The MscS-like channel YnaI has a gating mechanism based on flexible pore helices

Abstract

The mechanosensitive channel of small conductance (MscS) is the prototype of an evolutionarily diversified large family that fine-tunes osmoregulation but is likely to fulfill additional functions. Escherichia coli has six osmoprotective paralogs with different numbers of transmembrane helices. These helices are important for gating and sensing in MscS but the role of the additional helices in the paralogs is not understood. The medium-sized channel YnaI was extracted and delivered in native nanodiscs in closed-like and open-like conformations using the copolymer diisobutylene/maleic acid (DIBMA) for structural studies. Here we show by electron cryomicroscopy that YnaI has an extended sensor paddle that during gating relocates relative to the pore concomitant with bending of a GGxGG motif in the pore helices. YnaI is the only one of the six paralogs that has this GGxGG motif allowing the sensor paddle to move outward. Access to the pore is through a vestibule on the cytosolic side that is fenestrated by side portals. In YnaI, these portals are obstructed by aromatic side chains but are still fully hydrated and thus support conductance. For comparison with large-sized channels, we determined the structure of YbiO, which showed larger portals and a wider pore with no GGxGG motif. Further in silico comparison of MscS, YnaI, and YbiO highlighted differences in the hydrophobicity and wettability of their pores and vestibule interiors. Thus, MscS-like channels of different sizes have a common core architecture but show different gating mechanisms and fine-tuned conductive properties.

Structural Insights into the Venus flytrap Mechanosensitive Ion Channel Flycatcher1

“…This linker corresponds to the two helices extending from the TMD observed in the density map. Superposition of the C-terminal regions of FLYC1 to previously published MscS/MSL orthologs in various functional conformations 13,16,27,30,32 reveals that TM4-TM6a have an arrangement distinct from all other reported structures. Viewed from extracellular side, the outer TMs of FLYC1 are rotated clockwise relative to those in other structures (Supplementary Fig.…”

Biological insights from SMA-extracted proteins

“…It seems that the retained lipid environment when using SMA was important for preserving the complete protein structure. The mechanosensitive channel YnaI was isolated from E.coli using DIBMA rather than SMA, and it's structure determined by cryo-EM at a resolution of 3.0 Å [34] (Figure 1F). The overall structure was conserved with respect to the related MscS protein, but it was shown to have two additional transmembrane helices per subunit that extend the sensor paddle compared with MscS, although it should be noted that the resolution of these helices was only sufficient for the backbone to be modelled.…”

“…This suggests that these motions were exaggerated by the removal of the cavity lipids in structures of detergent-solubilised AcrB, and that the structural difference between wild-type and mutants of AcrB may be much more subtle [21]. Side view structure of YnaI (PDB ID 6ZYD) [34].Top view structure of GlyR showing bound partial agonist taurine (space filling grey) (PDB ID 6PM0) [46], alongside representative images of various types of functional assays. Bottom view of AcrB trimer showing central lipid filled cavity (space filling grey) (PDB ID 6BAJ) [21], alongside a representative mass spectrum for lipids co-purified with a protein from yeast.…”

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids

“…In DIBMA copolymers the styrene group is replaced with aliphatic moieties that are better suited to circular dichroism (CD) analysis of embedded proteins. These are also compatible with higher levels of divalent cations than conventional SMA [ 17 ], and have been used to solubilize channels [ 68 ] and GPCRs [ 98 ]. This backbone can also be derivatized with glucosamine and glycerol sidechains to expand solubility and utility.…”