Bacillus PS3 Research Articles

Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors

The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A3B3DF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor α3β3γ, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 μs resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M. mazei A3B3DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, because ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP binding to A3B3DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwell. In F-type motors, elevated ADP induces dwells 40° after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits.

On the Mg2+ binding site of the ε subunit from bacterial F-type ATP synthases

F-type ATP synthases, central energy conversion machines of the cell synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane and, reversely, can also hydrolyze ATP to pump ions across the membrane, depending on cellular conditions such as ATP concentration. To prevent wasteful ATP hydrolysis, mammalian and bacterial ATP synthases possess different regulatory mechanisms. In bacteria, a low ATP concentration induces a conformational change in the ε subunit from the down- to up-states, which inhibits ATP hydrolysis. Moreover, the conformational change of the ε subunit depends on Mg2+ concentration in some bacteria such as Bacillus subtilis, but not in others. This diversity makes the ε subunit a potential target for antibiotics. Here, performing molecular dynamics simulations, we identify the Mg2+ binding site in the ε subunit from B. subtilis as E59 and E86. The free energy analysis shows that the first-sphere bi-dentate coordination of the Mg2+ ion by the two glutamates is the most stable state. In comparison, we also clarify the reason for the absence of Mg2+ dependency in the ε subunit from thermophilic Bacillus PS3, despite the high homology to that from B. subtilis. Sequence alignment suggests that this Mg2+ binding motif is present in the ε subunits of some pathogenic bacteria. In addition we discuss strategies to stabilize an isolated ε subunit carrying the Mg2+ binding motif by site directed mutagenesis, which also can be used to crystallize Mg2+ dependent ε subunits in future.

ATP-induced dimerization of the F0F1 ε subunit from Bacillus PS3: a hydrogen exchange-mass spectrometry study.

F0F1 ATP synthase harnesses a transmembrane electrochemical gradient for the production of ATP. When operated in reverse, this multiprotein complex catalyzes ATP hydrolysis. In bacteria, the ε subunit is involved in regulating this ATPase activity. Also, ε is essential for coupling ATP hydrolysis (or synthesis) to proton translocation. The ε subunit consists of a β sandwich and two C-terminal helices, α1 and α2. The protein can switch from a compact fold to an alternate conformation where α1 and α2 are separated, resulting in an extended structure. ε from the thermophile Bacillus PS3 (Tε) binds ATP with high affinity such that this protein may function as an intracellular ATP level sensor. ATP binding to isolated Tε triggers a major conformational transition. Earlier data were interpreted in terms of an ATP + Tεextended → ATP·Tεcompact transition that may mimic aspects of the regulatory switching within F0F1 (Yagi et al. (2007) Proc. Natl. Acad. Sci. U.S.A., 104, 11233–11238). In this work, we employ complementary biophysical techniques for examining the ATP-induced conformational switching of isolated Tε. CD spectroscopy confirmed the occurrence of a large-scale conformational transition upon ATP binding, consistent with the formation of stable helical structure. Hydrogen/deuterium exchange (HDX) mass spectrometry revealed that this transition is accompanied by a pronounced stabilization in the vicinity of the ATP-binding pocket. Surprisingly, dramatic stabilization is also seen in the β8−β9 region, which is remote from the site of ATP interaction. Analytical ultracentrifugation uncovered a previously unrecognized feature of Tε: a high propensity to undergo dimerization in the presence of ATP. Comparison with existing crystallography data strongly suggests that the unexpected β8−β9 HDX protection is due to newly formed protein–protein contacts. Hence, ATP binding to isolated Tε proceeds according to 2ATP + 2Tεextended → (ATP·Tεcompact)2. Implications of this dimerization propensity for the possible role of Tε as an antibiotic target are discussed.

Basic Properties of Rotary Dynamics of the Molecular Motor Enterococcus hirae V1-ATPase

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe. EhV1 rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (Vmax) of 107 revolutions/s and a Michaelis constant (Km) of 154 μM at 26 °C. At all ATP concentrations tested, EhV1 showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V1-ATPase (TtV1). At 10 μM ATP (<<Km), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 × 10(6) M(-1) s(-1). At 40 mM ATP (>>Km), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV1. Our results identify the common properties of rotary catalysis of V1-ATPases that are distinct from those of F1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.

H+/ATP ratio of FoF1-ATP synthase from the thermophilic Bacillus PS3

H+/ATP ratio of FoF1-ATP synthase from the thermophilic Bacillus PS3

ATP-driven H+ pump activity of thermophilic Bacillus PS3 FoF1-ATP synthase reconstituted in a liposomal membrane measured by fluorescence ensemble and single liposome assays

ATP-driven H+ pump activity of thermophilic Bacillus PS3 FoF1-ATP synthase reconstituted in a liposomal membrane measured by fluorescence ensemble and single liposome assays

A point mutation, betaGln259Leu, relieves MgADP inhibition in Bacillus PS3 ATP synthase

A point mutation, betaGln259Leu, relieves MgADP inhibition in Bacillus PS3 ATP synthase

Kinetic Equivalence of Transmembrane pH and Electrical Potential Differences in ATP Synthesis

ATP synthase is the key player of Mitchell's chemiosmotic theory, converting the energy of transmembrane proton flow into the high energy bond between ADP and phosphate. The proton motive force that drives this reaction consists of two components, the pH difference (ΔpH) across the membrane and transmembrane electrical potential (Δψ). The two are considered thermodynamically equivalent, but kinetic equivalence in the actual ATP synthesis is not warranted, and previous experimental results vary. Here, we show that with the thermophilic Bacillus PS3 ATP synthase that lacks an inhibitory domain of the ε subunit, ΔpH imposed by acid-base transition and Δψ produced by valinomycin-mediated K(+) diffusion potential contribute equally to the rate of ATP synthesis within the experimental range examined (ΔpH -0.3 to 2.2, Δψ -30 to 140 mV, pH around the catalytic domain 8.0). Either ΔpH or Δψ alone can drive synthesis, even when the other slightly opposes. Δψ was estimated from the Nernst equation, which appeared valid down to 1 mm K(+) inside the proteoliposomes, due to careful removal of K(+) from the lipid.

Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic FoF1-ATP synthase expressed in Escherichia coli

FoF1-ATP synthase catalyzes ATP synthesis coupled with proton-translocation across the membrane. The membrane-embedded Fo portion is responsible for the H+ translocation coupled with rotation of the oligomeric c-subunit ring, which induces rotation of the γ subunit of F1. For solid-state NMR measurements, FoF1 of thermophilic Bacillus PS3 (TFoF1) was overexpressed in Escherichia coli and the intact c-subunit ring (TFoc-ring) was isolated by new procedures. One of the key improvement in this purification was the introduction of a His residue to each c-subunit that acts as a virtual His10-tag of the c-ring. After solubilization from membranes by sodium deoxycholate, the c-ring was purified by Ni–NTA affinity chromatography, followed by anion-exchange chromatography. The intactness of the isolated c-ring was confirmed by high-resolution clear native PAGE, sedimentation analysis, and H+-translocation activity. The isotope-labeled intact TFoc-ring was successfully purified in such an amount as enough for solid-state NMR measurements. The isolated TFoc-rings were reconstituted into lipid membranes. A solid-state NMR spectrum at a high quality was obtained with this membrane sample, revealing that this purification procedure was suitable for the investigation by solid-state NMR. The purification method developed here can also be used for other physicochemical investigations.

Kinetic Equivalence of pH Difference (ΔPh) and Electrical Potential (Δψ) across Membrane in ATP Synthesis by Bacillus PS3 FOF1-ATP Synthase

The ATP synthase is the key player of Mitchell's chemiosmotic theory, converting the energy of transmembrane proton flow into the high-energy bond between ADP and phosphate. The proton motive force that drives this reaction consists of two components, the pH difference (ΔpH) across the membrane and transmembrane electrical potential (Δψ). The two are considered thermodynamically equivalent, but kinetic equivalence in the actual ATP synthesis is not warranted and previous experimental results vary. Here we show that, in the thermophilic Bacillus PS3 ATP synthase that lacks an inhibitory domain of the e subunit, ΔpH imposed by acid-base transition and Δψ produced by valinomycin-mediated K+ diffusion potential contribute equally to the rate of ATP synthesis, within the experimental range examined (ΔpH −0.3 to 2.4, Δψ −30 to 140 mV, pH around the catalytic domain 8.0). Either ΔpH or Δψ alone can drive synthesis, even when the other slightly opposes.

3A1046 リポソーム膜中に再構成した好熱菌由来F_0F_1-ATP合成酵素によるATP駆動H^+輸送の定量(分子モーターIII:F1-ATPase,マイコプラズマ,口頭発表,日本生物物理学会第50回年会(2012年度))

3A1046 リポソーム膜中に再構成した好熱菌由来F_0F_1-ATP合成酵素によるATP駆動H^+輸送の定量(分子モーターIII:F1-ATPase,マイコプラズマ,口頭発表,日本生物物理学会第50回年会(2012年度))

1PS027 H^+/ATP ratio of F_OF_1-ATP synthase from the thermophilic Bacillus PS3(The 50th Annual Meeting of the Biophysical Society of Japan)

1PS027 H^+/ATP ratio of F_OF_1-ATP synthase from the thermophilic Bacillus PS3(The 50th Annual Meeting of the Biophysical Society of Japan)

Characterization of TFoF1 ATP Synthase C Subunit Ring in Membranes with HS-AFM and Solid-State NMR

FoF1-ATP synthase is located in the membranes and involved in ATP synthesis coupled with proton-translocation across the membrane. F1 carries out the rotational catalysis for the ATP synthesis and Fo is responsible for the H+ translocation coupled with the rotation of c subunit ring, which also rotates the gamma subunit of F1. We have established a new purification method of the c subunit rings from the FoF1 of thermophilic Bacillus PS3 (TFoF1) expressed in E. coli. They were reconstituted into membranes of dimyristoyl phosphatidylcholine with perdeuterated acyl chains. To examine the macroscopic architecture of the oligomeric TFoc in the DMPC membranes, successive high speed atomic force microscopy (HS-AFM) images of the reconstituted preparation on the mica in the presence of buffer were obtained at imaging rate of 2-5 frames per second at room temperature. The HS-AFM images of the TFoc oligomers expressed in E. coli and reconstituted into DMPC membranes revealed torus-shaped substances with the pores at the center, confirming that the intact c-ring structure is maintained in the purified smple. The similar images were obtained for the oligomeric TFoc synthesized in the wheat-germ-extract (WGE) cell-free system, showing that the ring can be formed with the c subunit produced with a WGE cell-free system. Then, solid-state NMR analysis was carried out. Two-dimensional 13C homonuclear correlation with DARR and 2D Ca(i+1)-Ca(i) correlation experiments were performed under MAS. The assignment was carried out, using fully 13C, 15N labeled TFoc and partially 13C, 15N labeled TFoc. The assignment strategy and the obtained structural information on the basis of the assignment and magnetization transfer will be discussed.

Pressure Sensitive Reaction of F1-ATPase

F1-ATPase is a rotational stepping molecular motor in which γ subunit rotates 120o against the α3β3 cylinder upon one ATP molecule hydrolysis. This 120o step is further divided into 80o and 40o substeps and each substep is triggered by ATP binding and ADP release and by ATP hydrolysis and Pi release, respectively. The stepping motion was sensitive against physical and chemical conditions, such as temperature and load Hydrostatic pressure is also a physical parameter to modulate the structure and function of protein molecules. Here, we developed a novel assay that monitored the stepping motion of single F1-ATPase molecules under various pressure conditions [1]. At ambient conditions, F1-ATPases derived from thermophilic Bacillus PS3 smoothly rotated with 9 Hz in the presence of 2 mM ATP. The rotational rate decreased with increased pressure, and then reached to 3 Hz and became stepping rotation by slowing a certain dwell at 140 MPa In order to identify which chemical state this dwell corresponds, the mutant F1(βE190D) which shows the pause of ATP catalytic dwell due to extremely slow ATP hydrolysis even under Vmax condition [2] was used. This pressure dependent dwell became obvious at +40o from catalytic dwell with applying pressure, i.e., it is the same position as ATP binding, where F1 executes ATP binding and ADP release on different catalytic sites. Thus, applied pressures seem to inhibit the ATP binding and/or ADP release reactions.[1] Nishiyama et al., Biophys J. 96(3) 1142-1150 (2009).[2] Shimabukuro et al.,Proc. Natl. Acad. Sci. U.S.A. 100, 14731–14736 (2003).

Mechanical modulation of catalytic power on F1-ATPase

The conformational fluctuation of enzymes has a crucial role in reaction acceleration. However, the contribution to catalysis enhancement of individual substates with conformations far from the average conformation remains unclear. We studied the catalytic power of the rotary molecular motor F(1)-ATPase from thermophilic Bacillus PS3 as it was stalled in transient conformations far from a stable pausing angle. The rate constants of ATP binding and hydrolysis were determined as functions of the rotary angle. Both rates exponentially increase with rotation, revealing the molecular basis of positive cooperativity among three catalytic sites: elementary reaction steps are accelerated via the mechanical rotation driven by other reactions on neighboring catalytic sites. The rate enhancement induced by ATP binding upon rotation was greater than that brought about by hydrolysis, suggesting that the ATP binding step contributes more to torque generation than does the hydrolysis step. Additionally, 9% of the ATP-driven rotary step was supported by thermal diffusion, suggesting that acceleration of the ATP docking process occurs via thermally agitated conformational fluctuations.

Characteristics of protection by MgADP and MgATP of α3β3Γ subcomplex of thermophilic Bacillus PS3 βY341W-mutant F1-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a Bi-site mechanism of catalysis

MgADP and MgATP binding to catalytic sites of βY341W-α(3)β(3)γ subcomplex of F(1)-ATPase from thermophilic Bacillus PS3 has been assessed using their effect on the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). It was assumed that NBD-Cl can inhibit only when catalytic sites are empty, and inhibition is prevented if a catalytic site is occupied with a nucleotide. In the absence of an activator, MgADP and MgATP protect βY341W-α(3)β(3)γ subcomplex from inhibition by NBD-Cl by binding to two catalytic sites with an affinity of 37 µM and 12 mM, and 46 µM and 15 mM, respectively. In the presence of an activator lauryldimethylamine-N-oxide (LDAO), MgADP protects βY341W-α(3)β(3)γ subcomplex from inhibition by NBD-Cl by binding to a catalytic site with a K(d) of 12 mM. Nucleotide binding to a catalytic site with affinity in the millimolar range has not been previously revealed in the fluorescence quenching experiments with βY341W-α(3)β(3)γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects βY341W-α(3)β(3)γ subcomplex from inhibition by NBD-Cl only partially, and the enzyme remains sensitive to inhibition by NBD-Cl even at MgATP concentrations that are saturating for ATPase activity. The results support a bi-site mechanism of catalysis by F(1)-ATPases.

Molecular motors in conservative and dissipative regimes

We present a theoretical study of a rotatory molecular motor under a conservative torque regime. We show that conservative and dissipative regimes present a different observable phenomenology. Our approach starts with a preliminary deterministic calculation of the motor cycle, which is complemented with stochastic simulations of a Langevin equation under a flashing ratchet potential. Finally, by using parameter values obtained from independent experimental information, our theoretical predictions are compared with experimental data of the F(1)-ATPase motor of the Bacillus PS3.

The β Subunit Loop That Couples Catalysis and Rotation in ATP Synthase Has a Critical Length

ATP synthase uses a unique rotational mechanism to convert chemical energy into mechanical energy and back into chemical energy. The helix-turn-helix structure in the C-terminal domain of the β subunit containing the conserved DELSEED motif, termed "DELSEED-loop," was suggested to be involved in coupling between catalysis and rotation. If this is indeed the role of the loop, it must have a critical length, the minimum length required to sustain its function. Here, the critical length of the DELSEED-loop was determined by functional analysis of mutants of Bacillus PS3 ATP synthase that had 7-14 amino acids within the loop deleted. A 10 residue deletion lost the ability to catalyze ATP synthesis, but was still an active ATPase. Deletion of 14 residues abolished any enzymatic activity. Modeling indicated that in both deletion mutants the DELSEED-loop was shortened by ∼10 Å; fluorescence resonance energy transfer experiments confirmed the modeling results. This appears to define the minimum length for DELSEED-loop required for coupling of catalysis and rotation. In addition, we could demonstrate that the loss of high-affinity binding to the catalytic site(s) that had been observed previously in two deletion mutants with 3-4 residues removed was not due to the loss of negative charged residues of the DELSEED motif in these mutants. An AALSAAA mutant in which all negative charges of the DELSEED motif were removed showed a normal pattern for MgATP binding to the catalytic sites, with a clearly present high-affinity site.

Efficient ATP synthesis by thermophilic Bacillus FoF1-ATP synthase.

FoF1-ATP synthase (FoF1) synthesizes ATP in the F1 portion when protons flow through Fo to rotate the shaft common to F1 and Fo. Rotary synthesis in isolated F1 alone has been shown by applying external torque to F1 of thermophilic origin. Proton-driven ATP synthesis by thermophilic Bacillus PS3 FoF1 (TFoF1), however, has so far been poor in vitro, of the order of 1 s−1 or less, hampering reliable characterization. Here, by using a mutant TFoF1 lacking an inhibitory segment of the ε-subunit, we have developed highly reproducible, simple procedures for the preparation of active proteoliposomes and for kinetic analysis of ATP synthesis, which was driven by acid–base transition and K+-diffusion potential. The synthesis activity reached ∼ 16 s−1 at 30 °C with a Q10 temperature coefficient of 3–4 between 10 and 30 °C, suggesting a high level of activity at the physiological temperature of ∼ 60 °C. The Michaelis–Menten constants for the substrates ADP and inorganic phosphate were 13 μm and 0.55 mm, respectively, which are an order of magnitude lower than previous estimates and are suited to efficient ATP synthesis.

Kinetic equivalence of membrane potential and pH difference across membrane in ATP synthesis by Bacillus PS3 FOF1-ATP synthase

Kinetic equivalence of membrane potential and pH difference across membrane in ATP synthesis by Bacillus PS3 FOF1-ATP synthase