The Closed Structure of the MscS Mechanosensitive Channel

Abstract

Mechanosensitive channels must make a large conformational change during the transition from the closed to the open state. The crystal structure of the open form of the Escherichia coli MscS channel was recently solved and depicts a homoheptamer (1). In this study, crosslinking of site-specific cysteine substitutions demonstrates that residues up to 10 -33 Å apart in the crystal structure readily form disulfide bridges in the closed form and can also be cross-linked by a 10-Å linker. Crosslinking between adjacent subunits stabilizes the heptameric form of the channel providing biochemical evidence to support the crystal structure. The data are consistent with the published model (1) in that the membrane domain is highly flexible and that the closed to open transition may involve a significant displacement of transmembrane helices 1 and 2, possibly by as much as 30 Å. The data are also consistent with significant flexibility of the cytoplasmic domain. Mechanosensitive (MS)1 channels underpin vital sensory processes in higher organisms and are responsible for structural integrity of bacterial cells during the transition from high to low osmolarity (downshock) (2-4). In the bacterial membrane closed MS channels must maintain the cation impermeability of the membrane, which is an essential requirement for energy transduction. Open MS channels in Escherichia coli exhibit conductances of 0.3-3 nanosiemens (5), consistent with the formation of large pores in the membrane of at least an 11-Å diameter (1, 6, 7). Clearly MS channels must undergo large conformational changes in the protein structure during the transition from the closed to the open state. The recent crystal structures of the bacterial MS channels, MscL and MscS, have been achieved for the closed and open states, respectively (1, 8). Model building, coupled with biochemical analysis of single cysteine mutants, has supported molecular dynamics-based analyses of the structural transitions that may open MscL (6, 7, 9 -11). However, the two proteins are so individual that there is little from the one structure that informs the possible mechanism of the other.The MscS protein has been much less intensively studied as a result of the relatively recent discovery of the structural gene (12). Genetic and biochemical analyses established that the protein has three transmembrane (TM) helices and a large carboxyl-terminal cytoplasmic domain (13). This was confirmed by resolution to 3.8 Å of a crystal structure of the MscS channel that also showed the protein to be a homoheptamer (1). The large cytoplasmic domain was demonstrated to form a 40-Å diameter chamber perforated by eight holes, seven of which arise at the boundary between pairs of monomers and the eighth from the formation of a ␤ barrel by the carboxyl-terminal 15 residues. In the crystal structure the TM3 helix bends at Gly 113 , such that the carboxyl-terminal part of this helix lies parallel to the membrane surface. The loop between TM2 and TM3 (residues 91-95) forms an extended chain that is part of the wa...

YbdG in Escherichia coli is a threshold-setting mechanosensitive channel with MscM activity

“…We have previously demonstrated the efficacy of cross-linking to estimate the oligomeric state of MscS channel complexes (28). A single native Cys residue (Cys102) is at the periplasmic end of the putative TM1 helix of YbdG (Fig.…”

“…Because the contribution of chromosomally expressed YbdG to protection is subtle, it is clear that the normal abundance of this channel protein is too low to afford the high levels of protection seen with MscS and MscL. This lack of channel abundance occurs despite increased transcription and Membrane fractions of MJF612 cells expressing the indicated mutant were isolated and cysteine cross-linking was performed using copper phenanthroline (Cu-phen) (28). A total of 75 μg of membrane proteins was incubated with 167 μM phenanthroline (+) or the solvent ethanol (−) and 13 μg was separated on SDS/PAGE under nonreducing (A) or reducing (B) conditions.…”

“…Restriction sites NcoI and XhoI were introduced in frame at the start and stop of the gene, respectively, by sitedirected mutagenesis using primers YbdGXhoI-1, YbdGXhoI-2, YbdGNcoI-1, and YbdGNcoI-2. To create a His 6 -tagged protein, the plasmid pTrcMscSH 6 was used for subcloning (28). Successful creation of pTrcYbdGH 6 was checked by PCR amplification and restriction analysis and confirmed by sequencing.…”

“…Cysteine cross-linking using copper phenanthroline was carried out on membrane samples as reported previously (28). Reactions were run on SDS gels and Western blot analysis was carried out as described above.…”

Structural Rearrangements of MscS during Activation Gating

“…At 25 kDa, MscS in SDS-PAGE gels runs smaller than its theoretically predicted molecular weight (31 kDa). This behavior was previously reported by a number of labs (Miller et al, 2003a;Miller et al, 2003b;Schumann et al, 2004;Sukharev, 2002). It is noteworthy that MscS, after purification, has some level of resistance to SDS, a strong anionic detergent that promotes oligomer dissociation in most membrane proteins.…”