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Science 321 (5893): 1210-1214

Copyright © 2008 by the American Association for the Advancement of Science

A Structural Mechanism for MscS Gating in Lipid Bilayers

Valeria Vásquez1,2, Marcos Sotomayor3, Julio Cordero-Morales1,2, Klaus Schulten4, and Eduardo Perozo2*

1 Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.
2 Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637, USA.
3 Howard Hughes Medical Institute and Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
4 Department of Physics, University of Illinois at Urbana–Champaign, and Beckman Institute for Advanced Science and Technology, Urbana, IL 61801, USA.

Figure 1 Fig. 1.. LPC incorporation permanently activates MscS. (A) (Top) MscS orientation in the inside-out patch-clamp configuration and perfusion of LPC micelles. {Delta}V, transmembrane voltage; lyso PC, lysophosphatidylcholine. (Bottom) Representative MscS macroscopic currents (~55 channels in the patch) activated by negative pressure (at –60 mmHg and +10 mV) reveal the presence of a time-dependent inactivation process. (B) Sequential incorporation of LPC (3 µM) into the internal leaflet of inside-out patches from Escherichia coli spheroplasts and in the absence of applied tension elicits spontaneous openings (after ~2 min). All the channels present in the patch (as determined from tension-induced macroscopic currents) are activated by LPC. The inset shows single-channel transitions. [View Larger Version of this Image (18K GIF file)]

Figure 2 Fig. 2.. Structural rearrangements underlying channel opening. (A) A single MscS monomer is represented as part of the heptamer according to the MscS closed-state model obtained from the EPR-based refinement (21). The amino acid residues subjected to cysteine-scanning mutagenesis in the present study are shown as black spheres. (B) Representative X-band EPR spectra of consecutively spin-labeled mutants along the permeation pathway (TM3). Black and red traces were obtained from channels in the closed and open conformations, respectively. All spectra were obtained from samples at the same protein-to-lipid ratio, and a dielectric resonator with the microwave power set to 2 mW was used. Channel opening was obtained in dioleoylphosphatidylcholine:palmitoylphosphatidylglycerol + 25 mole % LPC vesicles. (C) Residue-specific environmental parameter profiles obtained in the open and closed (black curve) conformations for the N-terminal and TM segments: mobility parameter {Delta}Ho–1 (top, green curve), O2 accessibility parameter {Pi}O2 (middle, red curve), and NiEdda accessibility parameter {Pi}NiEdda (bottom, blue curve) are shown. The black horizontal bar covers the region for which EPR spectra are shown in (B). Gray areas represent the TM segment assignment derived from the MscS crystal structure (12, 13). [View Larger Version of this Image (57K GIF file)]

Figure 3 Fig. 3.. Extent and direction of environmental parameter changes upon MscS opening. (A) Changes in local dynamics and solvent accessibilities mapped onto molecular surfaces of the closed-state EPR-based model (top) and the crystal structure (bottom). At left are ribbon representations of MscS (two subunits are shown for clarity), where individual TM segments are color-coded as follows: N terminus, green; TM1, yellow; TM2, blue; and TM3, red. From left to right, mobility ({Delta}{Delta}Ho–1), oxygen accessibility ({Delta}{Pi}O2), and NiEdda accessibility ({Delta}{Pi}NiEdda) changes are shown. (B) {Pi}NiEdda residue-specific environmental parameter profile for the TM3 helix obtained in the open (blue curve) and closed (black curve) conformations. (C) Vector analysis of TM3 environmental data in the open conformation. {Pi}NiEdda parameters have been superimposed in a polar coordinate. Resultant moments for the closed (black arrow) and open (red arrow) conformations were calculated from the accessibilities. [View Larger Version of this Image (58K GIF file)]

Figure 4 Fig. 4.. A structural model for MscS gating in lipid bilayers. (A) Side and extracellular views of the structural rearrangements leading to the open conformation. (Left) A single MscS subunit is highlighted in blue and gray for the open and closed states, respectively. (Middle) TM3a and TM3b helices (residues 94 to 128 and 91 to 128 for the closed and open models, respectively). (Right) Extracellular view of the pore. Helical movements are illustrated by red arrows. (B) GOF (blue) and LOF (red) mutants mapped onto two subunits of MscS closed (left) and open (right) conformation models. GOF: I39N and I78N (6); V40D (41); and T93R, A102P, and L109S (39). LOF: V6C and A19C (21); I48D/S49P (39); and A51N, L55N, F68N, A85N, and L86N (6). Residue L105 (arrows) is shown in stick representation. (C) Cross-sectional area of the MscS pore in the closed, open, and crystal conformations. Each cross section was obtained from the calculated surface with the use of the program HOLE (40). [View Larger Version of this Image (46K GIF file)]

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