Structural determinants of sensing and gating in MscS-like channels

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Mechanosensitive channels play an important role in osmoregulation, protecting bacteria against hypo-osmotic shock. Our research focusses on MscS-like channels in E.coli, which has six paralogues that promote a graded response to environmental changes. MscS-like channels have a common heptameric architecture comprising a fenestrated cytosolic vestibule that surrounds the entrance to the pore and membrane anchored paddles at the periphery which are contributed by each subunit. The size of the paddles varies between 2 and 10 helices depending on the paralogue. Based on our recently determined structures of the medium-sized YnaI and the large-sized YbiO, we hypothesize that the structural determinants for sensing, gating and conductance are encoded in the modular architecture of the channels. We think that the sensing module comprises the paddles. The pressure response is modulated by the interaction of positively charged residues in the paddles with lipids. Gating is probably encoded in a short stretch of 15 amino acids in the pore helices preceding the hydrophobic seal of the pore. YnaI gates by shortening the pore, which is markedly different to the mechanism of the prototypic small-sized MscS. We think that pore-shortening is encoded in a GGxGG motif specific to a subset of channels including YnaI. Finally, the conductance is encoded in the vestibule and the central pore following the gating motif. We suggest that the three modules are interchangeable units and if combined into chimeric channels will exhibit the functional characteristics of the respective donor channels. We propose to test these hypotheses in an YnaI background. Therefore, we will generate mutants that interrogate the importance of the positive charges in the paddles for lipid binding and pressure response. Other mutants will test the gating characteristics of naturally occurring Gly-rich motifs in an YnaI background with the aim to establish which motifs gate by pore shortening and which maintain the pore length as observed in MscS. Finally, we will generate chimeric channels, in which YnaI modules will be replaced with the respective modules from other MscS-like channels to proof that the modules are interchangeable and still assemble into functional channels. Mutations and chimeras will be characterized by measuring their ability to protect bacteria against hypo-osmotic shock. Patch-clamp experiments will quantify the pressure, threshold for channel opening, the conductance of the open channels and further gating characteristics. Structure determination by electron cryo microscopy and image processing will identify the conformational changes involved in the gating mechanism and will map functional changes to structural characteristics. Together this will generate a comprehensive understanding of the structure function relationship in MscS-like channels that will shed light on the role of this very diverse channel family in nature.