The N-Terminal Helix Acts as a Dynamic Membrane Coupler in the Gating Cycle of the Mechanosensitive Channel MscL

Abstract

The bacterial mechanosensitive channel of large conductance (MscL) gates in response to membrane tension as a result of force transmitted directly from the lipid bilayer. This channel represents an excellent model system to study the basic principles of mechanosensory transduction. However, despite extensive studies of this homopentameric channel, there is an incomplete understanding of the essential structural components that transduce bilayer tension into channel gating. We postulate that the amphipathic N-terminal helix, which is linked to the pore-lining TM1 helix, acts as a membrane-coupling element that transmits force from the bilayer to the channel pore. To examine this hypothesis we used a multi-scale computational approach, including all-atom molecular dynamics (MD) simulation and finite element (FE) modelling, supported by patch-clamp electrophysiology. Our simulations suggest a strong interaction between the five N-terminal helices of MscL and the lipid bilayer. Moreover, removal of the N-terminal helix, or extending the glycine linker with further (flexible) glycines between the N-terminus and the TM1 helix restricted opening of the channel pore by more than 50%. These computational results were supported by patch-clamp recordings of MscL channels in liposomes and E. coli spheroplasts. Extension of the glycine linker between the N-termini and TM1 helices increased the activation threshold of MscL and resulted in channels that almost exclusively gated in substates. Based on our MD results this is a due to greater conformational freedom of the TM1 helix. These results show that the N-terminus acts as a membrane-coupling element in the gating cycle of MscL. We suggest this element may represent a common recognisable structure amongst mechanosensitive channels coupling channel conformation to membrane strain.