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Published October 1, 2018 | Supplemental Material + Published
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Elucidating a role for the cytoplasmic domain in the Mycobacterium tuberculosis mechanosensitive channel of large conductance


Microbial survival in dynamic environments requires the ability to successfully respond to abrupt changes in osmolarity. The mechanosensitive channel of large conductance (MscL) is a ubiquitous channel that facilitates the survival of bacteria and archaea under severe osmotic downshock conditions by relieving excess turgor pressure in response to increased membrane tension. A prominent structural feature of MscL, the cytoplasmic C-terminal domain, has been suggested to influence channel assembly and function. In this report, we describe the X-ray crystal structure and electrophysiological properties of a C-terminal domain truncation of the Mycobacterium tuberculosis MscL (MtMscLΔC). A crystal structure of MtMscLΔC solubilized in the detergent n-dodecyl-β-D-maltopyranoside reveals the pentameric, closed state-like architecture for the membrane spanning region observed in the previously solved full-length MtMscL. Electrophysiological characterization demonstrates that MtMscLΔC retains mechanosensitivity, but with conductance and tension sensitivity more closely resembling full length EcMscL than MtMscL. This study establishes that the C-terminal domain of MtMscL is not required for oligomerization of the full-length channel, but rather influences the tension sensitivity and conductance properties of the channel. The collective picture that emerges from these data is that each MscL channel structure has characteristic features, highlighting the importance of studying multiple homologs.

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© The Author(s) 2018. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Received: 13 July 2018. Accepted: 31 August 2018. Published online: 01 October 2018. We thank Troy Walton, Chinenye (Chinny) Idigo, Jens Kaiser and Jeff Abramson for enlightening discussions and the Gordon and Betty Moore Foundation and the Beckman Institute for their generous support of the Molecular Observatory at Caltech. X-ray crystallography data was collected at the Stanford Synchrotron Radiation Lightsource (SSRL), a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated by the United States Department of Energy, by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P41GM103393) and the National Center for Research Resources, Biomedical Technology Program (P41RR001209). This research was funded in part by the National Institutes of Health National Institute of General Medical Sciences (5R01GM084211-07) and the Howard Hughes Medical Institute. N.H. was also independently funded by the Howard Hughes Medical Institute Gilliam Fellowship. Coordinates and structure factors have been deposited in the Protein Data Bank of the Research Collaboratory for Structural Bioinformatics, with PDBID 6CTD. Author Contributions: N.H., G.M., E.H., D.C.R. conceptualized the approach and developed the experimental protocols. N.H. purified, crystallized and solved the crystal structure and with D.C.R. analyzed the structure. G.M. performed and with E.H. analyzed the electrophysiology experiments. N.H. wrote the initial manuscript draft, and N.H., G.M., E.H., D.C.R. revised and edited the manuscript. All authors read and approved the final manuscript. The authors declare no competing interests.

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