This Article Full Text Full Text (PDF) Supplement All Versions of this Article: biophysj.107.110171v1 94/4/1252    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Anishkin, A. Articles by Sukharev, S. Search for Related Content PubMed PubMed Citation Articles by Anishkin, A. Articles by Sukharev, S.

Characterization of the Resting MscS: Modeling and Analysis of the Closed Bacterial Mechanosensitive Channel of Small Conductance

Department of Biology, University of Maryland, College Park, Maryland

Correspondence: Address reprint requests to Sergei Sukharev, Tel.: 301-571-9119; E-mail: sukharev{at}umd.edu.

Channels from the MscS family are adaptive tension-activated osmolyte release valves that regulate turgor in prokaryotes and volume in plant chloroplasts. The crystal structure of Escherichia coli MscS has provided a starting point for detailed descriptions of its mechanism. However, solved in the absence of the lipid bilayer, this structure may deviate from a native conformation. In this study, we utilized molecular dynamics simulations and a new iterative extrapolated-motion protocol to pack the splayed peripheral TM1 and TM2 transmembrane helices along the central TM3 shaft. This modification restored the tension transmission route between the membrane and the channel gate. We also modeled the structure of the 26-amino acid N-terminal segments that were unresolved in the crystals. The resulting compact conformation, which we believe approximates the closed resting state of MscS, matches the hydrophobic thickness of the lipid bilayer with arginines 46, 54, and 74 facing the polar lipid headgroups. The pore-lining helices in this resting state feature alternative kinks near the conserved G121 instead of the G113 kinks observed in the crystal structure and the transmembrane barrel remains stable in extended molecular dynamics simulations. Further analysis of the dynamics of the pore constriction revealed several moderately asymmetric and largely dehydrated states. Biochemical and patch-clamp experiments with engineered double-cysteine mutants demonstrated cross-linking between predicted adjacent residue pairs, which formed either spontaneously or under moderate oxidation. The L72C-V99C bridge linking more peripheral TM2 to TM3 caused a shift of channel activation to higher pressures. TM3 to TM3 cross-links through the A84C-T93C, S95C-I97C, and A106C-G108C cysteine pairs were shown to lock MscS in a nonconductive state. Normal channel activity in these mutants could be recovered upon disulfide reduction with dithiothreitol. These results confirmed our modeling predictions of a closed MscS channel featuring a TM3 barrel that largely resembles the crystal conformation though with more tightly packed peripheral helices. From this closed-resting conformation, the TM3 helices must expand to allow for channel opening.

This article has been cited by other articles:

				A. Anishkin, K. Kamaraju, and S. Sukharev Mechanosensitive Channel MscS in the Open State: Modeling of the Transition, Explicit Simulations, and Experimental Measurements of Conductance J. Gen. Physiol., July 1, 2008; 132(1): 67 - 83. [Abstract] [Full Text] [PDF]