Kinesin Motor Domain Research Articles

A new model for binding of kinesin 13 to curved microtubule protofilaments

Kinesin motor proteins use adenosine triphosphate hydrolysis to do work on microtubules (MTs). Most kinesins walk along the MT, but class 13 kinesins instead uniquely recognize MT ends and depolymerize MT protofilaments. We have used electron microscopy (EM) to understand the molecular interactions by which kinesin 13 performs these tasks. Although a construct of only the motor domain of kinesin 13 binds to every heterodimer of a tubulin ring, a construct containing the neck and the motor domain occupies alternate binding sites. Likewise, EM maps of the dimeric full-length (FL) protein exhibit alternate site binding but reveal density for only one of two motor heads. These results indicate that the second head of dimeric kinesin 13 does not have access to adjacent binding sites on the curved protofilament and suggest that the neck alone is sufficient to obstruct access. Additionally, the FL construct promotes increased stacking of rings compared with other constructs. Together, these data suggest a model for kinesin 13 depolymerization in which increased efficiency is achieved by binding of one kinesin 13 molecule to adjacent protofilaments.

Crystal Structure of Nucleotide-free Kinesin-1 Motor Domain Explains Coordinated Walking Mechanism

Kinesin is a highly processive motor that moves along microtubules in a hand-over-hand manner. To understand structural basis for the walking mechanism, atomic-detailed structural information of kinesin at various nucleotide states are essential, however the nucleotide-free crystal structure has been unavailable. Here we report the first crystal structure of kinesin-1 motor domain without bound nucleotide solved at a 2.8A resolution. The structure fits very well to the 9A cryo-EM density map of kinesin-microtubule complex without nucleotide (Sindelar et al. 2007), demonstrating that the structure represents the ADP-released microtubule-bound kinesin head. The nucleotide-free structure showed marked differences from the previously solved ATP- and ADP-like crystal structures. First, compared to ADP-like structure, the switch II helix extends and this extension is stabilized through the interactions with both switch I/II loops and tubulin subunits. Furthermore, switch I uncoils and extends toward the nucleotide pocket. We suggest that microtubule-binding is sensed via the extension of switch II helix, which then promotes ADP release through switch I/II loops. Another striking feature is that α6 helix that directly connects to the neck linker moved such that the beginning of the neck linker collides with the switch II helix. The neck linker itself is mobile but this steric hindrance prohibits extension of the neck linker to the forward direction. Using crystal structures docked to the EM density map, we modeled dimeric kinesin on microtubule and found that kinesin cannot adapt two-head-bound state when both heads are nucleotide-free due to the constraint posed on the neck linker, and the tethered head can bind only to the forward tubulin-binding site after the trailing head becomes ATP-like structure. These findings provide structural basis for the coordinated processive movement of two kinesin motor domains.

Expression and Characterization of Novel Rice Kinesin E15

Kinesin is an ATP driven motor protein that moves along microtubules. Kinesin plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. Kinesin species derived from vertebrates have been well characterized. In contrast, only a few kinesis have been characterized in plants. E15 is one of the kinesin encoded on rice genome. E15 has COOH-terminal motor domain and exhibits a high homology with the kinesin-14 family in Arabidopsis thaliana. However, this kinesin is not similar to other kinesin -14 family kinesins derived from animal, e.g., DmNcd, ScKar3, and CeKlp. Consequently, kinesin E15 may be plant-specific kinesin. In this study, we expressed the motor domain of a novel rice plant-specific kinesin, E15, in Escherichia coli. and studied its enzymatic characteristics and compared with other related kinesins. Molecular weight of the E15 motor domain was 37.6 kD. The MT-dependent ATPase activity was higher and the affinity for MT was weaker than rice kinesin K16 that we have previously reported. The optimum pH was pH 6.0–6.5, which is similar to K16. Interaction of E15 with fluorescent ATP analogues was also studied for the kinetic characterization. E15 showed weaker affinity for nucleotide than other kinesins. Currently, we are preparing E15 dimer for the motility assay.

Photo-activation Of Atpase Activity Of Caged-kinesin

Photo-responsive caged compounds have high potency in the application concerning functional biological molecules as photo-switching device. Kinesin is a motor protein that moves along microtubule by the energy generated from ATP hydrolysis. The structure of conventional kinesin has been well studied and the key regions related to the function were clarfied. In the present study, the photo-regulation of the catalytic activity of mouse brain and C. elegans kinesins were investigated by treating with a caging reagent, 4,5-dimethoxy-2-nitrobenzyl bromide (DMNBB). The mouse brain kinesin mutants that have a single reactive cysteine at 96C were prepared and modified by DMNBB in the presence and absence of ATP. For the kinesin modified in the absence of ADP, the ATPase activity was increased by 300% within 10 minutes. In the presence of ADP, the change of the ATPase activity was slower than that in the absence of ATP. Upon UV irradiation, the ATPase activity of the kinesin modified by DMNBB recovered to the level before modification. Wild type of C. elegans unc-116 kinesin motor domain derived from C. elegans, which has a single reactive cysteine residue. Modification with DMNBB and photo-irradiation on the wild type of C.elegans kinesin unc-116 showed also significant reduction and restoration of activity. We have identified the amino acid residue of kinesin unc-116, which affects activity by the modification with DMNBB as Cys16.

Photo-control Of Atpase Activity Of The Kinesin Motor Domain Intermolecularly Cross-linked By Bifunctional Photochromic Compound

Kinesin is an ATP driven dimeric motor protein carries cellular cargoes along microtubules. Azobenzene-dimaleimide (ABDM) is a bifunctional SH reactive photochromic compounds. We have previously demonstrated that ABDM was incorporated into the functional site of skeletal muscle myosin head (S1), and the global conformational change of S1 was induced by cis-trans isomerization of the cross-linked ABDM upon UV/VIS light irradiation, which may mimic the conformational change accompanied by energy transduction.

Analysis of Crystal Structure and Solution Structure of the Motor Domain of Rice Kinesin K16

The novel rice plant specific kinesin K16 has several unique enzymatic characteristics comparing with conventional kinesin. The most interesting property is that the ADP-free K16 motor domain is very stable, contrast to conventional kinesin that is very labile in ADP-free state. Recently, we have determined crystal structure of the novel rice kinesin K16 motor domain (K16MD) in complex with MgADP at 2.5A resolution. The overall structure of the K16MD is similar to that of conventional kinesin motor domains, as expected from the high similarity of amino acid sequence (43.2 %). However, the neck-linker of the ADP bound K16 motor domain showed an ordered conformation in a position quite different from that observed in conventional kinesin, which may reflect the unique enzymatic characteristics of rice kinesin K16. In the present study, we analyzed the inner structure of the K16 motor domain in detail and compared the structure with Eg5 and other related kinesins. It has been revealed that K16MD does not have interaction of amino acids side chains, which stabilizes the docking conformation of neck-linker. We have also analyzed the conformation of neck-linker in the solution using the K16 by FRET. Motor domain mutant G220C chimera protein fused with GFP at the neck-linker has been prepared and labeled IAE-DANS. The FRET efficiencies between the fluorescent probe DANS at C220 and GFP in the presence and absence of nucleotides were analyzed.

Configuration Of The Kinesin1 Motor Domains In The ATP-waiting State As Revealed By Fluorescence Polarization Microscopy

The molecular mechanism of coordination between kinesin1 heads during processive walking remains unclear, partly due to the lack of structural information on critical intermediates of the kinesin1 mechano-chemical cycle. To address this issue here we used ensemble and single molecule fluorescence polarization microscopy to determine the mobility and orientation of the kinesin motor domains. We first investigated conditions mimicking a state when only one head is bound to the microtubule and the other one is tethered. For this we made heterodimeric constructs with impaired microtubule binding in one head. Our results indicate that the tethered head is very mobile. We then investigated the orientation of the head domains in homodimeric constructs moving processively at saturating or limiting [ATP]. At saturating [ATP] both motor domains are well oriented relative to the microtubule but at limiting [ATP] there is an increase in mobility. This result indicates that before ATP-binding one motor domain is mobile.

Photo-regulation of Kinesin ATPase Activity using Photochromic Molecule

Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-forms in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Previously, we have introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the functional region of kinesin to reversibly regulate the ATPase activity of kinesin by photoirradiation. The five kinesin motor domain mutants, A247C, L249C, A252C, G272C and S275C, which contained a single reactive cysteine residue in loops L11 and L12 were prepared.

Location of Tethered Head of Kinesin-1 When Bound to a Microtubule

A dimer of kinesin-1 motor domains (heads), each with a bound ADP, looses only one of its 2 ADP molecules on binding to a microtubule (MT). The first equivalent of one kinesin dimer per two tubulin heterodimers (one head per tubulin heterodimer) binds tightly (K1), but additional kinesin binding can occur at higher kinesin concentration (K2). Whether the heads are arranged as in L1,2 or D1,2 is controversial. These cases can be distinguished by two additional criteria as indicated in the figure. One is that binding of a second outer layer of kinesin on going from L1 to L2 should not result in additional release of ADP, whereas additional ADP will be released on going from D1 to D2. Initial experiments indicate that no additional ADP is released and this favors states L1 and L2. These conformations can also be distinguished by oxygen isotopic methods because kinesin in L2 should continue to catalyze rapid medium Pi=water exchange, while conformation D2 should not catalyze this exchange reaction because none of the ADP containing heads are in contact with the MT.Supported by NSF grant MCB-0615549.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Identification and expression patterns of kif3bz during the zebrafish embryonic development

We are reporting the identification, expression patterns, and possible biological functions of zebrafish kif3b (kif3bz) encoding 475 amino acids. Kif3Bz contains the kinesin motor domain, catalytic domain, KISc domain, and one single coiled coil domain. Phylogenetic analysis indicates that kif3bz is a highly conserved gene among the tested vertebrates. First of all, both maternal and zygotic messages of kif3bz were evenly distributed in the blastomeres at 2‐cell stage. Its ubiquitous expression throughout the blastomeres continued till 40% epiboly. However, kif3bz transcripts became restricted in Kupffer's vesicle at tailbud and 6‐somite stages. At 13‐somite stage, kif3bz expression pattern became specific to the telencephalon, diencephalon, trigeminal placode, and somites. Such expression patterns were further intensified in the telencephalon, diencephalons, hind brain, pronephric ducts, optic vesicles, and spinal cord neurons in the 23‐somite stage embryos, and last till 24 hpf. We discussed possible functions of Kif3Bz related to the vertebrate embryonic development.

1P-146 キネシン1モータードメインのヌクレオチドフリー状態での結晶構造解析(分子モーター,第47回日本生物物理学会年会)

1P-146 キネシン1モータードメインのヌクレオチドフリー状態での結晶構造解析(分子モーター,第47回日本生物物理学会年会)

1TP4-06 キネシン1モータードメインのヌクレオチドフリー状態での結晶構造解析(分子モーター,第47回日本生物物理学会年会)

1TP4-06 キネシン1モータードメインのヌクレオチドフリー状態での結晶構造解析(分子モーター,第47回日本生物物理学会年会)

Mechanical Characterization of Motor Proteins: A Molecular Dynamics Approach

AbstractA new approach, based on molecular dynamics, is presented to measure the mechanical properties of motor proteins, which are important for accomplishing their intracellular functions. Two different set‐ups were designed to mimic the optical tweezers and surface force apparatus experiments. The results obtained show that the stiffness and elastic modulus of kinesin motor domain are 377 pN · nm−1 and 0.17 GPa, respectively, while the myosin motor domain is characterized by a stiffness of 271 pN · nm−1 and an elastic modulus of 0.26 GPa. These results suggest that the source of the very low stiffness detected for full‐length molecules is located outside of the globular part of the proteins.magnified image

Intramolecular Strain Coordinates Kinesin Stepping Behavior along Microtubules

Kinesin advances 8 nm along a microtubule per ATP hydrolyzed, but the mechanism responsible for coordinating the enzymatic cycles of kinesin's two identical motor domains remains unresolved. Here, we have tested whether such coordination is mediated by intramolecular tension generated by the "neck linkers," mechanical elements that span between the motor domains. When tension is reduced by extending the neck linkers with artificial peptides, the coupling between ATP hydrolysis and forward stepping is impaired and motor's velocity decreases as a consequence. However, speed recovers to nearly normal levels when external tension is applied by an optical trap. Remarkably, external load also induces bidirectional stepping of an immotile kinesin that lacks its mechanical element (neck linker) and fuel (ATP). Our results indicate that the kinesin motor domain senses and responds to strain in a manner that facilitates its plus-end-directed stepping and communication between its two motor domains.

Functional involvement of human discs large tumor suppressor in cytokinesis

Cytokinesis is the final step of cell division that completes the separation of two daughter cells. We found that the human discs large (hDlg) tumor suppressor homologue is functionally involved in cytokinesis. The guanylate kinase (GUK) domain of hDlg mediates the localization of hDlg to the midbody during cytokinesis, and over-expression of the GUK domain in U2OS and HeLa cells impaired cytokinesis. Mouse embryonic fibroblasts (MEFs) derived from dlg mutant mice contained an increased number of multinucleated cells and showed reduced proliferation in culture. A kinesin-like motor protein, GAKIN, which binds directly to the GUK domain of hDlg, exhibited a similar intracellular distribution pattern with hDlg throughout mitosis and localized to the midbody during cytokinesis. However, the targeting of hDlg and GAKIN to the midbody appeared to be independent of each other. The midbody localization of GAKIN required its functional kinesin-motor domain. Treatment of cells with the siRNA specific for hDlg and GAKIN caused formation of multinucleated cells and delayed cytokinesis. Together, these results suggest that hDlg and GAKIN play functional roles in the maintenance of midbody architecture during cytokinesis.

Direction and speed of microtubule movements driven by kinesin motors arranged on catchin thick filaments

Conventional kinesin (Kinesin-1) is a microtubule-based molecular motor that supports intracellular vesicle/organelle transport in various eukaryotic cells. To arrange kinesin motors similarly to myosin motors on thick filaments in muscles, the motor domain of rat conventional kinesin (amino acid residues 1-430) fused to the C-terminal 829 amino acid residues of catchin (KHC430Cat) was bacterially expressed and attached to catchin filaments that can attach to and arrange myosin molecules in a bipolar manner on their surface. Unlike the case of myosin where actin filaments move toward the center much faster than in the opposite direction along the catchin filaments, microtubules moved at the same speed in both directions. In addition, many microtubules moved across the filaments at the same speed with various angles between the axes of the microtubule and catchin filament. Kinesin/catchin chimera proteins with a shorter kinesin neck domain were also prepared. Those without the whole hinge 1 domain and the C-terminal part of the neck helix moved microtubules toward the center of the catchin filaments significantly, but only slightly, faster than in the opposite direction, although the movements in both directions were slower than those of the KHC430Cat construct. The results suggest that kinesin has substantial mechanical flexibility within the motor domain, possibly within the neck linker, enabling its interaction with microtubules having any orientation.

Localized Regulation of Axonal RanGTPase Controls Retrograde Injury Signaling in Peripheral Nerve

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.

Structural dynamics of the microtubule binding and regulatory elements in the kinesin-like calmodulin binding protein

Kinesins are molecular motors that power cell division and transport of various proteins and organelles. Their motor activity is driven by ATP hydrolysis and depends on interactions with microtubule tracks. Essential steps in kinesin movement rely on controlled alternate binding to and detaching from the microtubules. The conformational changes in the kinesin motors induced by nucleotide and microtubule binding are coordinated by structural elements within their motor domains. Loop L11 of the kinesin motor domain interacts with the microtubule and is implicated in both microtubule binding and sensing nucleotide bound to the active site of kinesin. Consistent with its proposed role as a microtubule sensor, loop L11 is rarely seen in crystal structures of unattached kinesins. Here, we report four structures of a regulated plant kinesin, the kinesin-like calmodulin binding protein (KCBP), determined by X-ray crystallography. Although all structures reveal the kinesin motor in the ATP-like conformation, its loop L11 is observed in different conformational states, both ordered and disordered. When structured, loop L11 adds three additional helical turns to the N-terminal part of the following helix alpha4. Although interactions with protein neighbors in the crystal support the ordering of loop L11, its observed conformation suggests the conformation for loop L11 in the microtubule-bound kinesin. Variations in the positions of other features of these kinesins were observed. A critical regulatory element of this kinesin, the calmodulin binding helix positioned at the C-terminus of the motor domain, is thought to confer negative regulation of KCBP. Calmodulin binds to this helix and inserts itself between the motor and the microtubule. Comparison of five independent structures of KCBP shows that the positioning of the calmodulin binding helix is not decided by crystal packing forces but is determined by the conformational state of the motor. The observed variations in the position of the calmodulin binding helix fit the regulatory mechanism previously proposed for this kinesin motor.

Leukocyte transmigration requires kinesin-mediated microtubule-dependent membrane trafficking from the lateral border recycling compartment

Diapedesis of leukocytes across endothelial cells is a crucial step in both the innate and adaptive immune responses. Surface molecules on leukocytes and endothelial cells critical for diapedesis have been identified, but the mechanisms underlying this process are not understood. Homophilic interaction between platelet/endothelial cell adhesion molecule (PECAM) on leukocytes and PECAM at the endothelial border triggers targeted recycling of membrane from a reticulum localized close to the endothelial cell lateral border. This membrane surrounds the transmigrating leukocyte (Mamdouh, Z., X. Chen, L.M. Pierini, F.R. Maxfield, and W.A. Muller. 2003. Nature. 421:748–753). How this process occurs and whether it is required for diapedesis independent of PECAM are not known. We now report that targeted recycling from this lateral border recycling compartment (LBRC) is required for diapedesis, is mediated by kinesin family molecular motors, and requires normally functioning endothelial microtubules. Selective disruption of microtubules or inhibition of kinesin motor domain blocked targeted recycling and diapedesis of monocytes. Furthermore, targeted recycling of membrane from the LBRC was required for transmigration of lymphocytes, which migrate independently of PECAM. Thus, trafficking of membrane from the LBRC to surround leukocytes may be a general requirement for migration of leukocytes across the endothelial cell border. Furthermore, these data provide the first demonstration of a role for endothelial microtubules and kinesins in promoting diapedesis, and a mechanism to explain targeted recycling.

Kinesin Motor Domain ofLeishmania donovanias a Future Vaccine Candidate

Visceral leishmaniasis (VL) is one of the important parasitic diseases, with approximately 350 million people at risk. Due to the nonavailability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. The present study was carried out to examine the immunological potential of kinesin protein from the microtubule locus of Leishmania donovani as a suitable vaccine candidate. In silico analysis of this region revealed clusters of major histocompatibility complex class I and II binding epitopes in its motor domain region. A recombinant protein was expressed from this region and named rLvacc. The antigenicity and immunogenicity studies of this protein by Western blot analysis revealed that rLvacc is strongly recognized by sera from acute VL patients. To evaluate its immunogenicity, peripheral blood mononuclear cells from cured VL patients were separated, and a lymphocyte proliferation assay was carried out in the presence of rLvacc. After lymphocyte proliferation, the pooled culture supernatant was assayed for anti-rLvacc antibody titers using an enzyme-linked immunosorbent assay. The results showed that immunoglobulin G2 (IgG2) subtype antibodies were predominant, while IgG1 subtype antibodies were produced in very low titers. On the basis of these ex vivo preliminary findings, its immunogenicity was studied in BALB/c mice. Vaccination with the DNA construct generated a good cellular immune response with significant increases in gamma interferon and interleukin-2 (IL-2) cytokine levels (Th1), but no increase in IL-4 levels (Th2). Taken together, our findings suggest the kinesin motor domain region of L. donovani as a potential vaccine candidate against visceral leishmaniasis.