The gating mechanism of the large mechanosensitive channel MscL

Abstract

The mechanosensitive channel of large conductance, MscL, is a ubiquitous membrane-embedded valve involved in turgor regulation in bacteria. The crystal structure of MscL from Mycobacterium tuberculosis provides a starting point for analysing molecular mechanisms of tension-dependent channel gating. Here we develop structural models in which a cytoplasmic gate is formed by a bundle of five amino-terminal helices (S1), previously unresolved in the crystal structure. When membrane tension is applied, the transmembrane barrel expands and pulls the gate apart through the S1-M1 linker. We tested these models by substituting cysteines for residues predicted to be near each other only in either the closed or open conformation. Our results demonstrate that S1 segments form the bundle when the channel is closed, and crosslinking between S1 segments prevents opening. S1 segments interact with M2 when the channel is open, and crosslinking of S1 to M2 impedes channel closing. Gating is affected by the length of the S1-M1 linker in a manner consistent with the model, revealing critical spatial relationships between the domains that transmit force from the lipid bilayer to the channel gate.

Conformational heterogeneity of the M2 proton channel and a structural model for channel activation

“…3 we display individual results for 15 Water Penetration Around Gly-34. Given the backbone flexibility of a Gly residue and its propensity to kink transmembrane helices (29)(30)(31), it was not surprising that helix kinking was observed in our simulations around Gly-34, the only Gly residue in the M2 TMD. The small size of the Gly side chain may allow for water access and hydrogen bonding with the backbone in the vicinity of the kink, thereby stabilizing it.…”

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

“…In order to evaluate to what extent our simulations reproduce the experimentally estimated MscL features, 7 we calculated the energy changes during the course of MscL opening and obtained an energy difference between closed and putative first-transition state. The obtained value, approximately 25 kcal/mol (42 k B T) in WT MscL, is comparable to the experimentally obtained value ca.…”

“…4,5 Purified MscL reconstituted into the lipid bilayer was found to retain its native mechanosensitive function, indicating that MscL activation is brought about exclusively by stress in the membrane and does not require any supporting proteins. 6,7 Patch-clamp experiments showed that MS channels are activated by tension in the membrane rather than by One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics.…”

“…[6][7][8][9] Ion channels in general, including MS channels, have two conducting states called open and closed, respectively. Detailed analyses of the conductance levels and the kinetics of state transitions in MscL have revealed that at least five subconductance states exist on the way to the fully open state.…”

“…As most of the experiments to date have been done with Eco-MscL, a molecular model for Eco-MscL was constructed based on the crystal structure of Tb-MscL to allow structurefunction investigation of MscL. 7 Eco-MscL (hereafter this will be denoted simply as MscL unless otherwise noted) forms a homopentamer, with a subunit having two transmembrane helices consisting of 136 amino acids (AAs), and with a molecular weight of 15 kDa. 4,6 The first transmembrane (TM1) helices line the pore and the second transmembrane (TM2) helices form the outer wall facing the lipids surrounding MscL (Fig.…”