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)

The ANGUSTIFOLIA (AN) gene is required for leaf hair (trichome) branching and is also involved in polarized expansion underlying organ shape. Here we show that the AN gene encodes a C-terminal binding proteins/brefeldin A ADP-ribosylated substrates (CtBP/BARS) related protein. AN is expressed at low levels in all organs and the AN protein is localized in the cytoplasm. In an mutant trichomes, the organization of the actin cytoskeleton is normal but the distribution of microtubules is aberrant. A role of AN in the control of the microtubule cytoskeleton is further supported by the finding that AN genetically and physically interacts with ZWICHEL, a kinesin motor molecule involved in trichome branching. Our data suggest that CtBP/BARS-like protein function in plants is directly associated with the microtubule cytoskeleton.
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PMID:The cell morphogenesis gene ANGUSTIFOLIA encodes a CtBP/BARS-like protein and is involved in the control of the microtubule cytoskeleton. 1188 34

Kinesin binds to microtubules with half-site ADP release to form a tethered intermediate with one attached head without nucleotide and one tethered head that retains its bound ADP. For DKH405 containing amino acid residues 1-405 of Drosophila kinesin, release of the remaining ADP from the tethered head is slow (0.05 s(-1)), but release is accelerated by added ADP or ATP. The maximum rate of ADP-stimulated dissociation of tethered DKH405 from the microtubule is approximately 12 s(-1) as determined by turbidity. Parallel measurements of ADP-stimulated release of 2'(3')-O-(N-methylanthraniloyl)-ADP (mantADP) from the tethered intermediate by fluorescence indicate that the reaction is biphasic with a fast phase that occurs at a rate that is similar to dissociation. The rate of the slow phase is dependent on the concentrations of salt and microtubules and is equal in each case to the rate for bimolecular stimulation of ADP release by microtubules as measured independently. These results are consistent with a scheme in which the fast phase, with approximately one-third of the total amplitude change, is due to ADP-stimulated release of mantADP from the tethered intermediate at approximately 6 s(-1). This direct release of mantADP continues until terminated by dissociation of DKH405 from the microtubule at approximately 12 s(-1). The majority of the amplitude change thus occurs through bimolecular recombination of DKH405.mantADP with microtubules following initial dissociation. Analysis of a simple scheme indicates that hydrolysis of ATP at the attached head before the tethered head can release its ADP and become tightly bound may be the principal limitation to processivity.
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PMID:Pathway of ADP-stimulated ADP release and dissociation of tethered kinesin from microtubules. Implications for the extent of processivity. 1191 91

Kinesin is a motor protein that transports organelles along a microtubule toward its plus end by using the energy of ATP hydrolysis. To clarify the nucleotide-dependent binding mode, we measured the unbinding force for one-headed kinesin heterodimers in addition to conventional two-headed kinesin homodimers under several nucleotide states. We found that both a weak and a strong binding state exist in each head of kinesin corresponding to a small and a large unbinding force, respectively; that is, weak for the ADP state and strong for the nucleotide-free and adenosine 5'-[beta,gamma-imido]triphosphate states. Model analysis showed that (i) the two binding modes in each head could be explained by a difference in the binding energy and (ii) the directional instability of binding, i.e., dependence of unbinding force on loading direction, could be explained by a difference in the characteristic distance for the kinesin-microtubule interaction during plus- and minus-end-directed loading. Both these factors must play an important role in the molecular mechanism of kinesin motility.
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PMID:Kinesin-microtubule binding depends on both nucleotide state and loading direction. 1195 22

Monastrol, a cell-permeable inhibitor of the kinesin Eg5, has been used to probe the dynamic organization of the mitotic spindle. The mechanism by which monastrol inhibits Eg5 function is unknown. We found that monastrol inhibits both the basal and the microtubule-stimulated ATPase activity of the Eg5 motor domain. Unlike many ATPase inhibitors, monastrol does not compete with ATP binding to Eg5. Monastrol appears to inhibit microtubule-stimulated ADP release from Eg5 but does not compete with microtubule binding, suggesting that monastrol binds a novel allosteric site in the motor domain. Finally, we established that (S)-monastrol, as compared to the (R)-enantiomer, is a more potent inhibitor of Eg5 activity in vitro and in vivo. Future structural studies should help in designing more potent Eg5 inhibitors for possible use as anticancer drugs and cell biological reagents.
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PMID:Evidence that monastrol is an allosteric inhibitor of the mitotic kinesin Eg5. 1232 73

Recent crystallographic studies of motor proteins showed that the structure of the motor domains of myosin and kinesin are highly conserved. Thus, these motor proteins, which are important for motility, may share a common mechanism for generating energy from ATP hydrolysis. We have previously demonstrated that, in the presence of ADP, myosin forms stable ternary complexes with new phosphate analogues of aluminum fluoride (AlF(4)(-)) and beryllium fluoride (BeF(n)), and these stable complexes mimic the transient state along the ATPase kinetic pathway [Maruta et al. (1993) J. Biol. Chem. 268, 7093-7100]. In this study, we examined the formation of kinesin.ADP.fluorometals ternary complexes and analyzed their characteristics using the fluorescent ATP analogue NBD-ATP (2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl]-ADP). Our results suggest that these ternary complexes may mimic transient state intermediates in the kinesin ATPase cycle. Thus, the kinesin.ADP.AlF(4)(-) complex resembles the kinesin.ADP state, and the kinesin.ADP.BeF(n) complex mimics the kinesin.ADP.P(i) state.
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PMID:Formation and characterization of kinesin.ADP.fluorometal complexes. 1235 72

The microtubule-dependent kinesin-like protein Eg5 from Homo sapiens is involved in the assembly of the mitotic spindle. It shows a three-domain structure with an N-terminal motor domain, a central coiled coil, and a C-terminal tail domain. In vivo HsEg5 is reversibly inhibited by monastrol, a small cell-permeable molecule that causes cells to be arrested in mitosis. Both monomeric and dimeric Eg5 constructs have been examined in order to define the minimal monastrol binding domain on HsEg5. NMR relaxation experiments show that monastrol interacts with all of the Eg5 constructs used in this study. Enzymatic techniques indicate that monastrol partially inhibits Eg5 ATPase activity by binding directly to the motor domain. The binding is noncompetitive with respect to microtubules, indicating that monastrol does not interfere with the formation of the motor-MT complex. The binding is not competitive with respect to ATP. Both enzymology and in vivo assays show that the S enantiomer of monastrol is more active than the R enantiomer and racemic monastrol. Stopped-flow fluorometry indicates that monastrol inhibits ADP release by forming an Eg5-ADP-monastrol ternary complex. Monastrol reversibly inhibits the motility of human Eg5. Monastrol has no inhibitory effect on the following members of the kinesin superfamily: MC5 (Drosophila melanogaster Ncd), HK379 (H. sapiens conventional kinesin), DKH392 (D. melanogaster conventional kinesin), BimC1-428 (Aspergillus nidulans BimC), Klp15 (Caenorhabditis elegans C-terminal motor), or Nkin460GST (Neurospora crassa conventional kinesin).
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PMID:Interaction of the mitotic inhibitor monastrol with human kinesin Eg5. 1252 61

The neck domain of fungal conventional kinesins displays characteristic properties which are reflected in a specific sequence pattern. The exchange of the strictly conserved Tyr 362, not present in animals, into Lys, Cys or Phe leads to a failure to dimerize. The destabilizing effect is confirmed by a lower coiled-coil propensity of mutant peptides. Whereas the Phe substitution has only a structural effect, the Lys and Cys replacements lead to dramatic kinetic changes. The steady state ATPase is 4- to 7-fold accelerated, which may be due to a faster microtubule-stimulated ADP release rate. These data suggest that an inhibitory effect of the fungal neck domain on the motor core is mediated by direct interaction of the aromatic ring of Tyr 362 with the head, whereas the OH group is essential for dimerization. This is the first demonstration of a direct influence of the kinesin neck region in regulation of the catalytic activity.
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PMID:A conserved tyrosine in the neck of a fungal kinesin regulates the catalytic motor core. 1255 46

Kinesin motors move on microtubules by a mechanism that involves a large, ATP-triggered conformational change in which a mechanical element called the neck linker docks onto the catalytic core, making contacts with the core throughout its length. Here, we investigate the thermodynamic properties of this conformational change using electron paramagnetic resonance (EPR) spectroscopy. We placed spin probes at several locations on the human kinesin neck linker and recorded EPR spectra in the presence of microtubules and either 5'-adenylylimidodiphosphate (AMPPNP) or ADP at temperatures of 4-30 degrees C. The free-energy change (DeltaG) associated with AMPPNP-induced docking of the neck linker onto the catalytic core is favorable but small, about 3 kJ/mol. In contrast, the favorable enthalpy change (DeltaH) and unfavorable entropy change (TDeltaS) are quite large, about 50 kJ/mol. A mutation in the neck linker, V331A/N332A, results in an unfavorable DeltaG for AMPPNP-induced zipping of the neck linker onto the core and causes motility defects. These results suggest that the kinesin neck linker folds onto the core from a more unstructured state, thereby paying a large entropic cost and gaining a large amount of enthalpy.
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PMID:Thermodynamic properties of the kinesin neck-region docking to the catalytic core. 1260 86

Kinesin is an ATP-driven molecular motor that moves processively along a microtubule. Processivity has been explained as a mechanism that involves alternating single- and double-headed binding of kinesin to microtubules coupled to the ATPase cycle of the motor. The internal load imposed between the two bound heads has been proposed to be a key factor regulating the ATPase cycle in each head. Here we show that external load imposed along the direction of motility on a single kinesin molecule enhances the binding affinity of ADP for kinesin, whereas an external load imposed against the direction of motility decreases it. This coupling between loading direction and enzymatic activity is in accord with the idea that the internal load plays a key role in the unidirectional and cooperative movement of processive motors.
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PMID:Loading direction regulates the affinity of ADP for kinesin. 1264 Apr 44

We have used site-directed spin-labeling and electron paramagnetic resonance spectroscopy to monitor a conformational change at the nucleotide site of kinesin. Cys-lite kinesin (K349 monomer) with the mutation S188C was spin labeled with MSL or MTSL. This residue is at the junction between the switch 1 region (which is a structure known to be sensitive to bound nucleotide in the G-proteins) and the alpha3-helix, adjacent to the nucleotide site. The spectra showed two or more components of mobility, which were independent of nucleotide in the absence of microtubules (MTs). The spectra of both labels showed a change of mobility upon binding to MTs. A more mobile spectral component became enhanced for all triphosphate analogs examined, AMPPNP, ADP.AlFx, or ADP.BeFx, in the presence of MTs, although the magnitude of the new component and the degree of mobility varied with nucleotide analog. The ADP state showed a much-reduced spectral change with a small shift to the more immobilized component in the presence of MTs. For kinesin.ADP.MT, a van't Hoff plot gave DeltaH degrees = -96 kJ/mol implying that the conformational change was extensive. We conclude there is a conformational change in the switch 1-alpha3-helix domain when kinesin binds to MTs.
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PMID:EPR spectroscopy shows a microtubule-dependent conformational change in the kinesin switch 1 domain. 1271 48


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