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

To understand the mechanism of kinesin movement we have investigated the relative configuration of the two kinesin motor domains during ATP hydrolysis using fluorescence polarization microscopy of ensemble and single molecules. We found that: (i) in nucleotide states that induce strong microtubule binding, both motor domains are bound to the microtubule with similar orientations; (ii) this orientation is maintained during processive motion in the presence of ATP; (iii) the neck-linker region of the motor domain has distinct configurations for each nucleotide condition tested. Our results fit well with a hand-over-hand type movement mechanism and suggest how the ATPase cycle in the two motor domains is coordinated. We propose that the motor neck-linker domain configuration controls ADP release.
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PMID:Configuration of the two kinesin motor domains during ATP hydrolysis. 1297 Jul 55

Molecular motors undergo conformational changes to produce force and move along cytoskeletal filaments. Structural changes have been detected in kinesin motors; however, further changes are expected because previous crystal structures are in the same or closely related conformations. We report here a 2.5 A crystal structure of the minus-end kinesin, Ncd, with the coiled-coil stalk/neck and one head rotated by approximately 75 degrees relative to the other head. The two heads are asymmetrically positioned with respect to the stalk and show asymmetry of nucleotide state: one head is fully occupied, but the other is unstably bound to ADP. Unlike previous structures, our new atomic model can be fit into cryoelectron microscopy density maps of the motor attached to microtubules, where it appears to resemble a one-head-bound motor with the stalk rotated towards the minus end. Interactions between neck and motor core residues, observed in the head that moves with the stalk, are disrupted in the other head, permitting rotation of the stalk/neck. The rotation could represent a force-producing stroke that directs the motor to the minus end.
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PMID:Rotation of the stalk/neck and one head in a new crystal structure of the kinesin motor protein, Ncd. 1453 11

A series of modifications of the junction of the neck linker and neck coil of dimeric Drosophila kinesin were constructed to determine the influence of head orientation and spacing on the ATPase kinetics. Ala(345) is the first residue in the coiled-coil of the neck, and its replacement with glycine or proline produces no significant change in the k(cat) or K(0.5(MT)) values for activation of their ATPase by microtubules (MTs) or in their k(bi(ratio)) value for the average number of ATP molecules hydrolyzed during a processive encounter with a MT. Addition or deletion of a single amino acid at the junction produces only modest changes with less than a 2-fold reduction in kinetic processivity. Insertion of a spacer of 6 or 12 additional amino acids at the neck linker junction increases the K(0.5(MT)) value by 3-4-fold with a corresponding decrease in kinetic processivity. The sliding velocities of all the mutant constructs under multimotor conditions are within 30% of the wild-type value. All the constructs with single residue changes exhibit half-site ADP release on binding to MTs. The constructs with long insertion, however, rapidly release both ADP molecules per dimer on binding to a MT, indicating that the steric constraints that prevent release of ADP from the tethered head of wild-type kinesin have been relieved by the long insertions. The constructs with long inserts have decreased kinetic processivity and dissociate from the MT during ATP hydrolysis 3-fold faster than wild-type.
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PMID:Modulation of kinesin half-site ADP release and kinetic processivity by a spacer between the head groups. 1455 32

Competing models for the coordination of processive stepping in kinesin can be tested by introducing a roadblock to prevent lead head attachment. We used T93N, an irreversibly binding mutant monomer, as a roadblock, and measured the rates of nucleotide-induced detachment of kinesin monomers or dimers with and without the T93N roadblock using microflash photolysis combined with stopped flow. Control nucleotide-induced monomer (rK340) unbinding was 73.6 s(-1) for ATP and 40.5 s(-1) for ADP. Control ADP-induced dimer (rK430) unbinding was 18.6 s(-1). Added 20 mM Pi slowed both monomer and dimer unbinding. With the roadblock in place, lead head attachment of dimers is prevented and ATP-induced trail head unbinding was then 42 s(-1). This is less than two-fold slower than the stepping rate of unimpeded rK430 dimers (50-70 s(-1)), indicating that during walking, lead head attachment induces at most only a slight (less than two-fold) acceleration of trail head detachment. As we discuss, this implies a coordination model having very fast (>2000 s(-1)) ATP-induced attachment of the lead head, followed by slower, strain-sensitive ADP release from the lead head.
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PMID:What kinesin does at roadblocks: the coordination mechanism for molecular walking. 1468 58

The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to a spatially periodic mechanical cycle (movement along a microtubule). To distinguish between different models of such chemical-to-mechanical coupling, we measured the speed of movement of conventional kinesin along microtubules in in vitro motility assays over a wide range of substrate (ATP) and product (ADP and inorganic phosphate) concentrations. In the presence and absence of products, the dependence of speed on [ATP] was well described by the Michaelis-Menten equation. In the absence of products, the K(M) (the [ATP] required for half-maximal speed) was 28 +/- 1 microM, and the maximum speed was 904 nm/s. P(i) behaved as a competitive inhibitor with K(I) = 9 +/- 1 mM. ADP behaved approximately as a competitive inhibitor with K(I) = 35 +/- 2 microM. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and/or mechanical changes. We found that the deviation from competitive inhibition by ADP was inconsistent with models in which P(i) is released before ADP. This is surprising because all known ATPases (and GTPases) with high structural similarity to the motor domains of kinesin release P(i) before ADP (or GDP). Our result is therefore inconsistent with models, such as one-headed and inchworm mechanisms, in which the hydrolysis cycle takes place on one head only. However, it is simply explained by hand-over-hand models in which ADP release from one head precedes P(i) release from the other.
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PMID:Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism. 1473 13

The flexible tubulin C-terminal tails (CTTs) have recently been implicated in the walking mechanism of dynein and kinesin. To address their role in the case of conventional kinesin, we examined the structure of kinesin-microtubule (MT) complexes before and after CTT cleavage by subtilisin. Our results show that the CTTs directly modulate the motor-tubulin interface and the binding properties of motors. CTT cleavage increases motor binding stability, and kinesin appears to adopt a binding conformation close to the nucleotide-free configuration under most nucleotide conditions. Moreover, C-terminal cleavage results in trapping a transient motor-ADP-MT intermediate. Using SH3-tagged dimeric and monomeric constructs, we could also show that the position of the kinesin neck is not affected by the C-terminal segments of tubulin. Overall, our study reveals that the tubulin C-termini define the stability of the MT-kinesin complex in a nucleotide-dependent manner, and highlights the involvement of tubulin in the regulation of weak and strong kinesin binding states.
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PMID:Modulation of kinesin binding by the C-termini of tubulin. 1497 55

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

Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: k(cat) = 5.5 s(-1), K(1/2,Mt) = 0.7 microm, and K(m,ATP) = 25 microm; and for Eg5-437: k(cat) = 2.9 s(-1), K(1/2,Mt) = 4.5 microm, and K(m,ATP) = 19 microm. 2'(3')-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 microm(-1)s(-1), followed immediately by ATP hydrolysis at 15 s(-1). ATP-dependent Mt.Eg5 dissociation was relatively slow and rate-limiting at 8 s(-1) with mantADP release at 40 s(-1). Surprisingly, Eg5-367 binds microtubules more effectively (11 microm(-1)s(-1)) than Eg5-437 (0.7 microm(-1)s(-1)), consistent with the steady-state K(1/2,Mt) and the mantADP release K(1/2,Mt). These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover.
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PMID:Mechanistic analysis of the mitotic kinesin Eg5. 1524 93

Cytoplasmic transport is mediated by a group of molecular motors that typically work in isolation, under conditions where they must move their cargos long distances without dissociating from their tracks. This processive behavior requires specific adaptations of motor enzymology to meet these unique physiologic demands. One of these involves the ability of the two heads of a processive motor to communicate their structural states to each other. In this study, we examine a processive motor from the myosin superfamily myosin V. We have measured the kinetics of nucleotide release, of phosphate release, and of the weak-to-strong transition, as this motor interacts with actin, and we have used these studies to develop a model of how myosin V functions as a transport motor. Surprisingly, both heads release phosphate rapidly upon the initial encounter with an actin filament, suggesting that there is little or no intramolecular strain associated with this step. However, ADP release can be affected by both forward and rearward strain, and under steady-state conditions it is essentially prevented in the lead head until the rear head detaches. Many of these features are remarkably like those underlying the processive movement of kinesin on microtubules, supporting our hypothesis that different molecular motors satisfy the requirement for processive movement in similar ways, regardless of their particular family of origin.
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PMID:A model of myosin V processivity. 1525 35


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