The Gating Mechanism of the Large Mechanosensitive Channel in Escherichia coli and Effects of Gain-of-Function Mutations

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The mechanosensitive channel of large conductance (MscL) in Escherichia coli is perhaps the best-characterized mechanosensitive protein. The structure of the Mycobacterium tuberculosis ortholog has been solved recently by X-ray crystallography, but the structural rearrangements associated with gating remain obscure. Based on the crystal structure, a homology model of E. coli MscL had been built and the gating process was proposed (Sukharev et al., 2001). I experimentally verified these working models. The result demonstrates that the iris-like expansion of M1 helixes does occur during the gating transition. In addition to the hydrophobic M1 gate, the S1 segments unresolved in the original crystal structure were easily cross-linked with pairs of cysteines and prevented opening, consistent with the proposed function of a second gate. Although the early models predicted a wide-open conformation with cytoplasmic S3 domains separated, the current data strongly suggest that S3 domains are in fact stably associated in both closed and open conformations. The open-state model predicts an in-plane expansion of the channel protein of about 23 nm2. The analysis of multiple MscL dose-response curves, accounting for non-homogeneity of channels in a population, (i.e. variable energy or area changes for individual channels), estimated the total channel expansion ~20 nm2 and the transition energy ~52 kT, consistent with molecular models of the open state. Gain-of-function (GOF) mutants with hydrophilic or charged substitutions in the main hydrophobic gate stably occupy low-conducting substates. The character of perturbations introduced in the main gate by GOF substitutions strongly supports the two-gate mechanism in which the first sub-transition (C->S) can be viewed as the opening of the M1 gate formed by the first transmembrane domains, resulting in an expanded leaky conformation (S). The second sub-transition (S->O) can be attributed to the separation of the N-terminal (S1) gate resulting in the fully conductive channel