List of plasmids used in the study.

<p>List of plasmids used in the study.</p

Twin-arginines are not essential for REMP binding.

<p>(<b>A</b>) SPR response curves obtained by injecting solutions containing 100 nM ssTorA-GFP (black line) or 100 nM ssTorA(KKK)-GFP (red line) over immobilized TorD. (<b>B</b>) Response curves obtained by injecting solutions of 100 nM ssDmsA-GFP (black line) or 100 nM ssDmsA(KK)-GFP (red line) over immobilized DmsD.</p

DmsD reduces membrane binding of ssDmsA-GFP.

<p>(<b>A</b>) SPR response curves of various proteins injected over a phospholipid bilayer that mimics the composition of the <i>E. coli</i> inner membrane. Injected solutions contain either 50 nM signal peptide-free Strep-GFP (orange line), 50 nM ssDmsA-GFP (blue line), 50 nM ssDmsA-GFP with 100 nM DmsD (red line), 50 nM ssDmsA-GFP with 200 nM DmsD (green line), 50 nM TorD (grey line), or 500 nM DmsD (black line). The red and green curves were corrected for the jump in refractive index by subtracting the response curves for injections of solutions containing 100 or 200 nM DmsD, respectively. (<b>B</b>) SPR response curve for an experiment in which DmsD was injected over a surface consisting of ssDmsA-GFP bound to a phospholipid bilayer. ssDmsA-GFP (100 nM) was injected over an immobilized phospholipid bilayer for a period of 100 s. The surface was washed with buffer for 500 s to remove weakly bound ssDmsA-GFP. Subsequently, buffer containing 25 nM DmsD was injected.</p

The signal sequence ensures specific REMP binding.

<p>Surface plasmon resonance sensorgrams for the injection of various proteins over immobilized TorD (<b>A</b>) and DmsD (<b>B</b>) are shown. The model pre-proteins ssDmsA-GFP (black lines), ssTorA-GFP (red lines) and signal sequence-free GFP (blue lines) were injected for 60 seconds at a concentration of 200 nM, a flow rate of 50 µl/min, and a temperature of 25°C.</p

Interactions of chimeric signal sequences with REMPs.

<p>The composition of the signal sequences is shown on the left as a three-letter code. This indicates whether the N-, H-, and C-regions originate from the DmsA (D) or the TorA (T) signal sequence, respectively. For example, DTT represents a signal sequence that consists of the N-region of DmsA followed by the H- and C-regions of TorA. For each chimeric pre-protein the SPR response curve is shown for an injection of a 200 nM solution over immobilized TorD and a DmsD, respectively, as indicated at the top of the graph. The response curves for the wild-type signal sequences and their corresponding REMPs are shown in red. A response curve for 200 nM signal peptide-free Strep-GFP is shown in the bottom panel.</p

Determination of the dissociation constant for ssTorA-GFP binding to immobilized TorD.

<p>(<b>A</b>) Raw SPR sensorgrams and (<b>B</b>) referenced sensorgrams of ssTorA-GFP binding to immobilized TorD at the following pre-protein concentrations (shown from dark to light grey): 39 nM, 78 nM, 156 nM, 313 nM, 625 nM, 1250 nM. The sensorgram for a buffer injection is shown in black. (<b>C</b>) Equilibrium SPR intensity for binding of ssTorA-GFP to TorD at various concentrations in a Scatchard plot (open symbols, see main text for details). The intensity values used are the average SPR response derived from the shaded area (22–28 s) in panel A. The data are fitted to a straight line (black). (<b>D</b>) The SPR intensity at equilibrium is plotted as a function of pre-protein concentration, for binding to immobilized TorD. The pre-proteins used are ssTorA-GFP (shown in black), ssTorA(RK)-GFP (shown in blue), ssTorA(KR)-GFP (shown in green) and ssTorA(KK)-GFP (shown in red). The best fit of the Langmuir binding isotherm (Equation 1) to the data is shown as a line in the corresponding color.</p

Dissociation constants for TorA signal peptides binding to TorD.

1<p>Dissociation constants from this work are obtained from SPR data by fitting the Langmuir equation (Equation 1) to the equilibrium SPR response as a function of pre-protein concentration. Errors are standard fitting errors.</p>2<p>ss, signal sequence; GFP, green fluorescent protein; MBP, maltose binding protein; SBP, streptavidin binding peptide.</p

TMC207 and its target mycobacterial ATP synthase.

<p>(<b>A</b>) Structure formula of TMC207. (<b>B</b>) ATP synthase subunit composition with subunit c in grey. A homology model of a subunit c monomer from <i>Mycobacterium tuberculosis</i> is shown enlarged. The acidic residue Glu61, essential for proton transport, is depicted in red. Point mutations that influence mycobacterial sensitivity for TMC207 are indicated in colour.</p

ATP synthesis inhibition by TMC207 at low proton motive force.

<p>(<b>A</b>) Inverted membrane vesicles from <i>Mycobacterium smegmatis</i> were diluted to 0.18 mg/ml in buffer containing 2 µM ACMA. To detect the proton motive force, quenching of ACMA fluorescence was investigated after addition of 5 mM succinate in the presence of increasing concentrations of the uncoupler SF6847. At the indicated time point, 1 µM of uncoupler SF6847 was added as control to collapse the proton gradient. (<b>B</b>) ATP synthesis by membrane vesicles of <i>M. smegmatis</i> (1 mg/ml) was measured in the presence of TMC207 and varying concentrations of uncoupler SF6847 to modulate the proton motive force. Samples were incubated at 37°C for 1 h in the presence of an ADP-regenerating system, and produced ATP was quantified spectrophotometrically by monitoring oxidation of glucose-6-phosphate with NADP⁺. As a control, 100 µM DCCD was added.</p

TMC207 binds to a defined binding site in ATP synthase.

<p>(<b>A</b>) The dose-dependency of ATP synthesis inhibition by TMC207 in inverted membrane vesicles of <i>Mycobacterium smegmatis</i> was fitted with a one-site binding hyperbola (Y = 104.9X/6.3+X, R²>0.99) (<b>B</b>) Binding of purified ATP synthase subunit c from <i>Mycobacterium tuberculosis</i> to an amine analog of TMC207 linked onto a BIAcore chip was fitted using mono-exponential binding models (Association = Req*(1−exp(−1*53737X)) and Dissociation = 165.654*exp(−1*0.002295*(X−45)) R²>0.99) and (<b>C</b>) Binding of purified ATP synthase from <i>Bacillus</i> PS3 to an amine analog of TMC207 linked onto a BIAcore chip was fitted using mono-exponential binding models (Association = Req*(1−exp(−1*153.7X)) and Dissociation = 8575.97*exp(−1*0.0001030*(X−1187)) R²>0.99).</p