Mechanosensitive ion channels play a critical role in transducing physical stresses at the cell membrane into an electrochemical response. The MscL family of large-conductance mechanosensitive channels is widely distributed among prokaryotes and may participate in the regulation of osmotic pressure changes within the cell. In an effort to better understand the structural basis for the function of these channels, the structure of the MscL homolog from Mycobacterium tuberculosis was determined by x-ray crystallography to 3.5 angstroms resolution. This channel is organized as a homopentamer, with each subunit containing two transmembrane α helices and a third cytoplasmic α helix. From the extracellular side, a water-filled opening approximately 18 angstroms in diameter leads into a pore lined with hydrophilic residues which narrows at the cytoplasmic side to an occluded hydrophobic apex that may act as the channel gate. This structure may serve as a model for other mechanosensitive channels, as well as the broader class of pentameric ligand-gated ion channels exemplified by the nicotinic acetylcholine receptor.

Materials and MethodsAs in the study of the previously determined MscL structure (S1), multiple homologs of MscS [encoded by the yggb gene (S2)] were identified by BLAST searching of the NCBI genome database. Ten homologs from prokaryotes and Archaea were identified and subsequently cloned. The channels were subcloned into expression vectors (pET system, Novagen) and expression screening was carried out. Cells expressing sufficient channels to be identified by Western blotting were subjected to extensive detergent screening utilizing ~50 detergents (Anatrace, Sigma, Aldrich) where both the ability of the channels to be extracted out of the membrane and the ability to remain as a homo-oligomer were determined. Subsequent large-scale expressions, extraction and purification produced sufficient amounts of protein for three channels (E. coli, B. subtilis and C. tepidum) for crystallization trials. Each of these channels was produced recombinantly (vector pet28b, Novagen) in 50-liter fermenter growths in a modified Terrific Broth media containing 1% glucose and 0.4% glycerol. Protein expression was initiated by the addition of 2% lactose and 2 mM IPTG for 2-4 hours, resulting in ~1.5 kg of wet cells. To obtain phase information, selenomethionine-derivatized protein was purified from cells grown in a modified M9 media containing 50 mg/l selenomethionine, and the remaining amino acids at 40 µg/l. Extraction of the E. coli MscS was carried out using sonication and solubilization with 1% Foscholine-14. Ni-affinity chromatography, anion exchange, and size exclusion chromatography in the presence of 0.05% Foscholine-14 were used to purify the protein to homogeneity. The apparent molecular mass of the protein, as indicated by size-exclusion chromatography, was in excess of 200 kD, similar to that reported for recombinant MscS by Sukharev (S3). Crystals were obtained with 10-15 mg/ml MscS by hanging drop vapor diffusion with 100 mM pH 7.2 Hepes buffer, 150 mM Na-formate, 8% glycerol, and 16% PEG-3350 as the precipitant. Crystals grew to ~200 µm in each dimension, and were assigned to space group P4 3 2 1 2 (a = b = 184.7 Å, c = 260.7 Å) with one MscS oligomer in the asymmetric unit (corresponding to ~71% solvent content). Only residues in the extramembrane (water-soluble) regions of MscS participated in lattice contacts.