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Functional visualization of viral molecular motor by hydrogen-deuterium exchange reveals transient states

Nature Structural & Molecular Biology volume 12, pages 460–466 (2005)Cite this article

Abstract

Molecular motors undergo cyclical conformational changes and convert chemical energy into mechanical work. The conformational dynamics of a viral packaging motor, the hexameric helicase P4 of dsRNA bacteriophage φ8, was visualized by hydrogen-deuterium exchange and high-resolution mass spectrometry. Concerted changes of exchange kinetics revealed a cooperative unit that dynamically links ATP-binding sites and the central RNA-binding channel. The cooperative unit is compatible with a structure-based model in which translocation is mediated by a swiveling helix. Deuterium labeling also revealed the transition state associated with RNA loading, which proceeds via opening of the hexameric ring. The loading mechanism is similar to that of other hexameric helicases. Hydrogen-deuterium exchange provides an important link between time-resolved spectroscopic observations and high-resolution structural snapshots of molecular machines.

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Figure 1: Mapping of peptic fragments on P4 structure.
Figure 2: Hydrogen-deuterium exchange measured by mass spectrometry.
Figure 3: Characterization of the L1-loop deletion mutant (P4ΔLKK).
Figure 4: Deuterium incorporation during RNA loading.
Figure 5: Comparison of exchange kinetics for selected regions.
Figure 6: Model of RNA loading and the cooperative unit.

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Acknowledgements

We thank A. Abu Ramadan for the software development. J.L. is supported by Viikki Graduate School in Biosciences, D.E.K is supported by the National Graduate School in Informational and Structural Biology. This work was supported by Academy of Finland grant 206926 (R.T.), the Finnish Centre of Excellence Program 2000–2005, the US National Science Foundation National High Field FT-ICR Mass Spectrometry Facility (CHE-94-13008), Florida State University and the National High Magnetic Field Laboratory.

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  1. TuKiet T Lam

    Present address: WM Keck Foundation Biotechnology Resource Laboratory, Yale University, 300 George Street, New Haven, Connecticut, 06511, USA

Authors and Affiliations

  1. Institute of Biotechnology and Department of Biological and Environmental Sciences, University of Helsinki, Helsinki, 00014, Finland

    Jiří Lísal, Denis E Kainov & Roman Tuma

  2. National High Magnetic Field Laboratory and Department of Chemistry and Biochemistry, Florida State University, 1800 E. Paul Dirac Drive, Tallahassee, 32310, Florida, USA

    TuKiet T Lam, Mark R Emmett & Alan G Marshall

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Supplementary information

Supplementary Fig. 1

Sequence alignment. (PDF 1989 kb)

Supplementary Fig. 2

Bimodal isotopic distributions during RNA loading. (PDF 50 kb)

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Lísal, J., Lam, T., Kainov, D. et al. Functional visualization of viral molecular motor by hydrogen-deuterium exchange reveals transient states. Nat Struct Mol Biol 12, 460–466 (2005). https://doi.org/10.1038/nsmb927

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