Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.4.4 (kinesin)
5,033 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Switch I and II are key active site structural elements of kinesins, myosins, and G-proteins. Our analysis of a switch I mutant (R210A) in Drosophila melanogaster kinesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule. To investigate the conserved arginine in switch I further, a lysine substitution mutant was generated. The R210K dimeric motor has lost the ability to hydrolyze ATP; however, it has rescued microtubule function. Our results show that R210K has restored microtubule association kinetics, microtubule affinity, ADP release kinetics, and motor domain cooperativity. Moreover, the active site at head 1 is able to distinguish ATP, ADP, and AMP-PNP to signal head 2 to bind the microtubule and release mantADP with kinetics comparable with wild-type. Therefore, the structural pathway of communication from head 1 to head 2 is restored, and head 2 can respond to this signal by binding the microtubule and releasing mantADP. Structural modeling revealed that lysine could retain some of the hydrogen bonds made by arginine but not all, suggesting a structural hypothesis for the ability of lysine to rescue microtubule function in the Arg210 mutant.
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PMID:A kinesin switch I arginine to lysine mutation rescues microtubule function. 1286 Sep 92

In order to investigate the microtubule-associated intracellular trafficking of the NH2-terminal cellular prion protein (PrPC) fragment [Biochem. Biophys. Res. Commun. 313 (2004) 818], we performed a real-time imaging of fluorescent PrPC (GFP-PrPC) in living cells. Such GFP-PrPC exhibited an anterograde movement towards the direction of plasma membranes at a speed of 140-180 nm/s, and a retrograde movement inwardly at a speed of 1.0-1.2 microm/s. The anterograde and retrograde movements of GFP-PrPC were blocked by a kinesin family inhibitor (AMP-PNP) and a dynein family inhibitor (vanadate), respectively. Furthermore, anti-kinesin antibody (alpha-kinesin) blocked its anterograde motility, whereas anti-dynein antibody (alpha-dynein) blocked its retrograde motility. These data suggested the kinesin family-driven anterograde and the dynein-driven retrograde movements of GFP-PrPC. Mapping of the interacting domains of PrPC identified amino acid residues indispensable for interactions with kinesin family: NH2-terminal mouse (Mo) residues 53-91 and dynein: NH2-terminal Mo residues 23-33, respectively. Our findings argue that the discrete N-terminal amino acid residues are indispensable for the anterograde and retrograde intracellular movements of PrPC.
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PMID:Anterograde and retrograde intracellular trafficking of fluorescent cellular prion protein. 1498 83

We have studied the structure of microtubules decorated with kinesin motor domains in different nucleotide states by 3D electron microscopy. Having docked the atomic coordinates of both dimeric ADP.kinesin and tubulin heterodimer into a map of kinesin dimers bound to microtubules in the presence of ADP, we try to predict which regions of the proteins interact in the weakly binding state. When either the presence of 5'-adenylyimidodiphosphate (AMP-PNP) or an absence of nucleotides puts motor domains into a strongly-bound state, the 3D maps show changes in the motor domains which modify their interaction with beta-tubulin. The maps also show differences in beta-tubulin conformation compared with undecorated microtubules or those decorated with weakly-bound motors. Strongly-bound ncd appears to produce an identical change.
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PMID:3D electron microscopy of the interaction of kinesin with tubulin. 1521 83

The motor protein kinesin moves along microtubules, driven by adenosine triphosphate (ATP) hydrolysis. However, it remains unclear how kinesin converts the chemical energy into mechanical movement. We report crystal structures of monomeric kinesin KIF1A with three transition-state analogs: adenylyl imidodiphosphate (AMP-PNP), adenosine diphosphate (ADP)-vanadate, and ADP-AlFx (aluminofluoride complexes). These structures, together with known structures of the ADP-bound state and the adenylyl-(beta,gamma-methylene) diphosphate (AMP-PCP)-bound state, show that kinesin uses two microtubule-binding loops in an alternating manner to change its interaction with microtubules during the ATP hydrolysis cycle; loop L11 is extended in the AMP-PNP structure, whereas loop L12 is extended in the ADP structure. ADP-vanadate displays an intermediate structure in which a conformational change in two switch regions causes both loops to be raised from the microtubule, thus actively detaching kinesin.
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PMID:KIF1A alternately uses two loops to bind microtubules. 1528 75

Interaction of kinesin-coated latex beads with a single microtubule (MT) was directly observed by fluorescence microscopy. In the presence of ATP, binding of a kinesin bead to the MT facilitated the subsequent binding of other kinesin beads to an adjacent region on the MT that extended for micrometers in length. This cooperative binding was not observed in the presence of ADP or 5'-adenylylimidodiphosphate (AMP-PNP), where binding along the MT was random. Cooperative binding also was induced by an engineered, heterodimeric kinesin, WT/E236A, that could hydrolyze ATP, yet remained fixed on the MT in the presence of ATP. Relative to the stationary WT/E236A kinesin on a MT, wild-type kinesin bound preferentially in close proximity, but was biased to the plus-end direction. These results suggest that kinesin binding and ATP hydrolysis may cause a long-range state transition in the MT, increasing its affinity for kinesin toward its plus end. Thus, our study highlights the active involvement of MTs in kinesin motility.
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PMID:Long-range cooperative binding of kinesin to a microtubule in the presence of ATP. 1573 63

KIF1A, a kinesin-related motor protein that transports pre-synaptic vesicles in neurons, was originally presumed to translocate along microtubules (MT) as a monomer. Protein structure predictions from its amino acid sequence failed to identify the long coiled-coil domains typical of kinesins, which led researchers to believe it does not oligomerize into the canonical kinesin dimer. However, mounting evidence using recombinant chimeric protein indicates that KIF1A, like conventional kinesin, requires dimerization for fast, unidirectional processive movement along MTs. Because these studies are somewhat indirect, we wished to test the oligomerization state of native KIF1A, and to compare that to full-length recombinant protein. We have performed hydrodynamic analyses to determine the molecular weights of the respective complexes. Our results indicate that most native KIF1A is soluble and indeed monomeric, but recombinant KIF1A is a dimer. MT-binding studies also showed that native KIF1A did not bind to MTs in either the presence of AMP-PNP, apyrase, or adenosine triphosphate (ATP), but recombinant KIF1A bound to MTs most stably in the presence of ATP, indicating very different motor functional states. To further characterize KIF1A's dimerization potential, we prepared peptides corresponding to the neck domains of MmKIF1A and CeUnc104, and by circular dichroism spectroscopy compared these peptides for their ability to form coiled-coils. Interestingly, both MmKIF1A and CeUnc104 neck peptides formed homodimeric coiled-coils, with the MmKIF1A neck coiled-coil exhibiting the greater stability. Collectively, from our data and from previous studies, we predict that native KIF1A can exist as both an inactive monomer and an active homodimer formed in part through its neck coiled-coil domain.
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PMID:Monomeric and dimeric states exhibited by the kinesin-related motor protein KIF1A. 1588 13

The kinesin-binding site on the microtubule has not been identified because of the technical difficulties involved in the mutant analyses of tubulin. Exploiting the budding yeast expression system, we succeeded in replacing the negatively charged residues in the alpha-helix 12 of beta-tubulin with alanine and analyzed their effect on kinesin-microtubule interaction in vitro. The microtubule gliding assay showed that the affinity of the microtubules for kinesin was significantly reduced in E410A, D417A, and E421A, but not in E412A mutant. The unbinding force measurement revealed that in the former three mutants, the kinesin-microtubule interaction in the adenosine 5'-[beta,gamma-imido]triphosphate state (AMP-PNP state) became less stable when a load was imposed towards the microtubule minus end. In parallel with this decreased stability, the stall force of kinesin was reduced. Our results implicate residues E410, D417, and E421 as crucial for the kinesin-microtubule interaction in the strong binding state, thereby governing the size of kinesin stall force.
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PMID:Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin. 1712 95

Kinesin-1 is a two-headed molecular motor that walks along microtubules, with each step gated by adenosine triphosphate (ATP) binding. Existing models for the gating mechanism propose a role for the microtubule lattice. We show that unpolymerized tubulin binds to kinesin-1, causing tubulin-activated release of adenosine diphosphate (ADP). With no added nucleotide, each kinesin-1 dimer binds one tubulin heterodimer. In adenylyl-imidodiphosphate (AMP-PNP), a nonhydrolyzable ATP analog, each kinesin-1 dimer binds two tubulin heterodimers. The data reveal an ATP gate that operates independently of the microtubule lattice, by ATP-dependent release of a steric or allosteric block on the tubulin binding site of the tethered kinesin-ADP head.
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PMID:An ATP gate controls tubulin binding by the tethered head of kinesin-1. 1741 43

To design safe, efficient synthetic gene therapy vectors, it is desirable to understand the intracellular mechanisms that facilitate their delivery from the cell surface to the nucleus. Elements of the cytoskeleton and molecular motor proteins are known to play a pivotal role in most intracellular active transport processes. The actin depolymerizer cytochalasin D and microtubule effectors colchicine and paclitaxel were used to evaluate the function of these components of the cytoskeleton in the trafficking of polyethylenimine (PEI)-DNA complexes. In addition, ATPase inhibitors erythro-9[3-(2-hydroxynonyl)] adenine (EHNA), vanadate, adenylylimidodiphosphate (AMP-PNP), and rose bengal lactone (RBL), which have inhibitory activity against dynein and kinesin, were used to examine to the effects of these molecular motors on PEI-DNA delivery. Disruption of microfilaments decreased the delivery efficiency of PEI polyplexes 60-80%, though cytochalasin D did not significantly inhibit uptake. Depolymerization of microtubules by colchicine decreased transfection efficiency by 75%. Microtubule stabilization with paclitaxel, however, facilitated a 20-fold increase in gene expression. Treatment with EHNA and vanadate caused 50% and 80% decreases in transfection efficiency, respectively. Transfection efficiency was also decreased by RBL (80%) and AMP-PNP (98%). Our findings confirm the importance of microfilament- and microtubule-based active transport of PEI-DNA complexes. Further, the strong decrease in transfection efficiency caused by ATPase inhibitors that possess inhibitory activity against kinesin implies an unexpected role for these motors in gene delivery.
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PMID:Biochemical investigation of active intracellular transport of polymeric gene-delivery vectors. 1771 50

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, M396-734 corresponding to amino acids Q396-N734 was fused with 6xHis-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of M396-734 is not activated by Ca2+, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 microM, much higher than that of other kinesins reported. The cosedimentation assay indicated that M396-734 could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.
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PMID:Cotton GhKCH2, a plant-specific kinesin, is low-affinitive and nucleotide-independent as binding to microtubule. 1792 6


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