The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels

The bacterial mechanosensitive channel MscL gates in response to membrane tension as a result of mechanical force transmitted directly to the channel from the lipid bilayer. MscL represents an excellent model system to study the basic biophysical principles of mechanosensory transduction. However, understanding of the essential structural components that transduce bilayer tension into channel gating remains incomplete. Here using multiple experimental and computational approaches, we demonstrate that the amphipathic N-terminal helix of MscL acts as a crucial structural element during tension-induced gating, both stabilizing the closed state and coupling the channel to the membrane. We propose that this may also represent a common principle in the gating cycle of unrelated mechanosensitive ion channels, allowing the coupling of channel conformation to membrane dynamics

The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels

The bacterial mechanosensitive channel MscL gates in response to membrane tension as a result of mechanical force transmitted directly to the channel from the lipid bilayer. MscL represents an excellent model system to study the basic biophysical principles of mechanosensory transduction. However, understanding of the essential structural components that transduce bilayer tension into channel gating remains incomplete. Here using multiple experimental and computational approaches, we demonstrate that the amphipathic N-terminal helix of MscL acts as a crucial structural element during tension-induced gating, both stabilizing the closed state and coupling the channel to the membrane. We propose that this may also represent a common principle in the gating cycle of unrelated mechanosensitive ion channels, allowing the coupling of channel conformation to membrane dynamics

Pulling MscL open via N-terminal and TM1 helices: A computational study towards engineering an MscL nanovalve

<div><p>There are great opportunities in the manipulation of bacterial mechanosensitive (MS) ion channels for specific and targeted drug delivery purposes. Recent research has shown that these ion channels have the potential to be converted into nanovalves through clever use of magnetic nanoparticles and magnetic fields. Using a combination of molecular dynamics (MD) simulations and the finite element (FE) modelling, this study investigates the theoretical feasibility of opening the MscL channel (MS channel of large conductance of <i>E</i>. <i>coli</i>) by applying mechanical force directly to its N-terminus. This region has already been reported to function as a major mechanosensor in this channel. The stress-strain behaviour of each MscL helix was obtained using all atom MD simulations. Using the same method, we simulated two models, the wild-type (WT) MscL and the G22N mutant MscL, both embedded in a POPE lipid bilayer. In addition to indicating the main interacting residues at the hydrophobic pore, their pairwise interaction energies were monitored during the channel gating. We implemented these inputs into our FE model of MscL using curve-fitting codes and continuum mechanics equations. In the FE model, the channel could be fully opened via pulling directly on the N-terminus and bottom of TM1 by mutating dominant van der Waals interactions in the channel pore; otherwise the stress generated on the channel protein can irreversibly unravel the N-secondary structure. This is a significant finding suggesting that applying force in this manner is sufficient to open an MscL nanovalve delivering various drugs used, for example, in cancer chemotherapy. More importantly, the FE model indicates that to fully operate an MscL nanovalve by pulling directly on the N-terminus and bottom of TM1, gain-of-function (GOF) mutants (e.g., G22N MscL) would have to be employed rather than the WT MscL channel.</p></div

Structural dynamics of the mscL C-terminal domain

The large conductance mechanosensitive channel (MscL), acts as an osmoprotective emergency valve in bacteria by opening a large, water-filled pore in response to changes in membrane tension. In its closed configuration, the last 36 residues at the C-terminus form a bundle of five α-helices co-linear with the five-fold axis of symmetry. Here, we examined the structural dynamics of the C-terminus of EcMscL using site-directed spin labelling electron paramagnetic resonance (SDSL EPR) spectroscopy. These experiments were complemented with computational modelling including molecular dynamics (MD) simulations and finite element (FE) modelling. Our results show that under physiological conditions, the C-terminus is indeed an α-helical bundle, located near the five-fold symmetry axis of the molecule. Both experiments and computational modelling demonstrate that only the top part of the C-terminal domain (from the residue A110 to E118) dissociates during the channel gating, while the rest of the C-terminus stays assembled. This result is consistent with the view that the C-terminus functions as a molecular sieve and stabilizer of the oligomeric MscL structure as previously suggested

Pore size and stress values along TM1 of several cases where pulling force was applied only to the N-terminus determined by FE modelling.

<p>Pore size and stress values along TM1 of several cases where pulling force was applied only to the N-terminus determined by FE modelling.</p

Activation of an MscL nanovalve by magnetic field.

<p><b>(A)</b> Magnetic nano-beads are chemically attached to MscL at N-terminus (±TM1), which then can be opened by application of static and/or oscillating magnetic field to release the drug cargo carried by a liposome in which the MscL nanovalves have been reconstituted. <b>(B)</b> <i>Left</i> and <i>Right</i> images depict the closed and open MscL channel respectively, in a liposome containing drug molecules.</p

Pore size and stress values along TM1 of several cases where pulling force was applied to both the N-terminus and TM1.

<p>Pore size and stress values along TM1 of several cases where pulling force was applied to both the N-terminus and TM1.</p

The mechanical response of EcMscL α-helices to force obtained from force-constant steered MD simulation.

<p>The stress-strain curves of <b>(A)</b> N-terminus, <b>(B)</b> TM1, <b>(C)</b> TM2, <b>(D)</b> C-terminus. <b>(E)</b> At 6% strain value, the C-terminal helix is the stiffest, whereas the N-terminal helix is the most compliant among all the α-helices. The transmembrane helices (TM1 and TM2) have similar responses, and both are more than 4 times stiffer than the N-terminus. These values are mean ± SEM of 3 different simulations.</p