The uniqueness of subunit α of mycobacterial F-ATP synthases: An evolutionary variant for niche adaptation

The F1F0 -ATP (F-ATP) synthase is essential for growth of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). In addition to their synthase function most F-ATP synthases possess an ATP-hydrolase activity, which is coupled to proton-pumping activity. However, the mycobacterial enzyme lacks this reverse activity, but the reason for this deficiency is unclear. Here, we report that a Mycobacterium-specific, 36-amino acid long C-terminal domain in the nucleotide-binding subunit α (Mtα) of F-ATP synthase suppresses its ATPase activity and determined the mechanism of suppression. First, we employed vesicles to show that in intact membrane-embedded mycobacterial F-ATP synthases deletion of the C-terminal domain enabled ATPase and proton-pumping activity. We then generated a heterologous F-ATP synthase model system, which demonstrated that transfer of the mycobacterial C-terminal domain to a standard F-ATP synthase α subunit suppresses ATPase activity. Single-molecule rotation assays indicated that the introduction of this Mycobacterium-specific domain decreased the angular velocity of the power-stroke after ATP binding. Solution X-ray scattering data and NMR results revealed the solution shape of Mtα and the 3D structure of the subunit α C-terminal peptide 521PDEHVEALDEDKLAKEAVKV540 of M. tubercolosis (Mtα(521–540)), respectively. Together with cross-linking studies, the solution structural data lead to a model, in which Mtα(521–540) comes in close proximity with subunit γ residues 104–109, whose interaction may influence the rotation of the camshaft-like subunit γ. Finally, we propose that the unique segment Mtα(514–549), which is accessible at the C terminus of mycobacterial subunit α, is a promising drug epitope.

The F 1 F 0 -ATP (F-ATP) synthase is essential for growth of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). In addition to their synthase function most F-ATP synthases possess an ATP-hydrolase activity, which is coupled to proton-pumping activity. However, the mycobacterial enzyme lacks this reverse activity, but the reason for this deficiency is unclear. Here, we report that a Mycobacterium-specific, 36-amino acid long C-terminal domain in the nucleotide-binding subunit ␣ (Mt␣) of F-ATP synthase suppresses its ATPase activity and determined the mechanism of suppression. First, we employed vesicles to show that in intact membrane-embedded mycobacterial F-ATP synthases deletion of the C-terminal domain enabled ATPase and protonpumping activity. We then generated a heterologous F-ATP synthase model system, which demonstrated that transfer of the mycobacterial C-terminal domain to a standard F-ATP synthase ␣ subunit suppresses ATPase activity. Single-molecule rotation assays indicated that the introduction of this Mycobacterium-specific domain decreased the angular velocity of the power-stroke after ATP binding. Solution X-ray scattering data and NMR results revealed the solution shape of Mt␣ and the 3D structure of the subunit ␣ C-terminal peptide 521 PDEHVEALDEDKLAKEAVKV 540 of M. tubercolosis (Mt␣(521-540)), respectively. Together with cross-linking studies, the solution structural data lead to a model, in which Mt␣(521-540) comes in close proximity with subunit ␥ residues 104 -109, whose interaction may influence the rotation of the camshaft-like subunit ␥. Finally, we propose that the unique segment Mt␣(514 -549), which is accessible at the C terminus of mycobacterial subunit ␣, is a promising drug epitope.
The F 1 F 0 -ATP synthase has been shown to be essential for growth in Mycobacterium smegmatis and Mycobacterium tuberculosis (Mt), with the latter causing tuberculosis (TB) 5 (1)(2)(3). This is different in other prokaryotes, where the enzyme is dispensable for growth on fermentable carbon sources and where increased glycolytic flux can compensate for the loss of oxidative phosphorylation (4). The difference was attributed to the extraordinarily high amount of ATP required to synthesize a mycobacterial cell (5). A special feature of the mycobacterial F-ATP synthase is its inability to establish a significant proton gradient during ATP hydrolysis, and its low or latent ATPase activity in the fast-or slow-growing form (6). Latency of ATP hydrolysis activity has also been observed for the thermoalkaliphile Bacillus sp. TA2.A1 (7)(8)(9) and the ␣-proteobacterium Paracoccus denitrificans F-ATP synthase (7, 10 -13), which results from distortion of the asymmetrical ␥-rotor within the ␣ 3 ␤ 3 -cylinder (13) and the -inhibitor protein, respectively (14).
The mycobacterial F 1 F 0 -ATP synthase is composed of nine subunits with a stoichiometry of ␣ 3 :␤ 3 :␥:␦:⑀:a:b:bЈ:c 9 , and organized as a membrane-embedded F 0 complex (abbЈc 9 ), and a water-soluble F 1 complex (␣ 3 ␤ 3 ␥␦⑀). The F 1 part contains three catalytic ␣␤-pairs that form an ␣ 3 ␤ 3 -hexamer, in which ATP synthesis or -hydrolysis takes place. This catalytic ␣ 3 ␤ 3 -headpiece is linked with the ion-pumping F 0 part via the two rotating central stalk subunits ␥ and ⑀, as well as the peripheral stalk subunits b, bЈ, and ␦ (3). The F 0 domain contains subunit a, and a ring structure consisting of 9 c subunits (15). The latter are proposed to form a helix-loop-helix structure, where the loops dock to the membrane facing, globular domain of the rotary ␥ subunit and enable the coupling to the F 1 portion to transfer torque, derived by ion transport, to the catalytic ␣ 3 ␤ 3 -headpiece (16). Thus, the rotational movement of the c-ring forces the central subunits ␥ and ⑀ to rotate (17), causing sequential conformational changes in the nucleotide-binding subunits ␣ and ␤ that result in the condensation of ADP ϩ P i to form ATP (18). In the case of ATP hydrolysis, the cleavage of ATP to ADP ϩ P i in the interfaces of three ␣␤-pairs leads to conformational changes in their C-terminal domains and subsequently to the stepwise counterclockwise 120°rotation of the central ␥ subunit (when viewed from the membrane side) (19). In prokaryotic F-ATP synthases described to date (20 -22), each 120°rotation is divided into 40°a nd 80°substeps, where ATP binding to an empty ␣␤-pair (␣␤) E induces the 80°substep, followed by ATP hydrolysis and ADP release in (␣␤) TP , and inorganic phosphate (P i ) release from (␣␤) DP that induces the 40°substep.
Amino acid sequence alignments of subunit ␣ (Fig. 1) reveal that the mycobacterial F-ATP synthase subunit ␣ has a unique extension of 36 amino acid residues at the very C terminus of subunit ␣, Mt␣(514 -549) (according to M. tuberculosis numbering), which is not present in any other known prokaryotic or eukaryotic ␣ subunit. Questions arise whether this extension contributes to important enzymatic differences of mycobacte- Novel regulatory role of mycobacterial subunit ␣ rial F-ATP synthases such as: (i) the latency of ATP hydrolysis activity and suppression of proton pumping. Thus, activation of the latent ATP hydrolysis driven H ϩ -pumping could reduce ATP reserves to alter the proton motive force (PMF) and to decrease the viability of the bacteria. (ii) Whether the C-terminal extension is related to possible mechanistic processes of the rotary F-ATP synthase motor, and (iii) whether this 36-amino acid extension can be employed as a potential drug target. Using a combination of recombineering, ensemble, and single molecule assays, solution X-ray scattering (SAXS), and NMR spectroscopy we demonstrated for the first time that this unique subunit ␣ C-terminal stretch is at least one of the regulatory elements inside mycobacterial F-ATP synthases that affects ATP hydrolysis and synthesis. Removal of this C-terminal extension was found to increase the rate of ATP cleavage and therefore provides energy to enable ATP-driven H ϩ -translocation. The ATP hydrolysis suppressing function of the mycobacterial C-terminal extension was confirmed by replacing the mycobacterial subunit ␣ with the subunit ␣ (502 residues) of Geobacillus stearothermophilus (formerly Bacillus PS3 or TF 1 ), which does not contain a C-terminal extension. Furthermore, by generating an ␣ 3 chi ␤ 3 ␥ complex, containing the chimeric ␣ consisting of subunit ␣ of the G. stearothermophilus F-ATP synthase (Gs␣(1-502)) and Mt␣(514 -549), we demonstrated, that the extension alters ATPase activity also in the chimeric complex. Single molecule rotation experiments revealed that the C-terminal stretch affects ATPase activity by reducing the angular velocity of the rotating ␣ 3 chi ␤ 3 ␥ complex after binding of ATP. In addition, solution structural and crosslinking studies lead to a model, in which the C-terminal extension of Mt␣ comes in close proximity with subunit ␥, whose interaction may influence its rotation.

The C terminus affects enzymatic activities
To gain insight into the enzymatic effect of the specific C-terminal domain of the mycobacterial F-ATP synthase subunit ␣ as well as the C-terminal helix (residues 521-540) inside this extension, the M. smegmatis F-ATP synthase mutants ⌬␣514 -548 and ⌬␣521-540 with deletion of amino acids 514 -548 or 521-540 of the respective C terminus of subunit ␣ were engineered. The effect of the C-terminal deletions of the ⌬␣514 -548 mutant on ATP hydrolysis was investigated using inverted membrane vesicles (IMVs). As demonstrated in Fig. 2, A and B, IMVs of wild type (wt) M. smegmatis revealed an ATPase activity of about 37.5 Ϯ 1.3 nmol min Ϫ1 (mg of total protein) Ϫ1 , confirming recent results that demonstrated that M. smegmatis, although at a relatively low level, hydrolyzes ATP (6,23). In contrast, when IMVs containing the F-ATP synthase mutant ⌬␣514 -548 were used, an ATPase activity of 61.4 Ϯ 0.4 nmol min Ϫ1 (mg of total protein) Ϫ1 was observed. Thus, deletion of the C-terminal domain caused a 64% increase in ATPase activity, which suggests that the C-terminal domain in the wt enzyme suppresses ATPase activity. To confirm that the additional C terminus causes suppression of ATPase hydrolysis, subunit ␣ of the G. stearothermophilus F-ATP synthase, which does not include the C-terminal extension, was engineered into M. smegmatis F-ATP synthase. IMVs of the Gs␣ replacement mutant showed also an increase in ATP hydrolysis (80.6 Ϯ 0.2 nmol min Ϫ1 (mg of total protein) Ϫ1 ) (data not shown) in comparison to the wt, supporting that the unique C-terminal extension of mycobacterial subunit ␣ suppresses ATPase activity. To determine whether the predicted helix inside the C-terminal extension of ␣ (Fig. 2, A and B) may be critical for this suppression, the ATPase activity of IMVs of the ⌬␣521-540 mutant was measured and found to be 56.0 Ϯ 0.3 nmol min Ϫ1 (mg of total protein) Ϫ1 (49% increase compared with wt activity), suggesting that the C-terminal helix contributes to this suppression.
To determine whether ATP hydrolysis is coupled with proton-pumping, IMVs containing M. smegmatis wt F-ATP synthase were tested for proton pumping activity in the presence of the fluorescent dye 9-amino-6-chloro-2-methoxyacridine (ACMA). As shown in Fig. 2C, adding the substrate ATP to the assay containing IMVs resulted in no further drop of fluorescence other than the typical slight ATP-induced signal quenching. This lack of ATP-dependent fluorescence quenching was not the result of leaky IMVs, because (i) addition of NADH caused a significant and fast quenching of ACMA due to the proton-pumping of the respiratory chain complexes (Fig. 2C), and (ii) upon addition of the uncoupler SF6847 the observed fluorescence was increased. These data correlate with recent results (23,24) that showed that ATP hydrolysis catalyzed by the M. smegmatis F-ATP synthase is not coupled to protonpumping. Addition of ATP to IMVs containing either the M. smegmatis ⌬␣(514 -548) or ⌬␣(521-540) mutant protein quenched ACMA fluorescence by about 10% (Fig. 2D) in a manner that was sensitive to the uncoupler SF6847. These data reveal that the increases in ATPase activity that resulted from the ⌬␣(514 -548) and ⌬␣(521-540) deletions were sufficient to create a small proton gradient across the membrane. Because the rate of H ϩ -conduction of both mutant proteins is similar, one can conclude that deletion of the C-terminal helix mainly causes the observed effect.
In parallel, ATP synthesis activities of the wt, the ⌬␣(514 -548) and ⌬␣(521-540) mutants in IMVs were measured. As shown in Fig. 2E, the IMVs of wt revealed an ATP synthesis activity of 2.27 Ϯ 0.05 nmol min Ϫ1 (mg of total protein) Ϫ1 , which is similar to the value (0.96 Ϯ 0.15 nmol min Ϫ1 (mg of total protein) Ϫ1 ) determined recently (6). In comparison, the ⌬␣(514 -548) and ⌬␣(521-540) mutants were active in ATP synthesis with values of 1.55 Ϯ 0.03 nmol min Ϫ1 (mg of total protein) Ϫ1 and 1.37 Ϯ 0.04 nmol min Ϫ1 (mg of total protein) Ϫ1 , respectively, demonstrating a decrease of about 40% in ATP synthesis activity for both mutant enzymes when compared with the wt.
In addition, the inhibitory effect of the TB drug bedaquiline (TMC207) (24) on ATP synthesis of wt and ⌬␣(514 -548) mutant IMVs was similar for both enzymes ( Fig. 2F and G), resulting in comparable IC 50 values of about 0.8 nmol. This is in line with those reported recently for M. smegmatis (2.5-12.9 nM) (25) and Mycobacterium phlei (20 -25 nM) (15) F-ATP synthase. These data as well as Western blot analyses (data not shown) confirm that the amount of F-ATP synthases located in the ⌬␣(514 -548) mutant vesicles is comparable with that of the wt vesicles.

ATPase activity decrease in the novel
The data above raise the question, whether the apparent ATPase activity suppressing function of the Mycobacteriumspecific ␣ domain can be transplanted in F-ATP synthases of other organisms. We used the ATPase proficient and mechanistically well understood ␣ 3 ␤ 3 ␥ complex of the G. stearothermophilus F-ATP synthase (Gs␣ 3 ␤ 3 ␥) as a prototype with an engineered cysteine at position ␥109 essential for single molecule rotation experiments (see below). An ␣ 3 chi ␤ 3 ␥ complex with a chimeric subunit ␣ (Gs␣(1-502)), and the very C-terminal ␣(514 -549)-segment of the M. tuberculosis F-ATP synthase (Mt␣(514 -549)), was compared with the Gs␣ 3 ␤ 3 ␥ complex. Highly purified complexes were produced (Fig. 3A) to deduce the effect of subunit ␣ chi on the ATP hydrolysis reaction (Fig. 3, B and C). At 37°C, the Gs␣ 3 ␤ 3 ␥ complex showed an ATPase activity of 4.9 Ϯ 0.04 mol min Ϫ1 (mg of protein) Ϫ1 , whereas ␣ 3 chi ␤ 3 ␥ revealed a 12% decreased ATPase activity (4.3 Ϯ 0.01 mol min Ϫ1 (mg of protein) Ϫ1 ), when the rates of the first 10 s of the reactions were compared, i.e. the time frames that were not affected by ADP inhibition of the enzyme. The effect of ADP inhibition is shown in Fig. 3D.
In an additional experiment we removed all nucleotides from the enzymes prior to ATP hydrolysis measurements. A similar reduction of ATP hydrolysis (10%) was observed for the nucleotide-depleted ␣ 3 chi ␤ 3 ␥ compared with -Gs␣ 3 ␤ 3 ␥ (Fig.  3E). These results show that the Mycobacterium-specific C-terminal extension is indeed able to suppress ATPase activity when transplanted onto the C terminus of an ATPase proficient prokaryotic enzyme. This concurs with Values are the mean of six determinations with two different IMV batches of wt and mutants. C and D, substrate driven proton-pumping in IMVs. C, M. smegmatis mc 2 155 wt membrane vesicles were diluted to 0.18 mg/ml. Fluorescence quenching of ACMA by wt IMVs was studied after the addition of a substrate (2 mM ATP (blue, profile 2)) or 2 mM NADH (purple, profile 3). The uncoupler (SF6847) was added at the indicated time point to collapse the proton gradient. In the control experiment, buffer was added in place for substrate (gray, profile 1). D, fluorescence quenching of ACMA by IMVs of the ⌬␣(514 -548) (red, profile 2) and ⌬␣(521-540) mutant (orange, profile 3) after addition of ATP in comparison to the wt IMVs (blue, profile 1) and the recently described ⌬␥(166 -179) mutant (green, profile 4) (23). Profile 5 (light blue) reveals the quenching of IMVs of the ⌬(␣514 -548) mutant in the presence of 2 mM NADH. Fluorescence quenching of ACMA with wt-and ␣-mutant IMVs was performed with four and two different batches of vesicles, respectively. E, ATP synthesis measured for wt (blue), ⌬␣(514 -548) (red) and ⌬␣(521-540) mutant (orange) IMVs of M. smegmatis. Effect of increasing concentrations of bedaquiline on ATP synthesis using the M. smegmatis ⌬␣(514 -548) mutant (F) and wt IMVs (G). the complementary finding above, showing the increase of ATPase activity of IMVs from the M. smegmatis deletion mutant ⌬␣514 -548.

Influence of the C terminus on ATP-dependent rotation
To further investigate the enzymatic influence of the C-terminal extension of mycobacterial subunit ␣, and to gain insight into a possible mechanistic event, we tested the Gs␣ 3 ␤ 3 ␥ and ␣ 3 chi ␤ 3 ␥ complexes in a single molecule rotation assay, where the rotation can be visualized directly (26). A fluorescently labeled 300-nm bead was attached to subunit ␥ of immobilized Gs␣ 3 ␤ 3 ␥ or ␣ 3 chi ␤ 3 ␥ complexes (Fig. 4A). Upon addition of saturating MgATP concentrations rotating beads were observed via TIRF microscopy. Fig. 4B shows the trajectory of a rotating Gs␣ 3 ␤ 3 ␥-bead complex, derived from a frame-by-frame analysis ( Fig. 4C) of the recorded video (26). The average rotational rate of the Gs␣ 3 ␤ 3 ␥ complex was 4.6 Ϯ 1.0 s Ϫ1 (n ϭ 9 particles). When a 600-nm bead duplex was attached to the protein, the average rotational rate under these conditions was 1.9 Ϯ 0.5 s Ϫ1 (n ϭ 8 particles) due to the high viscous drag. The rotation rates for this complex were similar to those reported previously (26,27).
The question was addressed, whether the C terminus of ␣ chi in the ␣ 3 chi ␤ 3 ␥ complex affects the rotation and dwell times of the ␣ 3 chi ␤ 3 ␥ complex. As demonstrated in a video (supplemental Video S1) and in Fig. 4D, all ␣ 3 chi ␤ 3 ␥-bead complexes rotated continuously counterclockwise without backward steps. At saturating ATP concentrations, the rotational rate measured by the bead assay was dominated by the dwell time of the catalytic dwell. A very prolonged catalytic dwell would decrease the overall rotational rate. The ATP-waiting dwell is not rate-limiting here, but again a largely increased dwell time at this position would also affect the overall rate. However, when the ␣ 3 chi ␤ 3 ␥ complex was used in the rotation assay, we observed the same rotational rates of 4.8 Ϯ 2.0 s Ϫ1 (n ϭ 13 particles) and 2.1 Ϯ 0.7 s Ϫ1 (n ϭ 6 particles) for complexes with single beads and bead duplexes, respectively. These experiments show that ATP hydrolysis results in the unidirectional counterclockwise chi ␥ and ATP hydrolytic activity of Gs␣ 3 ␤ 3 ␥ and ␣ 3 chi ␥. A, the chromatogram shows an elution profile of ␣ 3 chi ␤ 3 ␥ using a Resource-Q column (6 ml). The inset in the figure reveals a SDS gel, which corresponds to the shaded area (gray) of the elution peak. The eluted ␣ 3 chi ␤ 3 ␥ fractions were pooled and applied on the gel. Lanes 1 and 2 reveal the purified ␣ 3 chi ␤ 3 ␥ and protein markers, respectively. The protein size in kDa is indicated on the right. B, continuous ATPase activity of Gs␣ 3 ␤ 3 ␥ and ␣ 3 chi ␤ 3 ␥ measured at 2 mM MgATP, 37°C. Decrease in NADH absorption at 340 nm is plotted against time as dotted lines. The black lines show the linear least square fit for the first 10 s. C, specific ATPase activities of Gs␣ 3 ␤ 3 ␥ and ␣ 3 chi ␤ 3 ␥ determined from the slope of the least square fit. Values are the mean of 10 determinations. D, ATPase profile of nucleotide-depleted Gs␣ 3 ␤ 3 ␥ (-) in a continuous ATPase activity assay measured at 2 mM MgATP. Bound MgADP was removed prior to activity measurements by incubating the protein with 5 mM Na-P and 10 mM EDTA for 30 min at 4°C, and passing the enzyme samples through a Superdex 300 TM column (GE Healthcare). (⅐⅐⅐⅐) ATPase profile of Gs␣ 3 ␤ 3 ␥, which was preincubated with 100 M MgADP for 30 min, revealing the MgADP inhibition effect, and confirming that tightly bound MgADP was removed due to the nucleotide depletion process of nucleotide-depleted Gs␣ 3 ␤ 3 ␥ (-) described above. E, specific ATPase activities of the nucleotide-depleted Gs␣ 3 ␤ 3 ␥ and ␣ 3 chi ␤ 3 ␥. Values are the mean of three determinations.
rotation of the chimeric complex, and that the chimeric subunit ␣ does not alter the duration of the dwells significantly.
Changes in angular velocity as a function of rotational position during the power stroke cannot be investigated in detail with the bead assay because the movement of subunit ␥ is damped by the viscous drag of the large bead. In addition, the temporal and spatial resolution of the bead assay is too low to resolve the detailed motion during the power stroke. To overcome these limitations, we used a 40 ϫ 76-nm gold nanorod as a probe of rotation. The viscous drag on these nanorods is not rate-limiting to the power stroke of the Escherichia coli (30). The light scattered from the nanorod was detected by an avalanche photo diode (APD) after passing through a polarizing filter using dark-field microscopy ( Fig. 5A). Upon addition of 1 mM MgATP, sinusoidal variations of light intensities scattered from the nanorod were observed as a function of its position relative to the axis of polarization. At a sampling rate of 1 kHz, dwells in the rotation produced a peak in the intensity distribution, as reported previously for Ec␣ 3 ␤ 3 ␥ (28, 29), Gs␣ 3 ␤ 3 ␥, or MmA 3 B 3 DF (30). A series of 36 histograms of light intensities was recorded that were acquired when the rotational position of the polarizer was advanced successively by 10°. The observation of three off-set sinusoidal curves in the histograms, when the axis of rotation was close to the orthonormal position relative to the microscope slide, indicated the rotation of the ATP hydrolyzing complexes and the presence of three catalytic dwells separated by 120°steps (Fig. 5B).
The angular velocity profile for a 120°power stroke of the central stalk was calculated from the sinusoidal light intensity changes recorded with 200 kHz using the arcsin 1 ⁄2 function (30). The three off-set sinusoidal curves like that shown in Fig. 5B was observed for each molecule used to derive the angular velocity profile. Fig. 5C shows the angular velocity of Gs␣ 3 ␤ 3 ␥ (n ϭ 20 molecules, average number of transitions: 2153) for a 120°power stroke between two catalytic dwells, as described recently (30). Starting from a catalytic dwell, which here was defined to be at 0°, the angular velocity reached a plateau of about 200°ms Ϫ1 between 20°and 55°. This is the range of angular positions (phase 1) where ATP binds to the catalytic site (30). Following the plateau in phase 1, the angular velocity in phase 2 increased steadily to 500°ms Ϫ1 at 85°, reached a maximum of 1300°ms Ϫ1 at 90°, and then decreased rapidly to 200°m s Ϫ1 at 93°. After another small acceleration to 270°ms Ϫ1 at 98°, the rate declined to a minimum at 120°(i.e. at the next catalytic dwell). The plot for the ␣ 3 chi ␤ 3 ␥ complex (n ϭ 17 molecules, average number of transitions: 1860) showed a similar pattern. However, from 55°to 120°(i.e. after the ATP-waiting dwell), the angular velocity was reduced on average by 21%. Taking into account the same dwell time for the catalytic dwell and angular velocity for the first 55°of rotation, the larger percentile decrease for the power stroke is in line with the overall 16% decrease in ATPase activity for the chimeric complex.

Purification and nucleotide-binding of Gs␣ and ␣ chi
To gain insight into the location and structural traits of the extended C-terminal domain of mycobacterial subunit ␣, the recombinant ␣ chi composed of Gs␣(1-502) and the C-terminal Mt␣(514 -549) was compared with the wt Gs␣. Both proteins were generated, produced, and purified in high quality (Fig. 6, A  and B). The SDS gels of the purified proteins after size exclusion chromatography showed a single band that was running at a higher molecular weight in the case of ␣ chi .
Fluorescence correlation spectroscopy (FCS) was used to determine the ATP-and ADP-binding properties of Gs␣ and ␣ chi , and to determine whether the extension by the C-terminal Mt␣(514 -549) may have altered the nucleotide-binding traits of ␣ chi (Fig. 6, C and D).
A, schematic model of the microscope setup. Gold nanorods are illuminated by a dark-field condenser. Red polarized light scattered from a nanorod was recorded by an APD after passing through a polarizer. The model also shows the interaction of a gold nanorod with Gs␣ 3 ␤ 3 ␥ or ␣ 3 chi ␤ 3 ␥. The protein Gs␣ 3 ␤ 3 ␥ or ␣ 3 chi ␤ 3 ␥ (subunits ␣, ␤, and ␥ in orange, green, and yellow, respectively) is attached via its His 10 tags in subunit ␤ to a coverslide, whereas an avidin-coated nanorod is attached to the opposing biotin modified cysteine in subunit ␥. B, consecutive histograms of light intensities of a rotating nanorod attached to ␣ 3 chi ␤ 3 ␥ upon rotating the polarizer in 10°-steps. The three peak positions in the histograms, resulting from the catalytic dwells of protein, follow a sinusoidal curve over 360°rotation of the polarizer. The approximate courses of the three sine curves are indicated in gray. C, average angular velocity over angular position at 1 mM MgATP of Gs␣ 3 ␤ 3 ␥ (red) and    Fig. 6F) showed a bell-shaped peak at low angles indicating a well folded protein.
The radius of gyration (R g ) values determined from the Guinier approximation were 27.98 Ϯ 0.24 and 29.56 Ϯ 0.32 Å, for Gs␣ and ␣ chi , respectively. The determined distribution functions, p(r) (Fig. 6F), showed a maximum particle dimension, D max , of 89.5 Ϯ 10 and 102.5 Ϯ 10 Å for Gs␣ and ␣ chi , respectively, reflecting that ␣ chi is more elongated ( Table 1). The gross shapes of the proteins were ab initio reconstructed and had a good fit to the experimental data in the entire scattering range discrepancies of 2 ϭ 0.348 and 0.334 for Gs␣ and ␣ chi , respectively. Ten independent reconstructions produced similar envelopes with a normalized spatial discrepancy (NSD) of 0.611 Ϯ 0.06 and 0.603 Ϯ 0.04 for Gs␣ and ␣ chi , respectively. The average structure is shown in Fig. 6G. The theoretical scattering curve for the Gs␣ (PDB code 1SKY) was computed and compared with the experimental scattering curves of Gs␣ and ␣ chi using CRYSOL with resulting discrepancies of 2 ϭ 1.44 and 1.46, respectively (Fig. 6E). Superimposition of the Gs␣ and ␣ chi shapes with the crystal structure of Gs␣ indicated an extra domain of 15 ϫ 22 Å, reflecting the additional 36 residues of the chimera protein. Furthermore, the comparison demon-strated that the C-terminal Mt␣(514 -549) of ␣ chi is located below the very C terminus of Gs␣. The NSD between the Gs␣ and ␣ chi solution shapes is 0.53, whereas the NSD for the shapes of Gs␣ and ␣ chi with the crystal structure of Gs␣ (PDB code 1SKY) are 2.12 and 2.17, respectively.

Solution structure of the C-terminal peptide Mt␣(521-540)
According to the secondary structure prediction in Fig. 1, the extended C-terminal region 514 -549 of Mt␣ shows random coiled regions of the peptides 514 -525 and 540 -549 and a helical structure formed by the peptide 526 -539. To gain structural insight about this epitope and the ␣-helical feature in solution, the peptide 521 PDEHVEALDEDKLAKEAVKV 540 of Mt␣ (Mt␣(521-540)) was synthesized. The CD spectrum of Mt␣(521-540) showed ␣-helical and random-coiled formation of 43 and 52%, respectively (Fig. 7A). To determine the 3D structure of Mt␣(521-540), the primary sequences of ␣(521-540) were sequentially assigned as per standard procedures, using a combination of total correlation spectroscopy (TOCSY) and NOESY spectra. Secondary structure prediction was done using PREDITOR (31), which uses HA chemical shifts and showed the presence of an ␣-helical structure. The 20 lowestenergy structures of 100 were taken for further analysis. In total, an ensemble of nine calculated structures resulted in an overall root mean square deviation (r.m.s. deviation) of 0.65 Å for the backbone and 1.65 Å for heavy atoms. All the structures had energies lower than Ϫ100 kcal mol Ϫ1 , with no NOE-violations and no dihedral violations ( Table 2). Identified cross-peaks in the HN-HN and HA-NH regions are shown in Fig. 7B. From the assigned NOESY spectrum HN-HN, H␣-HN (i, iϩ3), H␣-HN (i, iϩ4), and H␣-H␤ (i, iϩ3) connectivities were plotted, which supported ␣-helical formation in the structure. The calculated structure has a total length of 20 Å and displays an ␣-helical region between residues 526 and 539 (Fig. 7C). Fig. 7D shows the molecular surface electrostatic potential of Mt␣(521-540).

Proximity of Mt␣(521-540) to subunit ␥ residues 104 -109
We further investigated whether the decreased hydrolysis rate correlated with a decreased angular velocity of the ␣ 3 chi ␤ 3 ␥ complex. A structural model of the ␣ 3 chi ␤ 3 ␥ complex was generated ( Fig. 8A) based on the G. stearothermophilus F 1 -ATPase structure (32) (PDB code 4XD7) and the structural insights derived from the present study (see "Experimental procedures" for details). To ascertain the influence of the extended C-terminal 36 residues of ␣ chi during hydrolysis, subunit ␥ was rotated counterclockwise (viewing from the membrane side) for the full 360°in steps of 20°in the ␣ 3 chi ␤ 3 ␥ complex model. At angles of 60 Ϯ 10°and in steps of 120°, the central globular domain of the ␥ subunit comes closer to the so-called "DELSEED" region, composed of residues 396 AQFGSDLDK 404 of subunit ␣, and to the extended C-terminal domain of ␣ chi . Particularly at 60°, the ␣-helical residues 90 AYNSNVLRLVYQT 102 of the ␥ subunit come into the vicinity of ␣ subunit residues 396 AQFGS-DLDK 404 . Interestingly, the polar residues Gln 104 and Arg 106 as well as Cys 109 are in proximity to the extended C-terminal residues Asp 522 , Glu 523 , and Val 525 . To test their closeness, Val 525 was substituted by a cysteine in a ␣ 3 chi ␤ 3 ␥ complex mutant called (␣ chi -V525C) 3 ␤ 3 ␥. The isolated protein was applied on a

Novel regulatory role of mycobacterial subunit ␣
9% SDS gel in the absence (Fig. 8B, lane 2) or presence (lane 3) of the reductant dithiothreitol (DTT). As shown in lane 2 of Fig.  8B, two cross-linked products I and II were observed. In comparison, these products were not formed by the DTT-treated protein and the band intensities of subunits ␣ chi -V525C and ␥ were stronger (lane 3). The bands of product I and II were cut out and incubated in the presence of 20 mM DTT before embedding them in a second SDS gel under reducing conditions. Fig. 8C demonstrates that product I is formed by ␣ chi -V525C dimers and product II by a cross-linked product of ␣ chi -V525C and ␥ that migrated aberrantly higher due to cross-link formation. The data revealed that C525 in ␣ chi -V525C and Cys 109 of subunit ␥ formed the disulfide bridge, which became cleaved after reduction. In parallel, ␣ 3 chi ␤ 3 ␥ was treated in the same way as (␣ chi -V525C) 3 ␤ 3 ␥. Fig. 8D shows that no cross-link product was formed in the presence or absence of DTT. Overall, these data confirmed the proximity of the extended C-terminal helix of ␣ chi with the subunit ␥ residues 104 -109, whose interaction may influence rotation of the camshaft-like subunit ␥.

Discussion
Activation of the latent ATP hydrolysis driven H ϩ -pumping of mycobacterial F-ATP synthase may alter the magnitude of the PMF and decrease the viability of the bacteria (33). An  amino acid sequence alignment of F-ATP synthase ␣ subunits reported here (Fig. 1) revealed that the mycobacterial subunit ␣, important for nucleotide-binding and catalysis, as well as the coupling and rotating subunits ⑀ and ␥ are different from any other pro-and eukaryotic counterparts (34,35). Here, the deletion of the mycobacterial C terminus of subunit ␣ increased ATP hydrolysis and H ϩ -pumping activity of about 63 and 10%, respectively, compared with the wt enzyme (Fig. 2B, 2D). These increases of ATP hydrolysis and H ϩ -pumping that were comparable for both the ⌬␣(514 -548) and ⌬␣(521-540) mutants demonstrated that the C-terminal helix plays a role in the regulation of catalytic and coupling events. In contrast, when this C-terminal extension was inserted into Gs␣ 3 ␤ 3 ␥ to form a chimeric protein, the ATPase rate was reduced by about 10%. These data reveal that the C-terminal extension of subunit ␣ contributes significantly to the reduced (inhibited) ATPase activity that does not provide sufficient energy to couple proton-conduction in mycobacteria. The extent of the reduced ATPase activity provided by the unique ␣ subunit extension occurs in addition to similar effects, like those reported in Fig. 2D, which result from the unique insertion of 14 amino acid residues of the M. tuberculosis subunit ␥ ( 165 TDNGEDQRSDSGEG 178 ) (23). This subunit ␥ insertion that forms a loop of polar residues has been proposed to be in vicinity to the polar residues Arg 41 , Gln 42 , Glu 44 , and Gln 46 of the c-ring (23, 24) (Fig. 9, A and B). Deletion of this loop in IMVs, containing the M. smegmatis ⌬␥(166 -179) mutant protein, led to ATP-driven H ϩ -pumping (about 61%) (23), as shown in Fig. 2D. It has been proposed that this unique polar loop of the mycobacterial subunit ␥ interacts with the c-ring such that it affects rotation and thereby inhibits proton pumping. Thus, the results reported here for the unique extension of subunit ␣, in addition to those reported previously for the unique insertion of subunit ␥, provide a novel inhibitory mechanism to regulate the PMF such that it enables the pathogenic bacterium to survive.
The structural model described here (Fig. 8A), which is confirmed by the formation of a cross-link between residues ␣Cys 525 and ␥Cys 109 of (␣ chi -V525C) 3 ␤ 3 ␥, clearly shows that the extended C-terminal stretch of mycobacterial subunit ␣ with its ␣-helical region between residues 526 and 539 and the rotating ␥ are in close proximity. Particularly at 60°, the ␣-helical residues 90 AYNSNVLRLVYQT 102 of subunit ␥ come into the vicinity of subunit ␣ residues 396 AQFGSDLDK 404 , and the polar residues Gln 104 , Arg 106 , and Cys 109 of subunit ␥ are in proximity to the extended C-terminal ␣ residues Asp 522 , Glu 523 , and Val 525 . The possible occurrence of additional interactions due to the extended ␣ subunit may also influence the rate of catalysis, which may explain the 12% reduction of the overall ATPase activity rate in bulk ATP hydrolysis of ␣ 3 chi ␤ 3 ␥ when compared with wt Gs␣ 3 ␤ 3 ␥. The effect of the C-terminal extension of subunit ␣ unique to mycobacteria on ATPase-dependent rotation is summarized in Fig. 9C. Hydrolysis of ATP occurs at the (␣␤) DP catalytic site during the catalytic dwell that ends upon release of phosphate (P i ) from the (␣␤) E catalytic site to initiate rotation (36). This initiates phase 1 of the power stroke (29) that rotates subunit ␥ by ϳ40°. If ATP has not become bound to (␣␤) E by the time that subunit ␥ reaches that position, an ATP-binding dwell is observed. Binding of ATP then induces phase 2 in which subunit ␥ rotates for 80°to complete the 120°power stroke and form the next catalytic dwell (29).
The single-molecule rotation assays with a fluorescent bead (Fig. 4B) indicate that ATPase-dependent rotation occurred in the counterclockwise direction without significant back-steps, and that the chimeric subunit ␣ does not alter the duration of the dwells significantly. This was corroborated by our FCS studies that showed that the binding affinities for ATP or ADP remained the same in the chimeric protein (Fig. 6, C and D). Using the gold nanorod assay capable of resolving the angular velocity of the power stroke under conditions in which not the viscosity but rather by the internal mechanism of the motor is rate-limiting, we found that the presence of the C-terminal extension of subunit ␣ decreased the angular velocity of the power stroke of the motor (Fig. 5C). Although the angular velocity profile of phase 1 remained unchanged, the final 65°of the power stroke (phase 2) was on average 21% slower with the chimeric subunit ␣. These results were consistent with the structural and cross-linking results presented here that showed an interaction between the C-terminal extension of subunit ␣ and subunit ␥.
The motion of the DELSEED sequence in subunit ␤ has been shown to contribute significantly to angular velocity of ATPase-dependent subunit ␥ rotation (29). We now show for the first time that the extension of the C terminus of mycobacterial subunit ␣ significantly limits the angular velocity of the rotor that is likely to result from steric hindrance between one of the ␣ chi subunits and subunit ␥ during the power stroke. An effect of steric interference due to electrostatic forces between subunit ␥ and the C-terminal domain of the catalytic ␤ E subunit of the thermoalkaliphilic Bacillus sp. TA2.A1 F-ATP synthase has been described to prevent the enzyme from rotating in the ATP hydrolysis direction (23). Our hypothesis is further strengthened by the observation that the ATP hydrolysis rate of the M. smegmatis ATP synthase deletion mutant ⌬␣(514 -548) Figure 9. Model of the interaction between subunits ␥ and ␣ chi in ␣ 3 chi ␤ 3 ␥ during rotation. A, the unique mycobacterial ␥-loop (red), which is in the vicinity to the M. phlei c-ring (brown, PDB code 4V1G) (15), was inserted in the Gs␥ (yellow). The arrow indicates the interaction between the 396 AQFGSDLDK 404 peptide (green) and the extra C-terminal domain of ␣ chi (light green) with the ␣-helical peptide 90 AYNSNVLRLVYQT 102 of Gs␥. B, the proximity of the C-terminal helix of Mt⑀ with the extended C-terminal stretch of ␣ chi . Subunit ⑀ (blue) inside the ␣ 3 chi ␤ 3 ␥ complex was modeled based on the recently determined solution structure of Mt⑀ (PDB code 5WY7), which was then superimposed onto the ␦ subunit of the bovine F 1 -ATPase. At 80°rotation of the central stalk, the compact C-terminal helix of Mt⑀ might interact with the extended C-terminal domain of ␣ chi on the way to form an extended conformation. C, subunits ␣ chi , ␤ with nucleotide occupancy, and ␥ with rotational position are shown in green, orange, and yellow, respectively, and labeled according to the native crystal structure (60). The extended C terminus of ␣ chi is shown in dark green. Catalytic events are based on the model by Watanabe et al. (63). The state on the left shows the catalytic dwell. During phase 1 (see Fig. 5C) P i release from ␤ E triggers a 40°rotation of subunit ␥, which leads the enzyme to adopt the ATP-waiting dwell. During phase 2, ATP binding and ADP release, accompanied by a change in the conformational states of the ␣ chi and ␤ subunits, causes subunit ␥ to further rotate by 80°. At a rotational position of 90°subunit ␥ is in close proximity to the extended C terminus of ␣ chi .
is enhanced when compared with its wt form (Fig. 2, A and B). This underlines that the C-terminal extension of the mycobacterial subunit ␣ contributes to the regulation of ATPase hydrolysis.
It is noteworthy that mycobacterial subunit ⑀, which also forms part of the drive shaft of the motor in the intact F-ATP synthase, has a truncated C-terminal domain compared with other organisms (Fig. 9B) and was predicted to be too short to reach into the upper region of the ␣-␤ interface close to the adenine-binding pocket (34). A recent study (34) extended our structural model to M. tuberculosis F-ATP synthase subunit ⑀ based on the recently determined NMR structure of Mt⑀ (PDB code 5WY7) (Fig. 9B). Application of similar rotational simulations applied to subunits ␥ and Mt⑀ in this model revealed that the C-terminal helix of Mt⑀ lies closer to the ␣ chi (Fig. 9B). At a rotational angle of 80 Ϯ 10°(in hydrolysis direction from a catalytic dwell position) the C terminus of Mt⑀ is in close proximity to the extended C-terminal domain of ␣ chi when the compact Mt⑀ is on the way to form an extended conformation. It is for future studies to reveal how these proposed interactions might influence the activity of the holoenzyme.
In summary, a novel combination of complementary approaches of genetic engineering, protein chemistry, ensemble enzymatic and single-molecule rotation assays, and structural biology has provided new insights into the unique mycobacterial C-terminal 36-amino acid residues extension of subunit ␣. The C-terminal ␣-helical extension of one ␣ subunit is suggested to interact with polar residues of subunit ␥, whereas a second ␣ (␣ TP ) is suggested to interact with the C-terminal helix of Mt⑀ to generate an inhibited state that reduces ATP hydrolysis. The low ATPase activity and the interaction of the ␥(166 -179)-loop with the c-ring significantly limit the extent of ATPase-driven H ϩ -pumping such that they alter the magnitude of PMF and decreases the viability of bacteria.

Genetic manipulation
DNA manipulations were done according to standard protocols (37). Plasmid and DNA isolations were performed according to Qiagen protocols. Mycobacterial DNA manipulations were done with some minor modifications according to published protocols (38). Primers and dsDNA oligonucleotides were synthesized by Integrated DNA Technologies (USA) and sequencing of DNA was done by AIT Biotech (Singapore).
Cultivation and preparation of electro-competent cells of M. smegmatis mc 2 155 was done according to the protocol described by Goude and Parish (39). For recombineering, M. smegmatis mc 2 155 cells containing the episomal plasmid pJV53, with the inducible proviral Che9c genes gp60 and gp61 under control of the acetamide promoter were used. Electrocompetent cells were prepared (39), washed, and cells after overnight growth were expanded in 7H9 medium supplemented with succinate (0.2% v/v) to an A 600 ϭ 0.6. Prior to cooling the cells on ice, recombineering genes were induced with 0.2% (v/v) acetamide (3 h, 37°C).

Recombineering assay with dsDNA in M. smegmatis mc 2 155
Recombineering was done according to van Kessel and Hatfull (40,41). To generate a M. smegmatis mc 2 155 F-ATP synthase mutant form with a deletion of amino acids 514 -548 of the C terminus of subunit ␣ (⌬␣(514 -548)), a stop codon TAG at position 514 in the double-stranded 100-bp DNA oligonucleotide of the gene atpA was synthesized by Integrated DNA Technologies, USA. This oligonucleotide was further expanded by 80 bp using PCR with the forward (forward ext ⌬␣1) and reverse extending primers (reverse ext ⌬␣1) as described in supplemental Table S1, which resulted in a 200-bp dsDNA oligonucleotide AES (allelic exchange substrate). In case of the M. smegmatis F-ATP synthase mutant ⌬␣(521-540), the deletion of the C-terminal helix was achieved by synthesizing a 90-bp junction dsDNA containing 45-bp DNA sequences upstream and downstream of the DNA coding for deleted helix 521-540 (dsDNA ⌬␣(521-540)). The junction dsDNA sequence was further extended to 210 bp by PCR using 90-bp extended primers (forward ext ⌬␣2/reverse ext ⌬␣2) (supplemental Table S1). The M. smegmatis F-ATP synthase recombinant strain with the gene atpA replaced by the gene atpA from G. stearothermophilus (GsatpA) was constructed using a 2.5-kb dsDNA AES and plasmid pSJ25 as a co-transformation vehicle. The 2.5-kb dsDNA AES contained the 1.47-kb GsatpA gene flanked at both 5Ј and 3Ј ends by a 500-bp DNA, homologous to the DNA upstream and downstream of atpA from M. smegmatis. The AES was generated by several PCRs. First, the upstream and downstream 500-bp homologous DNA was generated by separate PCR, using the primers Forward_Gs_A/Reverse_Gs_A (upstream homologous DNA) and Forward_Gs _B/Reverse_ Gs_B (downstream homologous DNA). The GsatpA gene was amplified using primers Forward_ext_Gs/Reverse_ext_Gs (see supplemental Table S1). Finally, the 2.5-kb AES was obtained by PCR using amplified DNA: 500-bp upstream/downstream homologous DNA and 1.47-kb GsatpA as templates and the pair of primers Forward_Gs_A/Reverse_Gs_B. The DNA sequence of the 2.5-kb AES was confirmed by sequencing.
Electrocompetent M. smegmatis mc 2 155 cells containing the plasmid pJV53 (Kan R ) were mixed with 200 ng of doublestranded DNA (210 bp dsDNA) and 100 ng of co-transforming plasmid pSJ25. Transformation was done using the following parameters: single pulse 2.5 kV, conductivity 25 F, resistance 1000 ⍀, and Gene pulser Xcell (Bio-Rad) as previously described (39 -41). Transformed cells were recovered (4 h, 37°C) in 7H9 supplemented with 10% ADC and 0.05% Tween 80, and plated on 7H10 agar supplemented with 10% oleic albumin dextrose catalase, kanamycin, and hygromycin. About 200 hygromycin-resistant transformants were streaked on master 7H10 agar plates and tested by colony PCR. Colony PCR was done as follows: cells from master agar plates were resuspended in 200 l of H 2 O, boiled (95°C, 20 min), and immediately cooled on ice. An aliquot a 1/10 of the reaction mixture was used as a template in PCR detection using forward atpA/ Reverse atpA-mama primers (supplemental Table S1). PCR parameters used in detecting recombinants were as follows: Taq DNA polymerase (Thermo Fischer), initial denaturation (95°C, 3 min), cycle denaturation (95°C, 30 s), annealing (65°C, Novel regulatory role of mycobacterial subunit ␣ 30 s), extension (72°C, 30 s), final extension (72°C, 5 min), and 30 cycles in total. MAMA-PCR (Mismatch Amplification Mutation Assay) of recombinants carrying the mutation resulted in a PCR product, whereas unchanged DNA of the wt failed to amplify DNA. Detected recombinants were re-streaked several times on non-selective 7H10 agar plates to lose the recombineering plasmid pJV53 (Kan R ). The targeted deletion of the DNA sequence in the atpA gene and inserted stop-codon of M. smegmatis of both mutants was confirmed by sequencing the DNA (AIT Singapore).

Preparation of inverted membrane vesicles from M. smegmatis
Inverted vesicles of M. smegmatis for ATP synthesis, hydrolysis, and proton-pumping assays, were generated (23,24).

Cloning, production, and purification of mutants
To amplify subunit ␣ of the G. stearothermophilus F-ATP synthase, the atpA gene was amplified using the plasmid DNA encoding for subcomplex Gs␣ 3 ␤ 3 ␥ of G. stearothermophilus as a template. NcoI and BamHI restriction sites were introduced in the forward and reverse primers, respectively: forward, 5Ј-GAGACCATGGCAATGAGCATTCGAGCGGAAGAA-3Ј and reverse, 5Ј-CCCGGGATCCTTATTGAGAAACGACAAA-CGTTTTCTT-3Ј.
To generate an ␣ 3 chi ␤ 3 ␥ complex, overlap extension PCR was performed, using templates of plasmid DNA for Gs␣␥␤ and Mt␣. The plasmid DNA of Gs␣␥␤ encodes for the ␣ 3 ␤ 3 ␥ complex, including mutations in ␣C193S and ␥S109C. The presence of a single cysteine residue enabled the selective biotin labeling for single molecule rotation studies. Restriction sites BamHI and HindIII were incorporated in the forward and reverse primers, respectively: forward, 5Ј-AGATGGATCCAA-TGAGCATTCGAGCGGA-3Ј and reverse, 5Ј-GAATAAGCT-TTCACACTTCGACACCCATCGC-3Ј. These served as the flanking primers (primer a and primer f). Primers b, c, d, and e were 5Ј-AGAGCCGCCTTGAGAAACGACAAACGT-3Ј, 5Ј-GTTTCTCAAGGCGGCTCTGTGGTGCCC-3Ј, 5Ј-CCGCC-GGCCTTATTTCTTCTTCTTCGG-3Ј, and 5Ј-AAGAAATA-AGGCCGGCGGGGGCATATC-3Ј, respectively. These were the chimeric primers, 27 nucleotides in length containing anchoring segments from the template and the adjoining anchoring segment. To amplify the individual fragments, overlap extension PCR was performed to allow insertion of the fragment encoding for the C terminus of Mt␣ at the end of the Gs␣ in the complex to yield ␣ chi ␥␤.
The plasmid containing ␣ chi ␥␤ genes was used as a template for site-directed mutagenesis and to generate the mutant (␣ chi -V525C) 3 ␤3␥. The entire plasmid (pET-24b vector) was amplified by KAPA HiFi DNA polymerase (KAPA BIOSYSTEMS) using forward, 5Ј-CGCCAAGGAAGCCTGCAAGGTCAAA-AAGCC-3Ј and the reverse, primer 5Ј-GGCTTTTTGACCTT-GCAGGCTTCCTTGGCG-3Ј. The amplified PCR product was purified and DpnI digestion was performed to remove the methylated parental plasmid. After DpnI digestion the PCR product of mutant (␣ chi -V525C) 3 ␤ 3 ␥ was purified and transformed into E. coli TOP10 cells. The DNA coding for the chimeric ␣ chi was amplified using the primers 5Ј-GAGACCA-TGGCAATGAGCATTCGAGCGGAAGAA-3Ј and 5Ј-CCG-GGATCCTTATTTCTTCTTCTTCGGCGC-3Ј as forward and reverse, respectively, which include the NcoI and BamHI restriction sites.
The amplified gene products for Gs␣ and ␣ chi were ligated into the pET-9d(ϩ) vector (42), and ␣ chi ␥␤ into the pET-24b vector, and transformed into E. coli DH5␣ cells for plasmid amplification. The generated plasmid with the Gs␣ and ␣ chi insert, respectively, was transformed into E. coli BL21(DE3) cells (Stratagene), and plasmids carrying the DNA for ␣ chi ␥␤ and mutant ␣ chi -V525C␥␤ were transformed into E. coli DK8 cells for protein production. Gs␣ and ␣ chi cells were grown in kanamycin-containing (30 g ml Ϫ1 ) LB-medium (37°C). The cells producing the mutants ␣ chi ␥␤ and (␣ chi -V525C) 3 ␤ 3 ␥ were grown in 2YT medium, supplemented with 30 g ml Ϫ1 of kanamycin. In all cases, protein production was induced by the addition of isopropyl ␤-D-thiogalactopyranoside (1 mM) at an A 600 ϭ 0.6, and cultivated at 37°C. Cells were harvested after overnight induction, frozen by liquid nitrogen, and stored at Ϫ80°C.

ATP hydrolysis activity measurements
A continuous ATP hydrolysis assay was applied to measure the ATPase activity of wt IMVs of M. smegmatis mc 2 155, ⌬␣(514 -548), and ⌬␣(521-540) mutants, and of the recombinant Gs␣ 3 ␤ 3 ␥ and ␣ 3 chi ␤ 3 ␥ complexes (26,44). In the case of IMVs the consumption of NADH by the type II NADH dehy-Novel regulatory role of mycobacterial subunit ␣ drogenase was inhibited by thioridazine. In addition, ATPase activity of IMVs was also measured in the absence of ATP and presence of thioridazine to identify any background NADH oxidation and MgATP hydrolysis.

ATP synthesis assay
ATP synthesis was measured as described recently (23).

Biotinylation of protein complexes
The cysteine at ␥109 in the Gs␣ 3 ␤ 3 ␥ and ␣ 3 chi ␤ 3 ␥ complexes was biotinylated for the attachment of beads or nanorods coated with streptavidin or neutravidin, respectively. Freshly prepared proteins in buffer C (50 mM Tris/HCl, pH 7.5, 250 mM NaCl) were incubated for 5 min on ice with a 1.2-fold molar excess of biotin-PEAC 5 -maleimide (Dojindo, Japan). The reaction was stopped by adding 1 mM acetylcysteine, and unbound biotin-maleimide was removed by adding buffer C, followed by filtration using Centricon YM-100 (100 kDa molecular mass cut off) spin concentrators (Millipore). Samples were frozen in liquid N 2 and stored at Ϫ80°C.

Single molecule rotation assay with beads
Gs␣ 3 ␤ 3 ␥ or ␣ 3 chi ␤ 3 ␥ complexes were fixed on a Ni 2ϩ -NTAcoated coverslide with their His 10 tag at the N termini of each ␤ subunit. On the opposing end the biotinylated cysteine at ␥109 was bound to a streptavidin-coated bead (Ø ϭ 300 nm) doped with biotinylated quantum dots 605 (Q-dots). After addition of 1 to 4 mM MgATP, videos of rotating protein-bead complexes were recorded with an inverted fluorescence microscope (IX83/Cell∧TIRF) and analyzed to determine the rotational rates as described before (26).

Single molecule rotation assay with nanorods
Biotinylated Gs␣ 3 ␤ 3 ␥ or ␣ 3 chi ␤ 3 ␥ complexes were fixed on a Ni 2ϩ -NTA-coated coverslide. The biotinylated cysteine at ␥109 was bound to a gold nanorods (40 ϫ 76 nm, Nanopartz) that was prepared by mixing 9 l of nanorods with 1 l of Neutravidin (5 mg/ml in H 2 O, Molecular Probes) for 1 h before adding 1 l of BSA (10% solution, Aurion, The Netherlands) for 1 min and 89 l of buffer RB (50 mM Tris, pH 8.0, 10 mM KCl). Solutions were infused in a flow cell (20 l, 5 min incubation) in the following order, with washing steps of 60 l of buffer RB in between: 1) Ni 2ϩ -NTA-horseradish peroxidase conjugate (Qiagen, Germany) in deionized H 2 O; 2) 1 nM biotinylated protein in buffer RB; 3) gold nanorods in buffer RB; 4) 2 mM ATP, 1 mM MgCl 2 in buffer RB. Rotation of protein-nanorod complexes via polarized darkfield microscopy and analysis of the data via customized software to obtain intensities and velocities at each angular position were performed as described before (28 -30). In brief, samples were illuminated by a mercury lamp through a dark field condenser in a confocal microscope (Axio-vert, Zeiss, Germany). When observed through a band pass filter and a polarizer the intensity of red light scattered from the nanorod, detected by an APD, changed sinusoidal from a maximum to a minimum when the long axis of the nanorod was parallel and perpendicular, respectively, to the axis of polarization. Data were collected for 10 and 30 s at 1 and 200 kHz, respectively. The obtained signals were evaluated with customized software (Matlab, MathWorks) to obtain intensities and velocities at each angular position of the rotating protein-nanorod complex (28 -30). From the light intensity fluctuations recorded by the APD, power strokes were identified by intensity changes from a minimum taken from the lowest 10 percentile of peaks to a maximum taken from the highest 5 percentile of peak, for which a linear least square fit gave an R-value of at least 0.9.

Synthesis of the peptide ␣(521-540) from M. tuberculosis
Peptide Mt␣(521-540) of the C-terminal amino acids 521-540 of Mt␣ (strain H37R V ) was synthesized at the peptide core facility, School of Biological Sciences, NTU on Liberty Automatic. The final product with at least 99% purity was confirmed by analytical high pressure liquid chromatography.

Circular dichroism spectroscopy of Mt␣(521-540)
Circular dichroism (CD) spectra of the peptide Mt␣ (521-540) were recorded with a CHIRASCAN spectrometer (Applied Photo-physics) using a 60-l quartz cell (Hellma, Germany) with 1-mm path length. Light of 190 -260 nm was used to record the far UV-spectra at 20°C with 1-nm resolution. The measurement was done three times for each sample. The CD-spectra were acquired in a buffer of 25 mM phosphate (pH 6.5), and 30% 2,2,2-trifluoroethanol with a peptide concentration of 2.0 mg/ml.

Solution structure determination of Mt␣(521-540)
For NMR spectroscopy experiments 2 mM Mt␣(521-540) were dissolved in 25 mM phosphate buffer (pH 6.5) and 30% deuterated 2,2,2-trifluoroethanol. All spectra were obtained at 298 K on a 700-MHz Avance Bruker NMR spectrometer, equipped with actively shielded cryoprobe and pulse field gradients. TOCSY and nuclear Overhauser effect spectroscopy (NOESY) spectra of the peptide were recorded with mixing times of 80 and 200 ms, respectively. 2,2-Dimethyl-2-silapentane-5-sulfonate sodium salt was used as an internal reference. Data acquisition and processing were done with the Topspin (Bruker Biospin) program. The Sparky suite was used for spectrum visualization and peak picking. Standard procedures based on spin system identification and sequential assignments were adopted to identify the resonances. Interproton distances were obtained from the NOESY spectra by using the caliba script included in the CYANA 2.1 package (45). Dihedral angle constraints were derived from TALOS. The predicted dihedral angle restraints were used for the structure calculation, with a variation of Ϯ30°from the average values. The CYANA 2.1 package (45) was used to generate the three-dimensional (3D) structure of the peptide. Several rounds of structural calculation were performed. Angle and distance constraints were adjusted until no NOE violations occurred. In total, 100 structures were calculated and an