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)

Bovine brain kinesin separates into two components on sucrose density gradient centrifugation. The predominant component is a heterotetramer of two 120 kDa alpha subunits and two 64 kDa beta subunits with an sedimentation coefficient of 9.6 S and a low Vm rate of microtubule-stimulated ATPase of 1.3 +/- 0.5 sec-1 at 25 degrees, pH 7.0. The minor element is a homodimer of two alpha subunits without beta subunits with a sedimentation coefficient of 6.9 S and a higher Vm rate of microtubule-stimulated ATPase of 7.0 +/- 1.9 sec-1. Microtubules stimulate the rate of release of ADP from the active site of the tetramer, but the rate of release is not fast enough to account for the rate of steady state ATP hydrolysis. Further complexity is indicated by biphasic release kinetics. In spite of the large difference in Vm ATPase rate for the two species, both drive the sliding of sea urchin axonemes over glass surfaces at the same velocity.
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PMID:Characterization of alpha 2 beta 2 and alpha 2 forms of kinesin. 182 68

The molecular motor kinesin is a homodimer containing two heads-globular domains each of which has an ATP- and a microtubule-binding site. It is argued by analogy to other proteins with coiled-coil dimerization domains that the kinesin dimer has an approximate axis of rotational symmetry. The path kinesin follows along the surface of the microtubule is parallel to the protofilaments, and the steps are likely separated by 8 nm, the length of the tubulin dimer. Micromechanical recordings from single kinesin molecules indicate that one motor can exert a force as great as 5 pN. The efficiency of kinesin probably is in the order of 50%, considering the free energy available from ATP hydrolysis. Structural, mechanical, and biochemical experiments suggest that in order not to let go of a microtubule, the two heads of kinesin might move in a coordinated manner, perhaps undergoing a rotary motion.
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PMID:The movement of kinesin along microtubules. 881 16

Kinesin is a processive motor protein: A single molecule can walk continuously along a microtubule for several micrometers, taking hundreds of 8-nm steps without dissociating. To elucidate the biochemical and structural basis for processivity, we have engineered a heterodimeric one-headed kinesin and compared its biochemical properties to those of the wild-type two-headed molecule. Our construct retains the functionally important neck and tail domains and supports motility in high-density microtubule gliding assays, though it fails to move at the single-molecule level. We find that the ATPase rate of one-headed kinesin is 3-6 s(-1) and that detachment from the microtubule occurs at a similar rate (3 s(-1)). This establishes that one-headed kinesin usually detaches once per ATP hydrolysis cycle. Furthermore, we identify the rate-limiting step in the one-headed hydrolysis cycle as detachment from the microtubule in the ADP.P(i) state. Because the ATPase and detachment rates are roughly an order of magnitude lower than the corresponding rates for two-headed kinesin, the detachment of one head in the homodimer (in the ADP.P(i) state) must be accelerated by the other head. We hypothesize that this results from internal strain generated when the second head binds. This idea accords with a hand-over-hand model for processivity in which the release of the trailing head is contingent on the binding of the forward head. These new results, together with previously published ones, allow us to propose a pathway that defines the chemical and mechanical cycle for two-headed kinesin.
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PMID:Kinesin's processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains. 1055 88

kinesin-II motor proteins are composed of two different kinesin-like motor proteins and one cargo binding subunit. Here we report the cloning of a new member of the kinesin-II superfamily, Xklp3A from Xenopus laevis, which forms a heterodimeric complex with Xklp3B. The heterodimer formation properties between Xklp3A and B have been tested in vitro using reticulocyte lysate expression and immunoprecipitation. To this end we produced a series of Xklp3A and B constructs of varying length and tested their propensity for heterodimer formation. We could demonstrate that, in contrast to conventional kinesin, the critical domains for heterodimer formation in Xklp3A/B are located at the C-terminal end of the stalk. Neither the neck nor the highly charged stretches after the neck region, which are typical of kinesins-II, are required for heterodimer formation, nor do they prevent homodimer formation. Dimerization is controlled by a cooperative mechanism between the C-terminal coiled-coil segments. Classical trigger sites were not identified. The critical regions for dimerization exhibit a very high degree of sequence conservation among equivalent members of the kinesin-II family.
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PMID:Analysis of heterodimer formation by Xklp3A/B, a newly cloned kinesin-II from Xenopus laevis. 1143 25

We have analysed the structural and physical properties of the carboxy-terminal stalk region of a kinesin-II, Xenopus kinesin-like protein 3A/B (Xklp3A/B), which we showed to be essential for heterodimerization in a previous work (De Marco et al., 2001). We expressed the corresponding A-stalk and B-stalk fragments and investigated their modes of interaction by analytical ultracentrifugation (AUC), circular dichroism spectroscopy, denaturation assays and electron microscopy. Co-expression of the A-stalk and B-stalk produced the properly folded, hetero-dimeric coiled coil at high yields. The dimeric nature of the complex was confirmed by AUC. We also found that the isolated A-stalk fragment forms a stable helix by itself and shows a significant tendency towards homodimer and higher-order complex formation. In the absence of the corresponding A-stalk fragment, the isolated B-stalk fragment remains partially unfolded, which suggests that the A-stalk provides a template structure for the B-stalk in order to recompose the complete heterodimeric coiled coil.
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PMID:Dimerization properties of a Xenopus laevis kinesin-II carboxy-terminal stalk fragment. 1283 58

KIF3A/B, a kinesin involved in intraflagellar transport and Golgi trafficking, is distinctive because it contains two nonidentical motor domains. Our hypothesis is that the two heads have distinct functional properties, which are tuned to maximize the performance of the wild-type heterodimer. To test this, we investigated the motility of wild-type KIF3A/B heterodimer and chimaeric KIF3A/A and KIF3B/B homodimers made by splicing the head of one subunit to the rod and tail of the other. The first result is that KIF3A/B is processive, consistent with its transport function in cells. Secondly, the KIF3B/B homodimer moves at twice the speed of the wild-type motor but has reduced processivity, suggesting a trade-off between speed and processivity. Third, the KIF3A/A homodimer moves fivefold slower than wild-type, demonstrating distinct functional differences between the two heads. The heterodimer speed cannot be accounted for by a sequential head model in which the two heads alternate along the microtubule with identical speeds as in the homodimers. Instead, the data are consistent with a coordinated head model in which detachment of the slow KIF3A head from the microtubule is accelerated roughly threefold by the KIF3B head.
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PMID:The two motor domains of KIF3A/B coordinate for processive motility and move at different speeds. 1899 Jul 83

We investigated the folding, stability, and specificity of dimerization of the neck regions of the kinesin-like proteins Kif3A (residues 356-416) and Kif3B (residues 351-411). We showed that the complementary charged regions found in the hinge regions (which directly follow the neck regions) of these proteins do not adopt any secondary structure in solution. We then explored the ability of the complementary charged regions to specify heterodimer formation for the neck region coiled-coils found in Kif3A and Kif3B. Redox experiments demonstrated that oppositely charged regions specified the formation of a heterodimeric coiled-coil. Denaturation studies with urea demonstrated that the negatively charged region of Kif3A dramatically destabilized its neck coiled-coil (urea1/2 value of 3.9 m compared with 6.7 m for the coiled-coil alone). By comparison, the placement of a positively charged region C-terminal to the neck coiled-coil of Kif3B had little effect on stability (urea1/2 value of 8.2 m compared with 8.8 m for the coiled-coil alone). The pairing of complementary charged regions leads to specific heterodimer formation where the stability of the heterodimeric neck coiled-coil with charged regions had similar stability (urea1/2 value of 7.8 m) to the most stable homodimer (Kif3B) with charged regions (urea1/2 value of 8.0 m) and dramatically more stable than the Kif3A homodimer with charged regions (urea1/2, value of 3.9 m). The heterodimeric coiled-coil with charged extensions has essentially the same stability as the heterodimeric coiled-coil on its own (urea1/2 values of 7.8 and 8.1 m, respectively) suggesting that specificity of heterodimerization is driven by non-specific attraction of the oppositely unstructured charged regions without affecting stability of the heterodimeric coiled-coil.
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PMID:Stability and specificity of heterodimer formation for the coiled-coil neck regions of the motor proteins Kif3A and Kif3B: the role of unstructured oppositely charged regions. 1570 65

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

Several lines of experimental evidence suggest that the conventional kinesin 1 walks by an asymmetric hand-over-hand mechanism, although it is a homodimer. In the previous study, we examined several important force-dependent features of the hand-over-hand mechanism of kinesin. In this study, we focus on the asymmetry in the hand-over-hand mechanism. We show that the experimentally observed kinesin limping can be explained in our model by the variation of the neck linker lengths in the kinesin stepping (which has also been suggested earlier by others). We also study the experimentally observed processive motion of a mutant heterodimer of kinesin, in which only one of the two heads has the capability of ATP hydrolysis, as well as the walking of wild-type kinesin in the presence of both ATP and its analogue AMPPNP. We show that the possible processive walking of the heterodimeric kinesin can be explained by introducing a force-generating intermediate, the kinesin-ATP complex, which is different from the posthydrolytic species, kinesin-ADP/Pi.
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PMID:Asymmetry in kinesin walking. 1763 Jul 71

A single molecule of the motor enzyme kinesin-1 keeps a tight grip on its microtubule track, making tens or hundreds of discrete, unidirectional 8 nm steps before dissociating. This high duty ratio processive movement is thought to require a mechanism in which alternating stepping of the two head domains of the kinesin dimer is driven by alternating, overlapped cycles of ATP hydrolysis by the two heads. The R210K point mutation in Drosophila kinesin heavy chain was reported to disrupt the ability of the enzyme active site to catalyze ATP P-O bond cleavage. We expressed R210K homodimers as well as isolated R210K heads and confirmed that both are essentially inactive. We then coexpressed tagged R210K subunits with untagged wild-type subunits and affinity purified R210K/wild-type heterodimers together with the inactive R210K homodimers. In contrast to the R210K head or homodimer, the heterodimer was a highly active (>50% of wild-type) microtubule-stimulated ATPase, and the heterodimer displayed high duty ratio processive movement in single-molecule motility experiments. Thus, dimerization of a subunit containing the inactivating mutation with a functional subunit can complement the mutation; this must occur either by lowering or by bypassing kinetic barriers in the ATPase or mechanical cycles of the mutant head. The observations provide support for kinesin-1 gating mechanisms in which one head stimulates the rate of essential processes in the other.
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PMID:Processive movement by a kinesin heterodimer with an inactivating mutation in one head. 1870 29


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