Disulfide trapping the mechanosensitive channel MscL into a gating-transition state

Abstract

The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock, and is to date the best characterized mechanosensitive channel. The N-terminal region of the protein has been shown to be critical for function by random, site-directed, and deletion mutagenesis, yet is structurally poorly understood. One model proposes that the extreme N-termini form a cluster of amphipathic helices that serves as a cytoplasmic second gate, separated from the pore-forming transmembrane domain by a "linker". Here, we have utilized cysteine trapping of single-cysteine mutated channels to determine the proximity, within the homopentameric complex, of residues within and just peripheral to this proposed linker. Our results indicate that all residues in this region can form disulfide bridges, and that the percentage of dimers increases when the channel is gated in vivo. Functional studies suggest that oxidation traps one of these mutated channels, N15C, into a gating-transition state that retains the capacity to obtain both fully open and closed states. The data are not easily explained by current models for the smooth transition from closed-to-open states, but predict that an asymmetric movement of one or more of the subunits commonly occurs upon gating.

FIGURE 1

Structural localization and conservation of S1-TM1 linker region. (A) Schematic representations of MscL based on the M. tuberculosis crystal structure (left) (14), which some evidence now suggests is in a “nearly closed” state (22,33), and the SG model for the closed structure of the E. coli MscL that was derived from it (right) (15,16). Lateral views of the homopentameric complex are shown with the residues that were cysteine substituted in this study (or the analog in M. tuberculosis) highlighted in black (R13–D18 in E. coli and R11–D16 in M. tuberculosis). (B) The result of an alignment of MscL residues R13–D18 from 129 homologs from different species is shown. Larger font sizes and darker boxes reflect more conserved residues. Black boxes are conserved in 80–99% of the aligned species (G14 99%, N15 98%, V16 80%, D18 91%). Dark boxes containing white fonts are conserved in 40–80% (R13 55%, K13 45%). Light gray boxes with white fonts 20–40% (V17 35%, I17 36%); lighter gray boxes with black font 5–20% (M17 14%, L17 11%), while very light gray boxes with letters in black fonts 2–5%, and no box reflects <2%. The alignment was generated using the ClustalW multiple sequence alignment program. A list of the species of the aligned proteins is available in online Supplementary Material.

FIGURE 2

Activity-dependent disulfide bridging between residues of the S1-TM1 linker region of MscL. (A) Western blots of MscL protein with single-cysteine substitutions; the sites of mutation range from residues R13–D18. Cells expressing each of the mutants were either diluted in a medium of the same osmolarity (mock-shock, upper panel) or osmotically downshocked (lower panel). MscL protein was found as either a monomer (1X arrows) or dimer (2X arrows) due to disulfide bridging between cysteines within the pentameric complex. The delayed migration of the monomers and dimers of D18C is consistent, and perhaps due to the removal of the charge. (B) Quantification of several experiments similar to the one shown in Fig. 2 A. The bars reflect the percentage of MscL protein existing as dimers, calculated from the optical density (represent the mean ± SE from at least 11 independent experiments). Gray bars correspond to mock-shocked whereas black bars correspond to osmotically shocked samples. Student's t-test was applied as two-tailed paired for each set (**) p < 0.00005, (*) p < 0.0005.

FIGURE 3

Disulfide bridging of MscL is mutant and oxidative-environment dependent. (A) Western blot analysis of samples from cells expressing N15C MscL protein after a mock-shock (MS) or an osmotic downshock (OS). N15C MscL protein exists as a monomer (1X arrow) or dimer (2X arrow), depending upon whether disulfide bonds are formed (left panel). The addition of β-mercapto-ethanol (βME) to a final concentration of 3% or DTT to 100 mM to the N15C samples was sufficient to completely reduce the disulfide bridges between cysteines from different subunits, thus leaving all the protein in its monomeric form (right panel). (B) Negative controls. Western blot analysis of samples from cells after an osmotic downshock. No MscL protein was detected in cells containing only vector (Ø), and dimers were absent in cells expressing either wild-type MscL or an independent TM1 MscL cysteine mutant, I24C.

FIGURE 4

The open probability of N15C MscL increases rapidly under oxidative conditions. (A) Representative trace of N15C channel activity recorded from an inside-out patch from E. coli giant spheroplasts. The reversible effects of oxidation on channel activity are shown. After the addition of H₂O₂, the open probability (P_o) of the channel increased dramatically (∼100-fold). This effect could be reversed by perfusion with a solution containing 1 mM of the reducing agent DTT, but absent of the H₂O₂ oxidant. Bars under the trace indicate the time in which the indicated drugs were present in the bath. Although a continuous trace, the double diagonal bars (//) reflect ∼15 s of the recording removed during the perfusion. A constant negative pressure of −200 mm Hg was applied to the patch throughout the entire trace shown. (B) Quantification of the change in NP_o upon oxidation. NP_o was binned in 5-s time spans with negative values corresponding to the time before the addition of H₂O₂ to the bath. Each recording was normalized independently to the average NP_o values before the treatment. Bars correspond to the mean ± SE of nine experiments. Note that the y axis is logarithmic, and that the full effect is seen within the shortest time accurately measured (5 s).

FIGURE 5

Oxidative conditions lead to increased open dwell times of the N15C MscL mutant. The histograms depict the open dwell distribution of N15C in a reduced (DTT, left) and oxidized (H₂O₂, right) state. Dwell times were fit with a three components model, as has been described before for wild-type channels and other mutants (19). The inserts within each graph are representative traces of channel activities observed in each condition.