Gating-associated conformational changes in the mechanosensitive channel MscL

Bacterial cells avoid lysis in response to hypoosmotic shock through the opening of the mechanosensitive channel MscL. Upon channel opening, MscL is thought to expand in the plane of the membrane and form a large pore with an estimated diameter of 3–4 nm. Here, we set out to analyze the closed and open structure of cell-free MscL. To this end, we characterized the function and structure of wild-type MscL and a mutant form of the protein (G22N MscL) that spontaneously adopts an open substate. Patch-clamp analysis of MscL that had been reconstituted into liposomes revealed that wild-type MscL was activated only by mechanical stimuli, whereas G22N MscL displayed spontaneous opening to the open substate. In accord with these results, Ca2+ influx into G22N MscL-containing liposomes occurred in the absence of mechanical stimulation. The electrophoretic migration of chemically cross-linked G22N MscL was slower than that of cross-linked wild-type MscL, suggesting that G22N MscL is in an expanded form. Finally, electron microscopy using low-angle rotary shadowing revealed the presence of a pore at the center of G22N MscL. No pore could be detected in wild-type MscL. However, wild-type MscL possessed a protrusion at one end, which was absent in G22N MscL. The deletion of carboxyl-terminal 27 residues resulted in the loss of protrusion and proper multimerization. The structures of wild-type and G22N MscL reveal that the opening of MscL is accompanied by the dissociation of a carboxyl-terminal protrusion and pore formation.

[1]  H. Guy,et al.  Structural models of the MscL gating mechanism. , 2001, Biophysical journal.

[2]  Boris Martinac,et al.  Open channel structure of MscL and the gating mechanism of mechanosensitive channels , 2002, Nature.

[3]  S. Sukharev,et al.  Gain-of-function Mutations Reveal Expanded Intermediate States and a Sequential Action of Two Gates in MscL , 2005, The Journal of general physiology.

[4]  C. Kung,et al.  One face of a transmembrane helix is crucial in mechanosensitive channel gating. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[5]  S. Sukharev,et al.  Stoichiometry of the Large Conductance Bacterial Mechanosensitive Channel of E. coli. A Biochemical Study , 1999, The Journal of Membrane Biology.

[6]  C Kung,et al.  Mechanosensitive channels of bacteria. , 1999, Methods in enzymology.

[7]  Yufeng Shen,et al.  Conformational pathways in the gating of Escherichia coli mechanosensitive channel , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[8]  J. Haiech,et al.  Xenopus laevis oocyte calmodulin in the process of meiotic maturation. , 1980, The Journal of biological chemistry.

[9]  D. Dougherty,et al.  Molecular dynamics simulations of wild-type and mutant forms of the Mycobacterium tuberculosis MscL channel. , 2001, Biophysical journal.

[10]  C Kung,et al.  Hydrophilicity of a single residue within MscL correlates with increased channel mechanosensitivity. , 1999, Biophysical journal.

[11]  H. Robert Guy,et al.  A large iris-like expansion of a mechanosensitive channel protein induced by membrane tension , 2002, Nature Structural Biology.

[12]  J. Betton,et al.  Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent. , 2004, Biochemistry.

[13]  Boris Martinac,et al.  A large-conductance mechanosensitive channel in E. coli encoded by mscL alone , 1994, Nature.

[14]  R. Minchin,et al.  Estimation of the pore size of the large-conductance mechanosensitive ion channel of Escherichia coli. , 1997, Biophysical journal.

[15]  K. Schulten,et al.  Gating of MscL studied by steered molecular dynamics. , 2003, Biophysical journal.

[16]  C. Kung,et al.  Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Frederick Sachs,et al.  Energetic and Spatial Parameters for Gating of the Bacterial Large Conductance Mechanosensitive Channel, MscL , 1999, The Journal of general physiology.

[18]  C Kung,et al.  Chemically charging the pore constriction opens the mechanosensitive channel MscL. , 2001, Biophysical journal.

[19]  R. Naik,et al.  Biomolecular stress-sensitive gauges: surface-mediated immobilization of mechanosensitive membrane protein. , 2003, Journal of the American Chemical Society.

[20]  D C Rees,et al.  Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. , 1998, Science.

[21]  P. Blount,et al.  Bacterial mechanosensitive channels: integrating physiology, structure and function. , 1999, Trends in microbiology.

[22]  H. Robert Guy,et al.  The gating mechanism of the large mechanosensitive channel MscL , 2001, Nature.

[23]  Boris Martinac,et al.  Physical principles underlying the transduction of bilayer deformation forces during mechanosensitive channel gating , 2002, Nature Structural Biology.

[24]  A. Mark,et al.  Simulation of MscL gating in a bilayer under stress. , 2003, Biophysical journal.

[25]  Y. Akao,et al.  Structural insight of human DEAD‐box protein rck/p54 into its substrate recognition with conformational changes , 2006, Genes to cells : devoted to molecular & cellular mechanisms.

[26]  H. Guy,et al.  On the Conformation of the COOH-terminal Domain of the Large Mechanosensitive Channel MscL , 2003, The Journal of general physiology.