MscL has been shown in previous studies to be, in general, quite tolerant of cysteine mutations.9
Perhaps partially because of this finding, cysteine trapping experiments have been utilized to address proximity of regions of the protein that are thought to interact. However, in some instances, researchers have been misled by utilizing targeted mutagenesis and disulfide trapping experiments, proposing models later shown to be false. For example, S1 was thought at one time to form a helical bundle that served as a second gate.18,19
The major form of evidence for this model was targeted disulfide trapping. However, when the entire region
was scanned and disulfide trapping assessed and compared,15
the results were consistent with the newly re-evaluated structure7
: S1 was an amphipathic helix running along the cytoplasmic membrane. There was no evidence that a S1 “second gate” was ever formed during the gating process. We have designed the experiments used in this study to avoid some of the many potential pitfalls of the disulfide trapping approach. First, we have not used a targeted approach. Instead, we have generated and screened 143 double cysteine mutants, which act as controls for each other; relative
amounts of disulfide bridging can easily be assessed. Second, the amount of oxidant used was carefully titrated to yield the maximum range of disulfide bridging; none of the interactions were forced by high concentrations of oxidant. Indeed, the amounts used in this study are 100 times less than that used to study TM1/TM2 interactions, where the interactions may be more insular and transient upon gating.17
Finally, we use an in vivo approach in which the membrane proteins are not solubilized prior to being resuspended in SDS running buffer; hence disulfide bridges cannot form in mild detergents that would preserve the pentameric complex while possibly allowing more dynamic interactions not reflecting any normal physiological state. We believe that the following of these practices when utilizing a disulfide trapping approach yields a more true and interpretable set of data.
Our current model for S1 function is that it serves as a stabilizer for the twisting and turning of the pore-forming first transmembrane domain.15
In addition, all current models for the structural changes that occur upon gating include a tilting of both transmembrane domains within the plane of the membrane. In addition to tilting, for TM1 several lines of evidence also suggest a clockwise “corkscrewing” of TM1 of almost 180°, as would be observed from the periplasm; these lines of evidence include EPR experiments,20
disulfide trapping experiments within the pore9
and between TM1 and TM2 upon gating,17
accessibility and gating influences of the sulfhydryl reagent MTSET+
on TM cysteine mutants,21,22
and the engineering of heavy metal binding sites within the pore region.8
The S1 amphipathic helix, with its two conserved phenylalanines, is joined to this dynamic TM1 by a conserved flexible glycine residue at position 14 that has been shown to have functional significance.9,23
Such a helix running along the membrane is observed in many channels including the other mechanosensitive channel in bacteria, MscS,7,24
the “slide helix” of KirBac1.1,25
a putative amphipathic “gate anchor” domain studied in TRPY1,26
and several others where they may also serve as stabilizers.
Although TM2 is not thought to “corkscrew” within the membrane, it is thought to tilt within it upon gating. Indeed, a recent study of the dynamics of the TM2ci
region has shown that channel gating can be influenced by modifications that make this region more hydrophobic.27
One residue, N103, was even shown to transiently insert into the membrane during the gating process in a piston-like manner. Hence, this region shows a dynamics within and along the membrane upon channel opening.
The proximity of the S1 and TM2ci regions is obvious in the closed M. tuberculosis MscL structure. An analysis of the equivalent residues to E. coli M12 and N103 is entirely consistent with our results that these residues, when mutated to cysteines, readily disulfide bridge and form a channel resistant to opening. On the other hand, several residue combinations, including the distal I3C and I96C, are not predicted to be close in the M. tuberculosis structure, do not lock the channel in the closed state when oxidized, but do stabilize substates. These data are consistent with the models for the dynamics of the S1 and TM2ci outlined above: upon gating the S1 helix slides along the cytoplasmic membrane, where it can interact with residues further up in the membrane portion of TM2 because of the tilting of this latter domain. Thus, overall the double cysteine mutants that bridge at high efficiency fall in two main categories: those that are predicted to be in close proximity from the closed structure and do lock the channel closed under oxidizing conditions, and those interactions consistent with the predicted sliding and tilting of these regions upon gating that show stabilized subconducting states when oxidized.
Collectively, the data from this study, as well as many others, have led to a cohesive model of how MscL senses and responds to mechanical forces. We know, for instance, that MscL senses the tension in the membrane3
; more specifically, changes within the lateral pressure profile.28
The observation that many other mechanosensors also have lower thresholds or are activated by amphipaths suggests this is a common stimulus for MS channels.29
Furthermore, the observation that the channel can function in a bilayer composed of phosphatidylcholine, which contains a zwitter-ionic headgroup not synthesized by E. coli
, demonstrates that neither interactions with negatively charged lipids nor native lipid headgroups are required for normal MS channel activity.3
Both transmembrane domains tilt within the membrane, and TM1 rotates in a corkscrew fashion clockwise, as observed from the periplasm.1
Finally, this study has helped to define the interactions between the S1 and the TM2ci
domains; the findings are consistent with the model of S1 maintaining its association with the cytoplasmic side of the membrane and thus serving as a stabilizer for the pore-forming TM1, and the dynamics of the TM2ci
subdomain and the predicted tilting of TM2 within the membrane.