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)

AMPPNP was found to be hydrolyzed by the motor domain of ncd (the product of a Drosophila gene, non-claret disjunctional), a kinesin-related protein. This hydrolysis could be monitored by 31P NMR spectroscopy and by an assay of phosphate, one of the products of the hydrolysis. The rate was approximately 0.00004 s(-1), 1% of the ATP turnover rate. The AMPPNP turnover was not stimulated by microtubules. Kinesin motor domain also turned over AMPPNP but at a somewhat lower rate. Although the turnover was slow, the present finding may present an important caveat, since AMPPNP has been widely used for investigations of kinesin and kinesin-related proteins as a non-hydrolyzable ATP analogue.
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PMID:Hydrolysis of AMPPNP by the motor domain of ncd, a kinesin-related protein. 919 97

The motor domains of kinesin and ncd contain ADP bound tightly at the enzymatic active sites even after purification. The microenvironments surrounding the phosphates of the tightly bound ADP molecules in these motor domains were studied using 31P-NMR. The alpha-phosphate and beta-phosphate of the tightly bound ADP molecules gave 31P-NMR signals at positions shifted to higher and lower field, respectively, from those of free MgADP in solution. On the other hand, the peak areas of the signals of the alpha-phosphates and beta-phosphates of bound ADP were much smaller than those of free MgADP. The temperature dependence of these signals was investigated to reveal that each signal of bound MgADP exhibited a large temperature dependence in the peak area from 0 to 20 degrees C with both proteins. This result implied that these motor domains would take multiple conformations in solution; one of these conformations gives the signals of bound MgADP observed by 31P-NMR as above, while another makes the signals of MgADP very broad and, hence, invisible in the 31P-NMR spectra. A possible reason for invisible signals would be strong chemical shift anisotropy effects of the phosphorus nuclei. In addition, we found a difference between kinesin and ncd motor domains in the chemical shift of the alpha-phosphate of bound MgADP, indicating that the electrostatic or magnetic microenvironments of this site of these motor domains differed from each other.
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PMID:Evidence for existence of multiple conformations of kinesin and ncd motor domains in solution revealed by 31P-NMR of the tightly bound ADP. 982 94

The conformation of a synthetic peptide, consisting of 30 amino acids spanning the neck and hinge regions of rat brain kinesin, was investigated by NMR spectroscopy. The peptide extends from K357 to D386 and has the sequence KSVIQHLEVELNRWRNGEAVPEDEQISAKD. A total of 82 distance range constraints and 23 dihedral angle constraints could be obtained from NOESY and E.COSY spectra, respectively. These were used to calculate 500 structures by applying the REDAC algorithm of the software package DYANA. The first half of the peptide matched the helical structure of the neck determined from an X-ray crystal structure of kinesin. This part normally dimerizes into a coiled-coil by virtue of a leucine zipper interaction, but it is alpha-helical even in the monomeric state. The second half (not visible in the X-ray structure because of disorder) contains locally defined structure elements (extended chain, helical loop) connected by flexible joints. This is consistent with the "hinge" function postulated for this domain which is important for kinesin's motility and orientation.
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PMID:Conformational preferences of a synthetic 30mer peptide from the interface between the neck and stalk regions of kinesin. 1102 35

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

Kinesin spindle protein (KSP/Eg5) is essential for the formation and maintenance of bipolar spindles during mitosis. Inhibition of this protein leads to cell cycle arrest and apoptosis without interfering other microtubule-dependent processes. Therefore, it is a potential target in cancer therapy. Here, a series of tetrahydro-beta-carboline derivatives 5a - k were synthesized as kinesin spindle protein inhibitor. Their structures were confirmed with 1H NMR, ESI-MS and elemental analysis. The synthesized compounds were evaluated for their inhibition of KSP.
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PMID:[Synthesis and biological evaluation of tetrahydro-beta-carline derivatives]. 1895 75

Calcium (Ca(2+)) is a key second messenger in eukaryotes where it regulates a diverse array of cellular processes in response to external stimuli. An important Ca(2+) sensor in both animals and plants is calmodulin (CaM). In addition to evolutionarily conserved CaM, plants possess a unique family of CaM-like (CML) proteins. The majority of these CMLs have not yet been studied, and investigation into their physical properties and cellular functions will provide insight into Ca(2+) signal transduction in plants. Here we describe the characterization of CML42, a 191-amino acid Ca(2+)-binding protein from Arabidopsis. Ca(2+) binding to recombinant CML42 was assessed by fluorescence spectroscopy, NMR spectroscopy, microcalorimetry, and CD spectroscopy. CML42 displays significant alpha-helical secondary structure, binds three molecules of Ca(2+) with affinities ranging from 30 to 430 nm, and undergoes a Ca(2+)-induced conformational change that results in the exposure of one or more hydrophobic regions. Gene expression analysis revealed CML42 transcripts at various stages of development and in many cell types, including the support cells, which surround trichomes (leaf hairs) on the leaf surface. Using yeast two-hybrid screening we identified a putative CML42 interactor; kinesin-interacting Ca(2+)-binding protein (KIC). Because KIC is a protein known to function in trichome development, we examined transgenic CML42 knockout plants and found that they possess aberrant trichomes with increased branching. Collectively, our data support a role for CML42 as a Ca(2+) sensor that functions during cell branching in trichomes.
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PMID:The calmodulin-related calcium sensor CML42 plays a role in trichome branching. 1972 Aug 24

Kinesin motor proteins transport a wide variety of molecular cargoes in a spatially and temporally regulated manner. Kinesin motor domains, which hydrolyze ATP to produce a directed mechanical force along a microtubule, are well conserved throughout the entire superfamily. Outside of the motor domains, kinesin sequences diverge along with their transport functions. The nonmotor regions, particularly the tails, respond to a wide variety of structural and molecular cues that enable kinesins to carry specific cargoes in response to particular cellular signals. Here, we demonstrate that intrinsic disorder is a common structural feature of kinesins. A bioinformatics survey of the full-length sequences of all 43 human kinesins predicts that significant regions of intrinsically disordered residues are present in all kinesins. These regions are concentrated in the nonmotor domains, particularly in the tails and near sites for ligand binding or post-translational modifications. In order to experimentally verify these predictions, we expressed and purified the tail domains of kinesins representing three different families (Kif5B, Kif10, and KifC3). Circular dichroism and NMR spectroscopy experiments demonstrate that the isolated tails are disordered in vitro, yet they retain their functional microtubule-binding activity. On the basis of these results, we propose that intrinsic disorder is a common structural feature that confers functional specificity to kinesins.
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PMID:Kinesin tail domains are intrinsically disordered. 2267 72

Fashioned through billions of years of evolution, biological molecular machines, such as ATP synthase, myosin, and kinesin, use the intricate relative motions of their components to drive some of life's most essential processes. Having control over the motions in molecules is imperative for life to function, and many chemists have designed, synthesized, and investigated artificial molecular systems that also express controllable motions within molecules. Using bistable mechanically interlocked molecules (MIMs), based on donor-acceptor recognition motifs, we have sought to imitate the sophisticated nanoscale machines present in living systems. In this Account, we analyze the thermodynamic characteristics of a series of redox-switchable [2]rotaxanes and [2]catenanes. Control and understanding of the relative intramolecular movements of components in MIMs have been vital in the development of a variety of applications of these compounds ranging from molecular electronic devices to drug delivery systems. These bistable donor-acceptor MIMs undergo redox-activated switching between two isomeric states. Under ambient conditions, the dominant translational isomer, the ground-state coconformation (GSCC), is in equilibrium with the less favored translational isomer, the metastable-state coconformation (MSCC). By manipulating the redox state of the recognition site associated with the GSCC, we can stimulate the relative movements of the components in these bistable MIMs. The thermodynamic parameters of model host-guest complexes provide a good starting point to rationalize the ratio of GSCC to MSCC at equilibrium. The bistable [2]rotaxanes show a strong correlation between the relative free energies of model complexes and the ground-state distribution constants (K(GS)). This relationship does not always hold for bistable [2]catenanes, most likely because of the additional steric and electronic constraints present when the two rings are mechanically interlocked with each other. Measuring the ground-state distribution constants of bistable MIMs presents its own set of challenges. While it is possible, in principle, to determine these constants using NMR and UV-vis spectroscopies, these methods lack the sensitivity to permit the determination of ratios of translational isomers greater than 10:1 with sufficient accuracy and precision. A simple application of the Nernst equation, in combination with variable scan-rate cyclic voltammetry, however, allows the direct measurement of ground-state distribution constants across a wide range (K(GS) = 10-10(4)) of values.
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PMID:Ground-state thermodynamics of bistable redox-active donor-acceptor mechanically interlocked molecules. 2274 9

CAP-Gly domain of dynactin, a microtubule-associated activator of dynein motor, participates in multiple cellular processes, and its point mutations are associated with neurodegenerative diseases. Recently, we have demonstrated that conformational plasticity is an intrinsic property of CAP-Gly. To understand its origin, we addressed internal dynamics of CAP-Gly assembled on polymeric microtubules, bound to end-binding protein EB1 and free, by magic angle spinning NMR and molecular dynamics simulations. The analysis of residue-specific dynamics of CAP-Gly on time scales spanning nano- through milliseconds reveals its unusually high mobility, both free and assembled on polymeric microtubules. On the contrary, CAP-Gly bound to EB1 is significantly more rigid. Molecular dynamics simulations indicate that these motions are strongly temperature-dependent, and loop regions are surprisingly mobile. These findings establish the connection between conformational plasticity and internal dynamics in CAP-Gly, which is essential for the biological functions of CAP-Gly and its ability to bind to polymeric microtubules and multiple binding partners. In this work, we establish an approach, for the first time, to probe atomic resolution dynamic profiles of a microtubule-associated protein assembled on polymeric microtubules. More broadly, the methodology established here can be applied for atomic resolution analysis of dynamics in other microtubule-associated protein assemblies, including but not limited to dynactin, dynein, and kinesin motors assembled on microtubules.
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PMID:Internal dynamics of dynactin CAP-Gly is regulated by microtubules and plus end tracking protein EB1. 2545 37

Mitotic kinesin Eg5 is an attractive anticancer drug target. Discovery of Eg5 inhibitors has been focused on targeting the 'monastrol-binding site'. However, acquired drug resistance has been reported for such inhibitors. Therefore, identifying new Eg5 inhibitors which function through a different mechanism(s) could complement current drug candidates and improve drug efficacy. In this study, we explored a novel allosteric site of Eg5 and identified new Eg5 inhibitors through structure-based virtual screening. Experiments with the saturation-transfer difference NMR demonstrated that the identified Eg5 inhibitor SRI35566 binds directly to Eg5 without involving microtubules. Moreover, SRI35566 and its two analogs significantly induced monopolar spindle formation in colorectal cancer HCT116 cells and suppressed cancer cell viability and colony formation. Together, our findings reveal a new allosteric regulation mechanism of Eg5 and a novel drug targeting site for cancer therapy.
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PMID:Discovery of Novel Allosteric Eg5 Inhibitors Through Structure-Based Virtual Screening. 2686 17


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