Lipid availability within transmembrane nano-pockets of ion channels is linked with mechanosensation. However, the effect of hindering lipid-chain penetration into nano-pockets on channel structure has not been demonstrated. Here we identify nano-pockets on the large conductance mechanosensitive channel MscL, the high-pressure threshold channel. We restrict lipid-chain access to the nano-pockets by mutagenesis and sulfhydryl modification, and monitor channel conformation by PELDOR/DEER spectroscopy. For a single site located at the entrance of the nano-pockets and distal to the channel pore we generate an allosteric response in the absence of tension. Single-channel recordings reveal a significant decrease in the pressure activation threshold of the modified channel and a sub-conducting state in the absence of applied tension. Threshold is restored to wild-type levels upon reduction of the sulfhydryl modification. The modification associated with the conformational change restricts lipid access to the nano-pocket, interrupting the contact between the membrane and the channel that mediates mechanosensitivity.

The mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis has been used as a structural model for rationalizing functional observations in multiple MscL orthologs. Although these orthologs adopt similar structural architectures, they reportedly present significant functional differences. Subtle structural discrepancies on mechanosensitive channel nanopockets are known to affect mechanical gating and may be linked to large variability in tension sensitivity among these membrane channels. Here, we modify the nanopocket regions of MscL from Escherichia coli and M. tuberculosis and employ PELDOR/DEER distance and 3pESEEM deuterium accessibility measurements to interrogate channel structure within lipids, in which both channels adopt a closed conformation. Significant in-lipid structural differences between the two constructs suggest a more compact E. coli MscL at the membrane inner-leaflet, as a consequence of a rotated TM2 helix. Observed differences within lipids could explain E. coli MscL’s higher tension sensitivity and should be taken into account in extrapolated models used for MscL gating rationalization.

Membrane proteins execute a wide variety of critical biological functions in all living organisms and constitute approximately half of current targets for drug discovery. As in the case of soluble proteins, the bacterium Escherichia coli has served as a very popular overexpression host for biochemical/structural studies of membrane proteins as well. Bacterial recombinant membrane proteins production, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low levels of final biomass and minute volumetric yields. In previous work, we generated the engineered E. coli strains SuptoxD and SuptoxR, which upon coexpression of the effector genes djlA or rraA, respectively, can suppress the cytotoxicity caused by membrane protein overexpression and produce enhanced membrane protein yields. Here, we systematically looked for gene overexpression and culturing conditions that maximize the accumulation of membrane-integrated and well-folded recombinant MPs in these strains. We have found that, under optimal conditions, SuptoxD and SuptoxR achieve greatly enhanced recombinant membrane protein production for a variety of membrane proteins, irrespective of their archaeal, eubacterial or eukaryotic origin. Furthermore, we demonstrate that the use of these engineered strains enables the production of well-folded recombinant MPs of high quality and at high yields, which are suitable for functional and structural studies. We anticipate that SuptoxD and SuptoxR will become broadly utilized expression hosts for recombinant membrane protein production in bacteria.

Highlights d Pocket delipidation of TM pockets stabilizes an expanded MscL state d HDX and EPR probe conformational transitions of MscL d Bilayer stretching MD independently generates a tensionactivated state d Structural analogy between these states has implications in MS channel regulation Authors

Escherichia coli is one of the most widely utilized hosts for recombinant protein production, including that of membrane proteins (MPs). We have recently engineered a specialized E. coli strain for enhanced recombinant MP production, termed SuptoxR. By appropriately co-expressing the effector gene rraA, SuptoxR can suppress the high toxicity, which is frequently observed during the MP-overexpression process, and, at the same time, enhance significantly the cellular accumulation of membrane-incorporated and properly folded recombinant MP. The combination of these two beneficial effects results in dramatically enhanced volumetric yields for various prokaryotic and eukaryotic MPs. Here, we engineered second-generation SuptoxR strains with further improved properties, so that they can achieve even higher levels of recombinant MP production. We searched for naturally occurring RraA variants with similar or improved MP toxicity-suppressing and production-promoting effects to that of the native E. coli RraA of the original SuptoxR strain. We found that the RraA proteins from Proteus mirabilis and Providencia stuartii can be even more potent enhancers of MP productivity than the E. coli RraA. By exploiting these two newly identified RraAs, we constructed two second-generation SuptoxR strains, termed SuptoxR2.1 and SuptoxR2.2, whose MP-production capabilities often surpass those of the original SuptoxR significantly. SuptoxR2.1 and SuptoxR2.2 are expected to become widely useful expression hosts for recombinant MP production in bacteria.