Chemically Stabilizing the fully open conformation of the bacterial mechanosensitive channel MscS

Authors

• EH (Chang)-Rabley
• EE Ricotta

Abstract

Structure determination for membrane proteins remains not only technically challenging but is further complicated by potentially non-native conformations resulting from removal of stabilizing lipids. This is especially applicable to mechanosensitive channels that gate in response to subtle changes in membrane tension, such as MscS. Cryo-EM structures of MscS fall into two categories depending on the method of solubilization: (1) nonconductive (lipid-reconstituted or mixed micelles) characterized by kinked pore-lining helices and splayed lipid-facing helices, or (2) semi-open (pure detergent or short-chain lipids) satisfying ~70% of experimental conductance. However, MscS has 3 functional states: open, closed, and inactivated. In patch-clamp, the closed to open transition occurs in response to abruptly applied membrane tensions of 7-9 mN/m, opening a 1.6 nm pore resulting in an 18 nm2 in-plane expansion. But under slowly applied moderate tensions of 5-6 mN/m, MscS transitions from closed to a nonconductive and tension insensitive inactivated state, producing an 8 nm2 expansion. We attempt to relate these functional states with existing MscS structures and hypothesize that MscS bears internal stress that is finely balanced by lateral pressure from lipids. This imparts the discrete tension sensitivities for the opening and inactivation transitions which leads to the two observed structural categories. Using MD simulations, we generated models for the missing states based on experimental conductance and in-plane area expansion and aim to capture these conformations through stabilizing mutations using cryo-EM. We present the first attempt to chemically stabilize the fully open state of MscS in cryo-EM, resulting in a 2.6 √Ö map.

Scientific Focus Area: Structural Biology

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