Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Structure of a tetrameric MscL in an expanded intermediate state

Nature volume 461, pages 120–124 (2009)Cite this article

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

Accession codes

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.

References

  1. Kung, C. A possible unifying principle for mechanosensation. Nature 436, 647–654 (2005)

    ADS  CAS  PubMed  Google Scholar 

  2. Sukharev, S. & Corey, D. P. Mechanosensitive channels: multiplicity of families and gating paradigms. Sci. STKE 2004, 1–24 (2004)

    Google Scholar 

  3. Booth, I. R., Edwards, M. D., Black, S., Schumann, U. & Miller, S. Mechanosensitive channels in bacteria: signs of closure? Nature Rev. Microbiol. 5, 431–440 (2007)

    CAS  Google Scholar 

  4. Sukharev, S. I., Blount, P., Martinac, B., Blattner, F. R. & Kung, C. A large-conductance mechanosensitive channel in E. coli encoded by mscL alone. Nature 368, 265–268 (1994)

    ADS  CAS  PubMed  Google Scholar 

  5. Chang, G., Spencer, R. H., Lee, A. T., Barclay, M. T. & Rees, D. C. Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. Science 282, 2220–2226 (1998)

    ADS  CAS  PubMed  Google Scholar 

  6. Steinbacher, S., Bass, R., Strop, P. & Rees, D. C. in Current Topics in Membranes. Mechanosensitive Ion Channels, Part A (ed. Hamill, O. P.) 1–24 (Academic, 2007)

    Google Scholar 

  7. Sukharev, S. I., Sigurdson, W. J., Kung, C. & Sachs, F. Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL. J. Gen. Physiol. 113, 525–540 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Sukharev, S., Betanzos, M., Chiang, C.-S. & Guy, H. R. The gating mechanism of the large mechanosensitive channel MscL. Nature 409, 720–724 (2001)

    ADS  CAS  PubMed  Google Scholar 

  9. Perozo, E., Cortes, D. M., Sompornpisut, P., Kloda, A. & Martinac, B. Open channel structure of MscL and the gating mechanism of mechanosensitive channels. Nature 418, 942–948 (2002)

    ADS  CAS  PubMed  Google Scholar 

  10. Anishkin, A. & Sukharev, S. State-stabilizing interactions in the bacterial mechanosensitive channel gating and adaptation. J. Biol. Chem. 284, 19153–19157 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Moe, P. C., Blount, P. & Kung, C. Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation. Mol. Microbiol. 28, 583–592 (1998)

    CAS  PubMed  Google Scholar 

  12. Levina, N. et al. Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity. EMBO J. 18, 1730–1737 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Blount, P., Sukharev, S. I., Schroeder, M. J., Nagle, S. K. & Kung, C. Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli. Proc. Natl Acad. Sci. USA 93, 11652–11657 (1996)

    ADS  CAS  PubMed  Google Scholar 

  14. Häse, C. C., Le Dain, A. C. & Martinac, B. Molecular dissection of the large mechanosensitive ion channel (MscL) of E. coli: mutants with altered channel gating and pressure sensitivity. J. Membr. Biol. 157, 17–25 (1997)

    PubMed  Google Scholar 

  15. Niegowski, D. & Eshaghi, S. The CorA family: structure and function revisited. Cell. Mol. Life Sci. 64, 2564–2574 (2007)

    CAS  PubMed  Google Scholar 

  16. Cogdell, R. J. et al. The structure and function of the LH2 (B800–850) complex from the purple photosynthetic bacterium Rhodopseudomonas acidophila strain 10050. Prog. Biophys. Mol. Biol. 68, 1–27 (1997)

    CAS  PubMed  Google Scholar 

  17. Stock, D., Leslie, A. G. & Walker, J. E. Molecular architecture of the rotary motor in ATP synthase. Science 286, 1700–1705 (1999)

    CAS  PubMed  Google Scholar 

  18. Sukharev, S., Durell, S. R. & Guy, H. R. Structural models of the MscL gating mechanism. Biophys. J. 81, 917–936 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Strop, P., Bass, R. & Rees, D. C. Prokaryotic mechanosensitive channels. Adv. Protein Chem. 63, 177–209 (2003)

    CAS  PubMed  Google Scholar 

  20. Beckstein, O. & Sansom, M. S. P. The influence of geometry, surface character, and flexibility on the permeation of ions and water through biological pores. Phys. Biol. 1, 42–52 (2004)

    ADS  CAS  PubMed  Google Scholar 

  21. Cruickshank, C. C., Minchin, R. F., Le Dain, A. C. & Martinac, B. Estimation of the pore size of the large-conductance mechanosensitive ion channel of Escherichia coli. Biophys. J. 73, 1925–1931 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Ajouz, B., Berrier, C., Garrigues, A., Besnard, M. & Ghazi, A. Release of thioredoxin via the mechanosensitive channel MscL during osmotic downshock of Escherichia coli cells. J. Biol. Chem. 273, 26670–26674 (1998)

    CAS  PubMed  Google Scholar 

  23. van den Bogaart, G., Krasnikov, V. & Poolman, B. Dual-color fluorescence-burst analysis to probe protein efflux through the mechanosensitive channel MscL. Biophys. J. 92, 1233–1240 (2007)

    CAS  PubMed  Google Scholar 

  24. Yoshimura, K., Usukura, J. & Sokabe, M. Gating-associated conformational changes in the mechanosensitive channel MscL. Proc. Natl Acad. Sci. USA 105, 4033–4038 (2008)

    ADS  CAS  PubMed  Google Scholar 

  25. Strop, P. & Brunger, A. T. Refractive index-based determination of detergent concentration and its application to the study of membrane proteins. Protein Sci. 14, 2207–2211 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Spencer, R. H. & Rees, D. C. The α-helix and the organization and gating of channels. Annu. Rev. Biophys. Biomol. Struct. 31, 207–233 (2002)

    CAS  PubMed  Google Scholar 

  27. Wang, W. et al. The structure of an open form of an E. coli mechanosensitive channel at 3.45 Å resolution. Science 321, 1179–1183 (2008)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Maurer, J. A., Elmore, D. E., Lester, H. A. & Dougherty, D. A. Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL). J. Biol. Chem. 275, 22238–22244 (2000)

    CAS  PubMed  Google Scholar 

  29. Tsai, I. J. et al. The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity. Eur. Biophys. J. 34, 403–412 (2005)

    CAS  PubMed  Google Scholar 

  30. Ajouz, B., Berrier, C., Besnard, M., Martinac, B. & Ghazi, A. Contributions of the different extramembranous domains of the mechanosensitive ion channel MscL to its response to membrane tension. J. Biol. Chem. 275, 1015–1022 (2000)

    CAS  PubMed  Google Scholar 

  31. Lusty, C. A gentle vapor-diffusion technique for cross-linking of protein crystals for cryocrystallography. J. Appl. Cryst. 32, 106–112 (1999)

    CAS  Google Scholar 

  32. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008)

    ADS  CAS  PubMed  MATH  Google Scholar 

  33. Read, R. Improved Fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr. A 42, 140–149 (1986)

    Google Scholar 

  34. Collaborative Computational Project, 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Google Scholar 

  35. Cowtan, K. DM: an automated procedure for phase improvement by density modification. Joint CCP4 ESF-EACBM Newslett. Protein Crystallogr. 31, 34–38 (1994)

    Google Scholar 

  36. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    PubMed  Google Scholar 

  37. Brunger, A. T. Version 1.2 of the crystallography and NMR system. Nature Protocols 2, 2728–2733 (2007)

    CAS  PubMed  Google Scholar 

  38. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993)

    CAS  Google Scholar 

  39. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

    CAS  PubMed  Google Scholar 

  40. Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–308 (1999)

    CAS  PubMed  Google Scholar 

  41. DeLano, W. L. The PyMOL User's Manual (Delano Scientific, 2002)

    Google Scholar 

  42. Smart, O. S., Neduvelil, J. G., Wang, X. N., Wallace, B. A. & Sansom, M. S. P. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. Model. 14, 354–360 (1996)

    CAS  Google Scholar 

  43. Hutchinson, E. G. & Thornton, J. M. PROMOTIF–a program to identify and analyze structural motifs in proteins. Protein Sci. 5, 212–220 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Iscla, I., Wray, R. & Blount, P. On the structure of the N-terminal domain of the MscL channel: helical bundle or membrane interface. Biophys. J. 95, 2283–2291 (2008)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  45. Häse, C. C., Le Dain, A. C. & Martinac, B. Purification and functional reconstitution of the recombinant large mechanosensitive ion channel (MscL) of Escherichia coli. J. Biol. Chem. 270, 18329–18334 (1995)

    PubMed  Google Scholar 

  46. Sukharev, S. I., Martinac, B., Arshavsky, V. Y. & Kung, C. Two types of mechanosensitive channels in the Escherichia coli cell envelope: solubilization and functional reconstitution. Biophys. J. 65, 177–183 (1993)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA,

    Zhenfeng Liu, Chris S. Gandhi & Douglas C. Rees

  2. Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA,

    Zhenfeng Liu & Douglas C. Rees

Authors
  1. Zhenfeng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chris S. Gandhi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Douglas C. Rees
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Douglas C. Rees.

Supplementary information

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)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08277

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing