The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

Abstract

The bacterial mechanosensitive channel of large conductance, MscL, is one of the best characterized mechanosensitive channels serving as a paradigm for how proteins can sense and transduce mechanical forces. The physiological role of MscL is that of an emergency release valve that opens a large pore upon a sudden drop in the osmolarity of the environment. A crystal structure of a closed state of MscL shows it as a homopentamer, with each subunit consisting of two transmembrane domains (TM). There is consensus that the TM helices move in an iris like manner tilting in the plane of the membrane while gating. An N-terminal amphipathic helix that lies along the cytoplasmic membrane (S1), and the portion of TM2 near the cytoplasmic interface (TM2(ci)), are relatively close in the crystal structure, yet predicted to be dynamic upon gating. Here we determine how these two regions interact in the channel complex, and study how these interactions change as the channel opens. We have screened 143 double-cysteine mutants of E. coli MscL for their efficiency in disulfide bridging and generated a map of protein-protein interactions between these two regions. Interesting candidates have been further studied by patch clamp and show differences in channel activity under different redox potentials; the results suggest a model for the dynamics of these two domains during MscL gating.

Figure 1. Schematic representation of MscL from the crystal structure of M. tuberculosis. (A) The scheme on the left represents a pentameric MscL in a closed state with the S1 and TM2_ci domains, in which the cysteine substitutions were performed, highlighted for clarity. The approximate location of the membrane is shown with horizontal gray lines. A single subunit is shown on the right to indicate all different MscL domains. (B)The same model with CPK representation of its residues shows how close the S1 and TM2_ci domains are predicted to be in the closed structure of MscL. In the bottom view it can be appreciated that the S1 and TM2_ci domains of alternating subunits, not direct neighbors, interact with each other.

Figure 2. Interactions between cytoplasmic domains. The graph summarizes the results of the in vivo disulfide trapping experiments and compares the efficiency of multimer formation between 143 MscL double cysteine mutants. The y and x axis reflect the sequence of amino acids of the S1 (residues 2 to 12) and TM2_ci (residues 93 to 105) regions respectively and the z axis reflects the percentage of total protein existing as multimers for each double cysteine mutant. The legend shows the color corresponding to each percentage value. All values represent the mean of at least three repeats.

Figure 3. Bridging M12C/N103C mutant locks the channel in a closed state (A). The scheme shows the corresponding position of the cysteine substituted residues M12 (red) and N103 (blue) in MscL M. tuberculosis structure, which reflects a nearly closed state. The insert is an amplification of the detail inside the square to show how close these two residues are predicted to be. (B) Western blot of M12C/N103C MscL derived from cells non-shocked (NS, left lane) or down-shocked in presence of 1.5 µM of the oxidizer copper-phenanthroline (S, right lane). Note that the protein derived from the shocked cells exists almost exclusively as a pentamer (97% of total protein). (C) Patch clamp analysis of M12C/N103C MscL in giant spheroplasts shows differential activities under different redox conditions. The traces show the M12C/N103C MscL channel activity elicited by applying negative pressure to the patch (in mmHg, below the traces). After adding the reducing agent DTT to the bath (upper trace), channel activity could be observed; but after washing the DTT from the bath and adding peroxide (lower trace) the channels were locked closed and could not be activated even at higher negative pressures.

Figure 4. Bridging I3C/I96C locks the channel into a sub-conducting state. (A) The scheme shows the corresponding position of the cysteine substituted residues I3 (red) and I96 (blue) in MscL M. tuberculosis structure. The insert is an amplification of the detail inside the square to show that I3 and I96 are not predicted to interact with each other in the closed MscL channel. (B) Western blot of I3C/I96C MscL derived from cells non-shocked (NS, left lane) or down-shocked in presence of 1.5 µM of the oxidizer copper-phenanthroline (S, right lane). Note that the protein derived from the shocked cells exists in multiple states from dimmers to pentamers, with multimers (3X to 5X) representing 54% of total protein. (C) Patch clamp analysis of I3C/I96C MscL in giant spheroplasts shows that the channel locks in a sub-conductive stated under oxidizing conditions. The traces show the I3C/I96C MscL channel activity elicited by applying negative pressure to the patch (in mmHg, below the traces). Under reducing conditions DTT (upper trace) channel activity could be observed with most openings reaching a single conductive state (o). After washing the DTT from the bath and adding peroxide (lower trace) the channels could still be activated but they stabilized in a sub-conductive state (s). (D) Amplitude histogram of traces from I3C/I96C patches under reducing (DTT red line) or oxidizing (peroxide blue line) conditions. Note that in the presence of peroxide a sub-conductive state is stabilized.

Figure 5. Correlation between multimer peaks and channel activity of MscL double mutants under different redox potentials. In a simplified version of the 3D graph shown in Figure 2, the multimer peaks for seven additional mutants are highlighted with red (mutants that lock close upon oxidation) or green (do not lock close upon oxidation). The inserts show the corresponding position in M. tuberculosis model of the substituted residues in E. coli MscL. Just two subunits are shown for clarity; residues in green are in S1 and those in red in TM2_ci. For each mutant the traces show single channel activity under reducing (black) or oxidizing (blue) conditions. The number under each trace is the negative pressure applied to the patch. Scale bars on the right bottom apply to all traces.