MscS is a ubiquitous mechanosensitive channel essential for bacterial adaptation to low osmolarity. E. coli MscS undergoes an adaptive functional cycle consisting of closed, open, and inactivated states. While opening occurs rapidly and is steeply tension-dependent, inactivation is a slower process driven by relatively low-tensions. Among the two classes of existing structures, splayed non-conductive conformations can reliably be identified as the inactivated state. To date, no experimental structures satisfy the criteria for either the closed or fully open states. To predict the closed conformation, we employed computational modeling in conjunction with a range of functional experimental tests. We observed that the MscS population is more active in cultures during early logarithmic growth phases than in lysozyme-treated giant spheroplasts. Taking this lead, we designed simulations that mimicked the forces involved in the recovery transition from the inactivated state, reflecting conditions in an actively growing cell. These forces included high turgor pressure, elevated lateral pressure of newly synthesized lipids in the cytoplasmic leaflet, and strong hyperpolarizing voltage. We combined these factors into a single simulation protocol in NAMD using ColVars, which led to the following structural changes in the channel: (1) upward displacement of the gate and a shift of the characteristic kink from G113 to G121, (2) formation of a hydrophobic contact between TM2 and TM3, (3) new interaction patterns with membrane lipids, and (4) creation of stabilizing salt bridges—both circumferential and vertical—interlinking TM1-TM2 pairs and the cytoplasmic cage domain. Functional patch-clamp and disulfide cross-linking experiments on mutants fully confirmed the predicted residue proximities and the salt bridge formations. While efforts to stabilize the compact closed state for cryo-EM visualization are ongoing, we have substantial evidence supporting the existence of this state.
Anishkin et al. (Sun,) studied this question.