Vanegas, J. M. | Search bioRxiv

bioRxiv2023

Tight hydrophobic core and flexible helices yield MscL with a high tension gating threshold and a membrane area mechanical strain buffer

The mechanosensitive (MS) channel of large conductance, MscL, is the high-tension threshold osmolyte release valve that limits turgor pressure in bacterial cells in the event of drastic hypoosmotic shock. Despite MscL from M. tuberculosis (TbMscL) being the first structurally characterized MS channel, its protective mechanism of activation at nearly-lytic tensions has not been fully understood. Here, we describe atomistic simulations of expansion and opening of wild-type (WT) TbMscL in comparison with five of its gain-of-function (GOF) mutants. We show that under far-field membrane tension applied to the edge of the periodic simulation cell, WT TbMscL expands into a funnel-like structure with trans-membrane helices bent by nearly 70 degrees, but does not break its hydrophobic seal within extended 20 s simulations. GOF mutants carrying hydrophilic substitutions in the hydrophobic gate of increasing severity (A20N, V21A, V21N, V21T and V21D) also quickly transition into funnel-shaped conformations but subsequently fully open within 1-8 us. This shows that solvation of the de-wetted (vapor-locked) constriction is the rate-limiting step in the gating of TbMscL preceded by area-buffering silent expansion. Pre-solvated gates in severe V21N and V21D mutants eliminate this barrier. We predict that the asymmetric shape-change of the periplasmic side of the channel during the silent expansion provides strain-buffering to the outer leaflet thus re-distributing the tension to the inner leaflet, where the gate resides.

biophysics↗

bioRxiv2020

Mechanical Activation of MscL Revealed by a Locally Distributed Tension Molecular Dynamics Approach

Membrane tension perceived by mechanosensitive (MS) proteins mediates cellular responses to mechanical stimuli and osmotic stresses, and it also guides multiple biological functions including cardiovascular control and development. In bacteria, MS channels function as tension-activated pores limiting excessive turgor pressure, with MscL (MS channel of large conductance) acting as an emergency release valve preventing cell lysis. Previous attempts to simulate gating transitions in MscL by either directly applying steering forces to the protein or by increasing the whole system tension were not fully successful and often disrupted the integrity of the system. We present a novel locally distributed tension molecular dynamics (LDT-MD) simulation method that allows application of forces continuously distributed among lipids surrounding the channel using a specially constructed collective variable. We report reproducible and reversible transitions of MscL to the open state with measured parameters of lateral expansion and conductivity that exactly satisfy experimental values. The LDT-MD method enables exploration of the MscL gating process with different pulling velocities and variable tension asymmetry between the inner and outer membrane leaflets. We use LDT-MD in combination with well-tempered metadynamics to reconstruct the tension-dependent free energy landscape for the opening transition in MscL. SIGNIFICANCEMembrane-embedded mechanosensitive (MS) proteins are essential for numerous biological functions including cardiovascular control and development, osmotic regulation, touch and pain sensing. In this work, we present a novel molecular dynamics simulation method that allows rapid and systematic exploration of structure, dynamics, and energetics of the mechanical transduction process in MS proteins under tightly controlled local tension distributed in the lipid rim around the protein. We provide a detailed description of the gating transition for the tension-activated bacterial mechanosensitive channel of large conductance, MscL, which is the best characterized channel of this type. MscL functions as a tension-activated emergency osmolyte release valve that limits excessive turgor pressure, prevents cell lysis and thus imparts environmental stability to most free-living bacteria.

biophysics↗