Thermophilic Bacillus PS3 Research Articles

Rotary properties of hybrid F1-ATPases consisting of subunits from different species

F1-ATPase (F1) is an ATP-driven rotary motor protein ubiquitously found in many species as the catalytic portion of FoF1-ATP synthase. Despite the highly conserved amino acid sequence of the catalytic core subunits: α and β, F1 shows diversity in the maximum catalytic turnover rate Vmax and the number of rotary steps per turn. To study the design principle of F1, we prepared eight hybrid F1s composed of subunits from two of three genuine F1s: thermophilic Bacillus PS3 (TF1), bovine mitochondria (bMF1), and Paracoccus denitrificans (PdF1), differing in the Vmax and the number of rotary steps. The Vmax of the hybrids can be well fitted by a quadratic model highlighting the dominant roles of β and the couplings between α-β. Although there exist no simple rules on which subunit dominantly determines the number of steps, our findings show that the stepping behavior is characterized by the combination of all subunits.

Direct identification of the rotary angle of ATP cleavage in F1-ATPase from Bacillus PS3

F1-ATPase is the world’s smallest biological rotary motor driven by ATP hydrolysis at three catalytic β subunits. The 120° rotational step of the central shaft γ consists of 80° substep driven by ATP binding and a subsequent 40° substep. In order to correlate timing of ATP cleavage at a specific catalytic site with a rotary angle, we designed a new F1-ATPase (F1) from thermophilic Bacillus PS3 carrying β(E190D/F414E/F420E) mutations, which cause extremely slow rates of both ATP cleavage and ATP binding. We produced an F1 molecule that consists of one mutant β and two wild-type βs (hybrid F1). As a result, the new hybrid F1 showed two pausing angles that are separated by 200°. They are attributable to two slowed reaction steps in the mutated β, thus providing the direct evidence that ATP cleavage occurs at 200° rather than 80° subsequent to ATP binding at 0°. This scenario resolves the long-standing unclarified issue in the chemomechanical coupling scheme and gives insights into the mechanism of driving unidirectional rotation.

Remotely controllable supramolecular rotor mounted inside a porphyrinic cage

Remotely controllable supramolecular rotor mounted inside a porphyrinic cage

Characterizing the Hydration Properties of Proton Binding Sites in the ATP Synthase c-Rings of Bacillus Species

The membrane-embedded domain of ATP synthases contains the c-ring, which translocates ions across the membrane, and its resultant rotation is coupled to ATP synthesis in the extramembranous domain. During rotation, the c-ring becomes accessible on both sides of the lipid bilayer to solvent via channels connected to the other membrane-embedded component, the a subunit, and thereby allows the ion to be released into the solvent environment. In recent times, many experimental structures of c-rings from different species have been solved. In some of these, a water molecule with a proposed "structural role" has been identified within the c-ring ion binding site, but in general, the requirement for high resolution to resolve specific water densities complicates their interpretation. In the present study, we use molecular dynamics (MD) simulations and rigorous free energy calculations to characterize the dynamics and energetics of a water molecule within the ion binding site of the c-ring from Bacillus pseudofirmus OF4, in its wild type (WT) and P51A mutant forms, along with the c-ring from thermophilic Bacillus PS3. Our data suggest that a water molecule stably binds to the P51A mutant, as well as helping to identify a bound water molecule in Bacillus PS3 whose presence was previously overlooked due to the limited resolution of the structural data. Sequence analysis further identifies a novel conserved sequence motif that is likely required to harbor a water molecule for stable ion coordination in the binding site of such proteins.

How Ligand Binding Affects the Dynamical Transition Temperature in Proteins.

The biochemical functions of proteins are activated at the protein glass transition temperature, which has been proposed to be dependent upon protein-water interactions. However, at the molecular level it is unclear how ligand binding to well-defined binding sites can influence this transition temperature. We thus report molecular dynamics (MD) simulations of the ϵ subunit from thermophilic Bacillus PS3 in the ATP-free and ligand-bound states over a range of temperatures from 20 to 300 K, to study the influence of ligand association upon the transition temperature. We also measure the protein mean square displacement (MSD) in each state, which is well established as a means to quantify this dynamical temperature dependence. We find that the transition temperature is largely unaffected by ligand association, but the MSD beyond the transition temperature increases more rapidly in the ATP-free state. Our data suggests that ligands can effectively "shield" a binding site from solvent, and hence stabilize protein domains with increasing temperature.

Single mutations in the ε subunit from thermophilic Bacillus PS3 generate a high binding affinity site for ATP.

The ε subunit from ATP synthases acts as an ATP sensor in the bacterial cell to prevent ATP hydrolysis and thus the waste of ATP under conditions of low ATP concentration. However, the ATP binding affinities from various bacterial organisms differ markedly, over several orders of magnitude. For example, the ATP synthases from thermophilic Bacillus PS3 and Escherichia coli exhibit affinities of 4 µM and 22 mM, respectively. The recently reported R103A/R115A double mutant of Bacillus PS3 ATP synthase demonstrated an increased binding affinity by two orders of magnitude with respect to the wild type. Here, we used atomic-resolution molecular dynamics simulations to determine the role of the R103A and R115A single mutations. These lead us to predict that both single mutations also cause an increased ATP binding affinity. Evolutionary analysis reveals R103 and R115 substitutions in the ε subunit from other bacillic organisms, leading us to predict they likely have a higher ATP binding affinity than previously expected.

The effect of culture conditions on the accumulation and activity of F0F1 ATP synthase in thermophilic bacteria BacillusPS3

AbstractBACKGROUNDThe proton‐translocating protein F0F1 ATP synthase is a transmembrane protein that catalyzes ATP synthesis and is important in artificial photosynthesis and other life science studies. The objective of this study was to understand the accumulation and ATPase activity of F0F1 ATP synthase in the thermophilic bacteria Bacillus PS3 affected by culture conditions, including the type of nitrogen source and the concentrations of polypeptone and sodium chloride in the culture medium, as well as medium pH and aeration rate.RESULTSThe highest yield and ATPase activity of purified enzymes were obtained when adding polypeptone as the nitrogen source at higher concentrations. Medium pH and aeration rate showed significant effects on the accumulation and activity of F0F1 ATP synthase. Contrarily, sodium chloride concentration had little effect on the growth of PS3 or F0F1 ATP synthase accumulation. The highest yield and specific activity of purified F0F1 ATP synthase achieved were 1.631 g L−1 and 7.99 µmol min−1 mg−1, under the culture conditions of pH 7, aeration of 3 L min−1 and 0.3% NaCl and 1.2% (w/w) polypeptone concentrations. The F0F1 ATP synthase purified was characterized by SDS‐PAGE and contained all expected subunits.CONCLUSIONAll of the studied culture conditions except for NaCl concentration significantly affected Bacillus PS3 cell growth and ATP synthase accumulation and reactivity. Polypeptone at higher concentrations in the culture medium along with neutral pH and greater aeration rates were desirable. Cell growth and ATPase activity of F0F1 ATP synthase were highly positively associated. © 2018 Society of Chemical Industry

Direct assignment of 13C solid-state NMR signals of TFoF1 ATP synthase subunit c-ring in lipid membranes and its implication for the ring structure.

FoF1-ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H+ translocation in membranes. The Fo c-subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state 13C NMR (ssNMR) signals of the Fo c-subunit ring of thermophilic Bacillus PS3 (TFo c-ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly 13C-labeled sample, the signal overlap has been a major bottleneck because most amino acid residues are hydrophobic. To overcome signal overlapping, we developed a method designated as COmplementary Sequential assignment with MInimum Labeling Ensemble (COSMILE). According to this method, we generated three kinds of reverse-labeled samples to suppress signal overlapping. To assign the carbon signals sequentially, two-dimensional Cα(i+1)-C'Cα(i) correlation and dipolar assisted rotational resonance (DARR) experiments were performed under magic-angle sample spinning. On the basis of inter- and intra-residue 13C-13C chemical shift correlations, 97% of Cα, 97% of Cβ and 92% of C' signals were assigned directly from the spectra. Secondary structure analysis predicted a hairpin fold of two helices with a central loop. The effects of saturated and unsaturated phosphatidylcholines on TFo c-ring structure were examined. The DARR spectra at 15ms mixing time are essentially similar to each other in saturated and unsaturated lipid membranes, suggesting that TFo c-rings have similar structures under the different environments. The spectrum of the sample in saturated lipid membranes showed better resolution and structural stability in the gel state. The C-terminal helix was suggested to locate in the outer layer of the c-ring.

The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase.

The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the ε subunit from bacterial ATP synthases.

Identification of aqueous access residues of the sodium half channel in transmembrane helix 5 of the Fo-a subunit of Propionigenium modestum ATP synthase.

The Fo-a subunit of the Na+-transporting FoF1 ATP synthase from Propionigenium modestum plays a key role in Na+ transport. It forms half channels that allow Na+ to enter and leave the buried carboxyl group on Fo-c subunits. The essential Arg residue R226, which faces the carboxyl group of Fo-c subunits in the middle of transmembrane helix 5 of the Fo-a subunit, separates the cytoplasmic side and periplasmic half-channels. To elucidate contributions of other amino acid residues of transmembrane helix 5 using hybrid FoF1 (Fo from P. modestum and F1 from thermophilic Bacillus PS3), 25 residues were individually mutated to Cys, and effects of modification with the SH-modifying agent N-ethylmaleimide (NEM) on ATP synthesis and hydrolysis activity were analyzed. NEM significantly inhibited ATP synthesis and hydrolysis as well as proton pumping activities of A214C, G215C, A218C, I223C (cytoplasmic side from R226), and N230C (periplasmic side from R226) mutants and inhibited ATP synthesis activity of the K219C mutant (cytoplasmic side from R226). Thus, these residues contribute to the integrity of the Na+ half channel, and both half channels are present in the Fo-a subunit.

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.

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

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.

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)

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.

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.