Structures of kinesin motor proteins

Hoenger, Andreas; Mandelkow, Eckhard

doi:10.1002/cm.20392

Cited by 74 publications

(61 citation statements)

References 71 publications

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“…Numerous X-ray structures of kinesins detached from their track protein are known; these kinesins are mostly bound to ADP (for example, see refs 5-7; for a review, see ref. 8) but in a few cases ATP-like structures have been determined 9,10 . There is also a recent structure of ATP-like kinesin bound to tubulin 11 (ATP-like complex).…”

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confidence: 99%

The structure of apo-kinesin bound to tubulin links the nucleotide cycle to movement

et al. 2014

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Kinesin-1 is a dimeric ATP-dependent motor protein that moves towards microtubules ( þ ) ends. This movement is driven by two conformations (docked and undocked) of the two motor domains carboxy-terminal peptides (named neck linkers), in correlation with the nucleotide bound to each motor domain. Despite extensive data on kinesin-1, the structural connection between its nucleotide cycle and movement has remained elusive, mostly because the structure of the critical tubulin-bound apo-kinesin state was unknown. Here we report the 2.2 Å structure of this complex. From its comparison with detached kinesin-ADP and tubulin-bound kinesin-ATP, we identify three kinesin motor subdomains that move rigidly along the nucleotide cycle. Our data reveal how these subdomains reorient on binding to tubulin and when ATP binds, leading respectively to ADP release and to neck linker docking. These results establish a framework for understanding the transformation of chemical energy into mechanical work by ( þ ) end-directed kinesins.

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confidence: 99%

The structure of apo-kinesin bound to tubulin links the nucleotide cycle to movement

et al. 2014

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“…A plot of the rate constants (k obs ) determined versus the mantATP concentration is shown in Figure 3C. Fitting these data to a linear function (i.e., k obs = k 1 [mantATP] + k -1 ) allows deconvolution of the rate constants that contribute to k obs (Step 2.8). Therefore, enabling the determination of both the association and the dissociation rate constant for mantATP (Figure 1, k 1 and k -1 respectively).…”

Section: Representative Resultsmentioning

confidence: 99%

“…Deconvolve the association and dissociation rate constants by plotting k obs against mantATP concentration (e.g., Figure 3C). Fit these data to a linear function (i.e., k obs = k 1 [mantATP] + k -1 ) to obtain the gradient and y-axis intercept. The gradient represents the rate constant for mantATP association (Figure 1, k 3.…”

Section: Measurement Of the Association And Dissociationmentioning

confidence: 99%

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Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

Patel

Belsham

Rathbone

et al. 2014

JoVE

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The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin's ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.

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“…Most kinesin complexes have two motor domains formed by a tetramer consisting of a homodimer of two kinesin heavy chains. Two identical kinesin light chains in the kinesin complex contribute to kinesin regulation and cargo attachment (Marx et al, 2009). …”

Section: Kinesinmentioning

confidence: 99%

Molecular interactions of proteins associated with surface membrane biogenesis and renewal in the tegument of Schistosoma mansoni

Schulte¹

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The tegument of Schistosoma mansoni serves a variety of critical biological roles for the parasite, including uptake of nutrients, osmoregulation, immune evasion and immunomodulation. Dynein light chains (DLCs) and tetraspanins (TSPs) are prominent proteins in the tegument and appear to be intimately linked with other molecules present in the apical, host-interactive, membrane of the parasites. In other eukaryotic cells, cytoplasmic DLCs are a subunit of motor complexes involved in such diverse cellular translocation events as protein trafficking, mitosis and ciliary beating. DLCs of the schistosome tegument likely have similar roles and are likely to be important in the development and renewal of the tegument membrane, thereby contributing to parasite survival in its host. There is evidence that S. mansoni DLCs bind to a group of proteins unique to the tegument, the tegument allergen-like antigens (TALs) suggesting that schistosome DLCs may form complexes outside of the dynein complex. TSPs are membrane-spanning proteins that act as scaffolds for the formation of membraneassociated protein complexes comprising a wide variety of proteins. These TSP-enriched microdomains are known to play a wide range of roles within human cells, including maintenance of cell morphology and cell signalling.The biology of DLCs, TALs and TSPs in schistosome tegument is investigated in this study. Two DLCs, SmDLC1 and SmDLC5, three TALs, Sm20.8, Sm21.7 and Sm22.6 along with SmTSP-2 are the focus of this study. The tegumental localisation and transcription throughout the schistosome life cycle are reported for each transcript. By indirect immunocytochemistry the localisation within the tegument of SmTSP-2, SmDLC1 and Sm22.6 was investigated using parasites prepared by high pressure freezing for electron microscopy and cryosubstitution in uranyl acetate in solvent. SmTSP-2 was strongly membrane associated but little labelling was associated with the apical tegument membranes. SmDLC1 labelling was abundant in the entire tegument cytoplasm, close to the apical tegument membranes, surface invaginations and on the membranes on some tegumental vesicles. Sm22.6 had a more diffuse localisation pattern across the cytoplasm and labelling was apparent close to the apical surface membranes. Dual labelling immunocytochemistry of SmDLC1 and Sm22.6 did not show labelling patterns that confirmed the interaction of these SmDLCs and SmTALs. The transcript abundance of SmDLCs and SmTALs were assessed in the schistosome life cycle by real-time PCR. A similar pattern was seen for most transcripts, with transcript abundance increasing as the parasite developed from free-living life cycle stages (egg, miracidia, cercariae) to parasitic ii stages (schistosomula and adults). The most striking exception to this pattern is Sm21.7, which has a consistently high abundance across all life cycle stages.Protein interactions in the tegument were explored using Blue Native polyacrylamide gel electrophoresis and protein crosslinkers, coupled with in-line liqui...

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Structures of kinesin motor proteins

Cited by 74 publications

References 71 publications

The structure of apo-kinesin bound to tubulin links the nucleotide cycle to movement

The structure of apo-kinesin bound to tubulin links the nucleotide cycle to movement

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

Molecular interactions of proteins associated with surface membrane biogenesis and renewal in the tegument of Schistosoma mansoni

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