Structural Rearrangements of MscS during Activation Gating

Vásquez, Valeria, Department of Molecular Physiology and Biological Physics, University of Virginia
Perozo, Eduardo, Department of Molecular Physiology and Biological Physics, University of Virginia

The mechanosensitive channel of small conductance (MscS) is part of a coordinated response to osmotic challenges in E. coli. MscS is particularly interesting since it is activated by changes in membrane tension and its inactivation is modulated by transmembrane voltage. Its crystal structure depicts a homoheptamer, in which each of the subunits has three TM segments and a large cytoplasmic domain. At first glance, the crystal structure seemed to be trapped in an open state based on the calculated diameter of the permeation pathway that appeared to agree with MscS single channel conductance. In this work using electrophysiological experiments and MD simulations we were able to determine that the crystal represents a non-conductive conformation. The crystal structure also suggested that several arginines were exposed to the lipids in TM1 and TM2, thus, it was proposed that these charged residues could act as voltage sensors. We have experimentally tested this proposal and we have found that none of the TM charges are involved in the MscS voltage sensing process. On the other hand, we established that an inter-subunit salt-bridge connecting the TM domain with the cytoplasmic basket influences the inactivation process, but the voltage sensor entity still remains ambiguous. In an effort to understand the molecular basis of MscS gating mechanism, we have improved the strategies for MscS spectroscopical analysis by optimizing the available purification and reconstitution protocols. We also have combined different approaches such as spin-labeling-EPR, high throughput functional assays and patch-clamp methods with classical molecular biology and biochemical procedures. By manipulating the location of reactive cysteines, we have introduced probes along MscS transmembrane domain. With these reporters, we obtained information on the topology, secondary, and tertiary structure of the closed and open conformations, using EPR analysis of spin labeled mutants under native-like conditions. In the closed conformation, MscS shows a more compact TM domain than in the crystal structure, described by a realignment of the TM segments towards the normal of the membrane, with the previously unresolved NH2- terminus placed at the periplasmic interface of the lipid bilayer. Whereas, the transition to the open state is characterized by an increase in overall dynamics, that involves major rearrangements such as burying of the NH2-terminus in the membrane, tilting of TM1- TM2 segments, expansion, rotation, and wetting of the TM3 helices. The present threedimensional models of membrane-embedded MscS in the closed state and the open conformation (in progress) represent key steps in determining the molecular mechanism of MscS gating.

PHD (Doctor of Philosophy)
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