Hydrophilicity of a Single Residue within MscL Correlates with Increased Channel Mechanosensitivity

Abstract

Mechanosensitive channel large (MscL) encodes the large conductance mechanosensitive channel of the Escherichia coli inner membrane that protects bacteria from lysis upon osmotic shock. To elucidate the molecular mechanism of MscL gating, we have comprehensively substituted Gly(22) with all other common amino acids. Gly(22) was highlighted in random mutagenesis screens of E. coli MscL (, Proc. Nat. Acad. Sci. USA. 95:11471-11475). By analogy to the recently published MscL structure from Mycobacterium tuberculosis (, Science. 282:2220-2226), Gly(22) is buried within the constriction that closes the pore. Substituting Gly(22) with hydrophilic residues decreased the threshold pressure at which channels opened and uncovered an intermediate subconducting state. In contrast, hydrophobic substitutions increased the threshold pressure. Although hydrophobic substitutions had no effect on growth, similar to the effect of an MscL deletion, channel hyperactivity caused by hydrophilic substitutions correlated with decreased proliferation. These results suggest a model for gating in which Gly(22) moves from a hydrophobic, and through a hydrophilic, environment upon transition from the closed to open conformation.

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

“…Simulations for the G22N mutant was performed without applying negative pressure and only during the equilibrating calculation for 5 ns, because the G22N mutant undergoes spontaneous opening without mechanical stimulation (membrane stretch). 13,16 Estimation of the pore size. The minimum pore radius of MscL was calculated by the HOLE program using a spherical probe.…”

“…In the present study, pore radii were calculated in the plane where AA 22 (G22) is located, which has been suggested to be the most constricted part of the pore called gate. 13 …”

“…12 Many studies have been performed using site-directed mutagenesis in order to better understand the structure-function of MscL. [13][14][15][16][17][18][19] One of these studies produced an estimation of the tension-sensing site in MscL using random scanning mutagenesis, where individual hydrophobic AAs facing the lipids were replaced with the hydrophilic AA asparagine to identify any "loss-of-function" mutants lacking mechanosensitivity. 15 As the result, it was found that replacement of one of seven amino residues located at the periplasmic end of the transmembrane helices caused the loss of MscL mechanosensitivity, suggesting that one or some of them may act as a tension sensor in MscL.…”

“…On the other hand, when Gly22, located near the most constricted part of the ion permeation pore that is considered to be a compartment of the mechanosensitive gate of MscL, is substituted to another AA, typically asparagine (G22N), the resulting mutants could more easily be opened (gain-of-function) in comparison with the wild-type (WT) MscL. 13,16 Escherichia coli (Eco-MscL), with a comparable conductance as Eco-MscL. 10 The structure of Tb-MscL suggests that it is mostly in a closed form, and its open structure has not been resolved yet.…”

“…Finally, to evaluate whether the model and the condition set in the simulation are appropriate for analyzing the MscL opening process, we constructed molecular models for two mutants that are known to open more easily (G22N) or with greater difficulty (F78N) than WT MscL, and examined whether they were able to reproduce the essence of experimentally observed features. 13,15,16 …”