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Nucleotide binding and hydrolysis induces a disorder-order transition in the kinesin neck-linker region

Nature Structural & Molecular Biology volume 13, pages 648–654 (2006)Cite this article

Abstract

Kinesin translocation is thought to occur by a conformational change in a region of the motor domain called the neck linker. However, most evidence supporting this hypothesis comes from monomeric constructs unable to move processively. To address this issue, we investigated the neck-linker configuration on microtubule-bound monomeric and dimeric kinesin constructs using single-molecule fluorescence polarization microscopy. We found that the neck-linker region (i) is very mobile in the absence of nucleotides and during steady walking, (ii) decreases mobility and aligns along the microtubule axis in the presence of AMPPNP or ADP+AlF−4, (iii) is mostly ordered in the monomeric constructs in the presence of ADP+AlF−4, and (iv) is closer to parallel to the microtubule axis in the dimeric constructs. These results support the proposed role of the neck linker and suggest a coordination mechanism between the two motor domains in the dimer.

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Figure 1: Probe locations.
Figure 2: Ensemble fluorescence anisotropy measurements, LD0, for the three kinesin constructs investigated in the presence of different nucleotides.
Figure 3: Single-molecule fluorescence intensity traces.
Figure 4: Single-molecule relative frequency distributions.
Figure 5: Neck-linker configuration models.

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Acknowledgements

We thank D. Buster and D. Sharp for discussions and critical reading of the manuscript, R. Fenton for technical help and H. Deng and E. Nieves for mass spectrometry and analysis. This project was supported by a US National Institutes of Health grant (RO1-AR48620) to H.S.

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Authors and Affiliations

  1. Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, 10461, New York, USA

    Ana B Asenjo, Yonatan Weinberg & Hernando Sosa

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  1. Ana B Asenjo
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  2. Yonatan Weinberg
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  3. Hernando Sosa
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Correspondence to Hernando Sosa.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

KM330_335 single-molecule frequency distributions (PDF 108 kb)

Supplementary Table 1

Kolmogorov-Smirnov statistical tests (PDF 104 kb)

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Asenjo, A., Weinberg, Y. & Sosa, H. Nucleotide binding and hydrolysis induces a disorder-order transition in the kinesin neck-linker region. Nat Struct Mol Biol 13, 648–654 (2006). https://doi.org/10.1038/nsmb1109

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