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Straightening and sequential buckling of the pore-lining helices define the gating cycle of MscS

Nature Structural & Molecular Biology volume 14, pages 1141–1149 (2007)Cite this article

Abstract

We describe a mechanism connecting the adaptive behavior of the bacterial mechanosensitive channel MscS to the flexibility of the pore-lining helix TM3. Simulated expansion of the channel structure revealed straightening of a characteristic kink near Gly113 in the open state; return to the closed state produced an alternative kink at Gly121. Patch-clamp experiments showed that higher helical propensity introduced by a G113A mutation prevented inactivation. A similar mutation, G121A, kinetically impeded both closure and inactivation. Duplicating the glycines at each of these sites to increase flexibility produced directly opposite effects. The severely toxic G113A G121A mutation resulted in channels that could not inactivate or close with the release of tension. These data suggest that the open MscS features straight TM3 helices, which act as collapsible 'struts'. Closure and desensitization rely on buckling at Gly121, whereas the crystal-like kink at Gly113 is a feature of the inactivated state.

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Figure 1: Analysis of the MscS crystal structure and modeling of its different functional states.
Figure 2: Characterization of the adaptive behaviors of WT MscS and two mutants with altered TM3b flexibility near Gly113.
Figure 3: Mutations in TM3b at Gly121 greatly affect channel responses to pressure.
Figure 4: Adaptive current decline is a result of two processes, desensitization and inactivation.
Figure 5: Tension dependence of inactivation for WT MscS and mutants.
Figure 6: High helical propensity in TM3b impairs closure of MscS.

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Acknowledgements

We thank L. Shirinian for generating some of the MscS mutants, M. Kiyatkin for compiling a library of MscS and MscK homologs and conducting the multiple sequence alignments, and I.R. Booth (University of Aberdeen) and P. Blount (University of Texas, Dallas) for kindly providing the MJF465 and PB113 strains. This work was supported by the US National Institutes of Health.

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  1. Bradley Akitake and Andriy Anishkin: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology, University of Maryland, Building 144, College Park, 20742, Maryland, USA

    Bradley Akitake, Andriy Anishkin, Naili Liu & Sergei Sukharev

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Contributions

B.A., A.A. and S.S. designed the experiments and analyzed the models and data. B.A. conducted all of the patch-clamp experiments and generated some of the MscS mutants. A.A. performed all of the computations (extrapolated-motion analysis and molecular dynamics simulations). N.L. performed the growth-curve analysis and generated some of the MscS mutants. The paper was written and edited by B.A., A.A. and S.S.

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Correspondence to Sergei Sukharev.

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Akitake, B., Anishkin, A., Liu, N. et al. Straightening and sequential buckling of the pore-lining helices define the gating cycle of MscS. Nat Struct Mol Biol 14, 1141–1149 (2007). https://doi.org/10.1038/nsmb1341

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