Structure of a tetrameric MscL in an expanded intermediate state

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Abstract

The ability of cells to sense and respond to mechanical force underlies diverse processes such as touch and hearing in animals, gravitropism in plants, and bacterial osmoregulation1,2. In bacteria, mechanosensation is mediated by the mechanosensitive channels of large (MscL), small (MscS), potassium-dependent (MscK) and mini (MscM) conductances. These channels act as ‘emergency relief valves’ protecting bacteria from lysis upon acute osmotic down-shock3. Among them, MscL has been intensively studied since the original identification and characterization 15 years ago4. MscL is reversibly and directly gated by changes in membrane tension. In the open state, MscL forms a non-selective 3 nS conductance channel which gates at tensions close to the lytic limit of the bacterial membrane. An earlier crystal structure at 3.5 Å resolution of a pentameric MscL from Mycobacterium tuberculosis represents a closed-state or non-conducting conformation5,6. MscL has a complex gating behaviour; it exhibits several intermediates between the closed and open states, including one putative non-conductive expanded state and at least three sub-conducting states7. Although our understanding of the closed5,6 and open8,9,10 states of MscL has been increasing, little is known about the structures of the intermediate states despite their importance in elucidating the complete gating process of MscL. Here we present the crystal structure of a carboxy-terminal truncation mutant (Δ95–120) of MscL from Staphylococcus aureus (SaMscL(CΔ26)) at 3.8 Å resolution. Notably, SaMscL(CΔ26) forms a tetrameric channel with both transmembrane helices tilted away from the membrane normal at angles close to that inferred for the open state9, probably corresponding to a non-conductive but partially expanded intermediate state.

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Figure 1: SaMscL(CΔ26) is a pressure-sensitive channel.
Figure 2: Structures of SaMscL(CΔ26) and MtMscL.
Figure 3: Alignment of SaMscL(CΔ26) (yellow) and MtMscL (blue).
Figure 4: The two-step helix-pivoting model of SaMscL gating.

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Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors have been deposited at the Protein Data Bank with accession numbers 3HZQ.

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Acknowledgements

We thank A. Shih for early cloning work, T. Walton for biochemical analysis, A. Lee for initial efforts on the MscL project, J. Choe for expression and purification protocols, O. Lewinson for manuscript reading, Y. Poon and J. Lai for treating MJF465 with λDE3 and the osmotic down-shock assay protocol, J. Kaiser for suggestions on structure refinement, R. Phillips, E. Haswell and P. Pal for discussions, P. Blount for the MJF465 strain, and the staff at SSRL, the Advanced Light Source (ALS) and the Advanced Photon Source (APS) for technical support with crystal screening and data collection. We would like to acknowledge the Gordon and Betty Moore Foundation for support of the Molecular Observatory at Caltech. Operations at SSRL, ALS and APS are supported by the US Department of Energy and NIH. This work was supported in part by grants from the Howard Hughes Medical Institute and the National Institutes of Health (GM084211). C.S.G. was supported in part by postdoctoral fellowships from the National Institutes of Health and the Beckman Foundation. D.C.R. is an Investigator in the Howard Hughes Medical Institute.

Author Contributions Z.L. designed and performed the experiments in molecular biology, biochemistry, crystallography and the structure analysis. Z.L. and C.S.G. conducted the down-shock assays. C.S.G. was responsible for the protein reconstitution and electrophysiology. D.C.R. coordinated the project and contributed to the structure analysis. The manuscript was written by Z.L., C.S.G. and D.C.R.

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Correspondence to Douglas C. Rees.

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Supplementary Information

This file contains Supplementary Figures 1-4 with Legends and Supplementary Table 1. (PDF 550 kb)

Supplementary Movie 1

This movie file shows proposed two-step helix pivoting model for SaMscL gating. (MOV 1680 kb)

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Liu, Z., Gandhi, C. & Rees, D. Structure of a tetrameric MscL in an expanded intermediate state. Nature 461, 120–124 (2009) doi:10.1038/nature08277

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