent with kinesin-5 acting as a microtubule polymerase. To understand this polymerase mechanism, TIRF microscopy was used to visualize individual GFP-labeled kinesin-5 dimers on immobilized microtubules. Motors walked to the ends of microtubules and remained bound there for 7 seconds, much longer than the 0.1 s motor step time. We hypothesize that kinesin-5 promotes microtubule polymerization by stabilizing longitudinal interactions between tubulin subunits on a single protofilament. Consistent with this protofilament stabilization hypothesis, fluorescence analysis suggests that microtubule plus-ends are more tapered in the presence of kinesin-5. Furthermore, curved and looped ends were observed, which occasionally resolved into straight microtubules, consistent with kinesin-5 stabilizing long protofilament bundles. In cells, these end-binding and polymerase activities should enhance the ability of kinesin-5 to establish and maintain spindle pole separation during mitosis.