Three-Dimensional Architecture of Membrane-Embedded MscS in the Closed Conformation

The mechanosensitive channel of small conductance (MscS) is part of a coordinated response to osmotic challenges in Escherichia coli. MscS opens as a result of membrane tension changes, thereby releasing small solutes and effectively acting as an osmotic safety valve. Both the functional state depicted by its crystal structure and its gating mechanism remain unclear. Here, we combine site-directed spin labeling, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations with novel energy restraints based on experimental electron paramagnetic resonance data to investigate the native transmembrane (TM) and periplasmic molecular architecture of closed MscS in a lipid bilayer. In the closed conformation, MscS shows a more compact TM domain than in the crystal structure, characterized by a realignment of the TM segments towards the normal of the membrane. The previously unresolved NH₂-terminus forms a short helical hairpin capping the extracellular ends of TM1 and TM2 and is in close interaction with the bilayer interface. The present three-dimensional model of membrane-embedded MscS in the closed state represents a key step in determining the molecular mechanism of MscS gating.