On the structure of the N-terminal domain of the MscL channel: helical bundle or membrane interface.
The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock and is to date the best characterized mechanosensitive channel. A well-recognized and supported model for Escherichia coli MscL gating proposes that the N-terminal 11 amino acids of this protein form a bundle of amphipathic helices in the closed state that functionally serves as a cytoplasmic second gate. However, a recently reexamined crystal structure of a closed state of the Mycobacterium tuberculosis MscL shows these helices running along the cytoplasmic surface of the membrane. Thus, it is unclear if one structural model is correct or if they both reflect valid closed states. Here, we have systematically reevaluated this region utilizing cysteine-scanning, in vivo functional characterization, in vivo SCAM, electrophysiological studies, and disulfide-trapping experiments. The disulfide-trapping pattern and functional studies do not support the helical bundle and second-gate hypothesis but correlate well with the proposed structure for M. tuberculosis MscL. We propose a functional model that is consistent with the collective data.
Department of Physiology, University of Texas, Southwestern Medical Center at Dallas, Dallas, Texas 75390-9040, USA.
Irene
I
Iscla
Robin
R
Wray
Paul
P
Blount
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004594:Electrophysiology; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D015640:Ion Channel Gating / Q000502:physiology*; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D008954:Models, Biological; D009997:Osmotic Pressure; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion
D001426:Bacterial Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D007473:Ion Channels; C086596:MscL protein, E coli; C116377:Tb-MscL protein, Mycobacterium tuberculosis; D003545:Cysteine