Bacterial ion channels as the model structures

Eukaryotic ion channels have been studied over the last fifty years and an extensive knowledge has been accumulated on their function. We know little, however, about their molecular structures. Most of the structural information on eukaryotic ion channels currently available results from homology modeling based on the structures of their bacterial homologues. The crystal structures of two prokaryotic potassium channels from S. lividans and M. thermautotrophicum served as good models for determining the molecular principles of gating in potassium channels. The channel from S. lividans provides also the best model of K+ selectivity. Based on the structure of another potassium channel that from Aeropyrum pernix, several models of voltage sensing have been proposed. The two bacterial mechanosensitive channels from E. coli are to date the best-characterized mechanosensitive channel molecules in terms of structure and function and they may provide an excellent model for understanding the general principles of mechanotransduction in biological membranes

Cytoplasmic Domain of MscS Interacts with Cell Division Protein FtsZ: A Possible Non-Channel Function of the Mechanosensitive Channel in Escherichia Coli.

Bacterial mechano-sensitive (MS) channels reside in the inner membrane and are considered to act as emergency valves whose role is to lower cell turgor when bacteria enter hypo-osmotic environments. However, there is emerging evidence that members of the Mechano-sensitive channel Small (MscS) family play additional roles in bacterial and plant cell physiology. MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel. Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein. We identify point mutations in the MscS C-terminal domain that reduce binding to FtsZ and show that bacteria expressing these mutants are compromised in growth on sublethal concentrations of β-lactam antibiotics. Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics

Surface Changes of the Mechanosensitive Channel MscS upon Its Activation, Inactivation, and Closing

MscS is a bacterial mechanosensitive channel that shows voltage dependence. The crystal structure of MscS revealed that the channel is a homoheptamer with a large chamber on the intracellular site. Our previous experiments indicated that the cytoplasmic chamber of the channel is not a rigid structure and changes its conformation upon the channel activation. In this study, we have applied various sized cosolvents that are excluded from protein surfaces. It is well known that such cosolvents induce compaction of proteins and prevent thermal fluctuations. It is also known that they shift channel equilibrium to the state of lower volume. We have found that large cosolvents that cannot enter the channel interior accelerate channel inactivation when applied from the cytoplasmic side, but they slow down inactivation when applied from the extracellular side. We have also found that small cosolvents that can enter the channel cytoplasmic chamber prevent the channel from opening, unlike the large ones. These data support our idea that the channel cytoplasmic chamber shrinks upon inactivation but also give new clues about conformational changes of the channel upon transitions between its functional states

ABDOM binds FtsZ but does not interfere with its polymerization neither <i>in vitro</i> nor <i>in vivo</i>.

<p>A. Purified MBP-ABDOM bound assembled FtsZ <i>in vitro</i>. The MBP-ABDOM fusion protein, but not MBP alone, co-sedimented with polymerized FtsZ. Fixed concentration of FtsZ (16 μM) was co-sedimented by addition of 1mM GTP in the presence of increasing concentration of MBP or MBP-ABDOM. For reference, equal amounts of MBP-ABDOM and MBP supernatant without FtsZ are shown in rightmost lane. Proteins were detected with anti-MBP antibody. <b>B.</b> MBP-ABDOM had no effect on FtsZ polymerization/depolymerization <i>in vitro</i>, as detected by 90° angle light scattering. FtsZ was polymerized after addition of 1 mM GTP (arrow) in the presence of either MBP-ABDOM (black line) or MBP (red line) <b>C.</b> Filaments produced by MscSΔ266–286 expression contain multiple nonfunctional Z-rings (upper row), which resemble Z-rings arrested by the cephalexin inhibition of PBP3 (middle row), control cells (lower row). FtsZ-YFP was expressed to visualize Z-rings. Scale bar represents 10 μm. <b>D.</b> Peptidoglycan synthesis detected by immunofluorescent D-cysteine labeling. Overexpression of MscSΔ266–286 resulted in cell filamentation, with clear dark rings of newly synthesized murein in septal areas (left). A similar pattern of murein segregation was observed in cells treated with 1μg/ml aztreonam, a selective inhibitor of PBP3 whose septal localization depends on functional Z-rings (right). Experiments were carried out over one cell cycle in D-cysteine, followed by 30 min. chase. The labeled, older murein is seen as bright spots, while newly made murein is seen as dark areas. Scale bar represents 1 μm.</p

Alanine substitutions of K258 and R259 in ABDOM impair its binding to FtsZ <i>in vitro</i>.

<p>A. A crystal structure of nonconductive MscS (PDB ID: 2OAR), amino acids K258 and R259 are shown in red spacefill. <b>B</b>. Double alanine substitution K258A/R259A impaired ABDOM binding to FtsZ. Co-sedimentation experiments were carried out similarly to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127029#pone.0127029.g002" target="_blank">Fig 2</a>, panel A. The data were fitted with Hill model (y = xⁿ/(Kd+xⁿ) with Kd = 5.3 μM, n = 2.6 and Kd = 14.6 μM, n = 4.3 for MBP-ABDOM and MBP-ABDOM-K258A/R259A respectively.</p

MscS protects cells from β-lactam antibiotics.

<p>Experiments were conducted on plasmid transformed MJF429 strain in two different concentrations of ampicillin: 1.6 μg/ml (<b>A</b>, <b>C</b>, <b>E</b>) and 4.1μg/ml ampicillin (<b>B</b>, <b>D</b>, <b>F</b>). <b>A, B.</b> Overexpression of MscS but not MscS-K258A/R259A or MscS-YFP supports the cell growth in the presence of subMIC of ampicillin. Inset in <b>B</b>: growth of ampicillin treated cells shown using expanded Y axis scale. <b>C, D.</b> The number of elongated cells estimated from forward scatter (FSC) was reduced for bacteria expressing MscS (green) as compared to MscS-K258A/R259A (pink) or MscS-YFP (blue). Sample FSC histograms are presented in insets, for complete FSC analysis see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127029#pone.0127029.s008" target="_blank">S8 Fig</a>, panels A-C. <b>E, F.</b> The number of cells with compromised membrane integrity estimated using PI staining was decreased for bacteria expressing MscS (green) as compared to MscS-K258A/R259A (pink) or MscS-YFP (blue). Sample scatter plots are presented for complete FC analysis see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127029#pone.0127029.s008" target="_blank">S8 Fig</a>, panels D-F. Cells grown in A, B were then used in experiments presented in <b>C</b>, <b>D</b>, <b>E</b>, <b>F</b>. For statistically different results p-values < 0.05 of paired Student`s t-test are shown above the graphs (n = 3).</p

Hypothetical model of MscS acting as a dual-action protective gate.

<p>A. Under non-stress conditions MscS remains in the closed state and the ABDOM domain of MscS is inaccessible to interaction with FtsZ. <b>B.</b> Under severe osmotic downshocks the channel opens and jettisons osmolytes rapidly returning to the closed state. The short-lived open state does not result in formation of the cell-wall remodeling complex. <b>C.</b> Under slowly rising and prolonged local increase of tension within the bulges of the membrane the channel performs transition into the inactivated state. In this channel conformation ABDOM becomes accessible and binds FtsZ.</p

MscS, the structure and its mutations affecting cell shape.

<p><b>A.</b> Crystal structure of the MscS homoheptamer (PDB ID:2OAR) representing the nonconductive state of the channel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127029#pone.0127029.ref036" target="_blank">36</a>]. ABDOM (aa 175–265) of each subunit is shown in orange and the C-terminal fragment (aa 266–286), which is deleted in the MscSΔ266–286 mutant, is shown in red. White arrow indicates the thickness of the membrane. <b>B.</b> Overexpression of <b>ABDOM</b> leads to cell filamentation, while cells expressing <b>wt-MscS</b> did not differ in shape from control cells (<b>empty vector</b>). The highest level of cell filamentation was observed when truncated <b>MscSΔ266–286</b> was expressed (note the presence of branched filaments). The amount of regular cell filaments induced by MscSΔ266–286 was unaffected by double mutation <b>A51N/F68N</b>, which prevents ion conduction through the channel pore. Scale bar represents 10 μm. Expression of the proteins was confirmed by Western blotting variants tagged with HA epitope (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127029#pone.0127029.s001" target="_blank">S1 Fig</a>).</p