Masasuke Yoshida

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.

Evaluation of intramitochondrial ATP levels identifies G0/G1 switch gene 2 as a positive regulator of oxidative phosphorylation

Significance We developed a sensitive method to assess the activity of oxidative phosphorylation in living cells using a FRET-based ATP biosensor. We then revealed that G0/G1 switch gene 2, a protein rapidly induced by hypoxia, increases mitochondrial ATP production by interacting with FoF1-ATP synthase and protects cells from a critical energy crisis. The oxidative phosphorylation (OXPHOS) system generates most of the ATP in respiring cells. ATP-depleting conditions, such as hypoxia, trigger responses that promote ATP production. However, how OXPHOS is regulated during hypoxia has yet to be elucidated. In this study, selective measurement of intramitochondrial ATP levels identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS. A mitochondria-targeted, FRET-based ATP biosensor enabled us to assess OXPHOS activity in living cells. Mitochondria-targeted, FRET-based ATP biosensor and ATP production assay in a semiintact cell system revealed that G0s2 increases mitochondrial ATP production. The expression of G0s2 was rapidly and transiently induced by hypoxic stimuli, and G0s2 interacts with OXPHOS complex V (FoF1-ATP synthase). Furthermore, physiological enhancement of G0s2 expression prevented cells from ATP depletion and induced a cellular tolerance for hypoxic stress. These results show that G0s2 positively regulates OXPHOS activity by interacting with FoF1-ATP synthase, which causes an increase in ATP production in response to hypoxic stress and protects cells from a critical energy crisis. These findings contribute to the understanding of a unique stress response to energy depletion. Additionally, this study shows the importance of assessing intramitochondrial ATP levels to evaluate OXPHOS activity in living cells.

Functional analysis of membranous Fo-a subunit of F1Fo-ATP synthase by in vitro protein synthesis

The a subunit of F(1)F(o) (F(1)F(o)-ATP synthase) is a highly hydrophobic protein with five putative transmembrane helices which plays a central role in H(+)-translocation coupled with ATP synthesis/hydrolysis. In the present paper, we show that the a subunit produced by the in vitro protease-free protein synthesis system (the PURE system) is integrated into a preformed F(o) a-less F(1)F(o) complex in Escherichia coli membrane vesicles and liposomes. The resulting F(1)F(o) has a H(+)-coupled ATP synthesis/hydrolysis activity that is approximately half that of the native F(1)F(o). By using this procedure, we analysed five mutations of F(1)F(o), where the conserved residues in the a subunit (Asn(90), Asp(112), Arg(169), Asn(173) and Gln(217)) were individually replaced with alanine. All of the mutant F(o) a subunits were successfully incorporated into F(1)F(o), showing the advantage over conventional expression in E. coli by which three (N90A, D112A, and Q217A) mutant a subunits were not found in F(1)F(o). The N173A mutant retained full activity and the mutants D112A and Q217A had weak, but detectable, activity. No activity was observed for the R169A and N90A mutants. Asn(90) is located in the middle of putative second transmembrane helix and likely to play an important role in H(+)-translocation. The present study exemplifies that the PURE system provides an alternative approach when in vivo expression of membranous components in protein complexes turns out to be difficult.

Ribosomal protein L2 associates with E. coli HtpG and activates its ATPase activity

Although eukaryotic Hsp90 has been studied extensively, the function of its bacterial homologue HtpG remains elusive. Here we report that 50S ribosomal protein L2 was found as an associated protein with His-tagged HtpG from Escherichia coli cultured in minimum medium at 45 °C. L2 specifically activated ATPase activity of HtpG, but other denatured proteins did not. The analysis using domain derivatives of HtpG and L2 showed that C-terminal domain of L2 and the middle to C-terminal domain of HtpG are important for interaction. At physiological salt concentration, L2 was denatured state and was recognized by HtpG as well as other chaperones, DnaK/DnaJ/GrpE and GroEL/GroES. The ATPase of HtpG at increasing concentration of L2 indicated that an L2 molecule bound to a dimer HtpG with apparent K(D) of 0.3 μM at 100mM KCl and 3.3 μM at 200 mM KCl.

Torque Generation and Utilization in Motor Enzyme F0F1-ATP Synthase: HALF-TORQUE F1 WITH SHORT-SIZED PUSHROD HELIX AND REDUCED ATP SYNTHESIS BY HALF-TORQUE F0F1*

Background: ATP synthase (F0F1) is a rotary motor enzyme. Results: F1 with a short-sized helix-1 in β subunit rotates with half of the normal torque and supports reduced ATP synthesis activity. Conclusion: Helix-1 acts as a “pushrod” to generate torque, and torque-reduced F0F1 retains the catalytic ability of ATP synthesis. Significance: Generation and utilization of the torque are crucial for motor enzymes. ATP synthase (F0F1) is made of two motors, a proton-driven motor (F0) and an ATP-driven motor (F1), connected by a common rotary shaft, and catalyzes proton flow-driven ATP synthesis and ATP-driven proton pumping. In F1, the central γ subunit rotates inside the α3β3 ring. Here we report structural features of F1 responsible for torque generation and the catalytic ability of the low-torque F0F1. (i) Deletion of one or two turns in the α-helix in the C-terminal domain of catalytic β subunit at the rotor/stator contact region generates mutant F1s, termed F1(1/2)s, that rotate with about half of the normal torque. This helix would support the helix-loop-helix structure acting as a solid “pushrod” to push the rotor γ subunit, but the short helix in F1(1/2)s would fail to accomplish this task. (ii) Three different half-torque F0F1(1/2)s were purified and reconstituted into proteoliposomes. They carry out ATP-driven proton pumping and build up the same small transmembrane ΔpH, indicating that the final ΔpH is directly related to the amount of torque. (iii) The half-torque F0F1(1/2)s can catalyze ATP synthesis, although slowly. The rate of synthesis varies widely among the three F0F1(1/2)s, which suggests that the rate reflects subtle conformational variations of individual mutants.

Activation and Stiffness of the Inhibited States of F1-ATPase Probed by Single-molecule Manipulation

F1-ATPase (F1), a soluble portion of FoF1-ATP synthase (FoF1), is an ATP-driven motor in which γϵ subunits rotate in the α3β3 cylinder. Activity of F1 and FoF1 from Bacillus PS3 is attenuated by the ϵ subunit in an inhibitory extended form. In this study we observed ATP-dependent transition of ϵ in single F1 molecules from extended form to hairpin form by fluorescence resonance energy transfer. The results justify the previous bulk experiments and ensure that fraction of F1 with hairpin ϵ directly determines the fraction of active F1 at any ATP concentration. Next, mechanical activation and stiffness of ϵ-inhibited F1 were examined by the forced rotation of magnetic beads attached to γ. Compared with ADP inhibition, which is another manner of inhibition, rotation by a larger angle was required for the activation from ϵ inhibition when the beads were forced to rotate to ATP hydrolysis direction, and more torque was required to reach the same rotation angle when beads were forced to rotate to ATP synthesis direction. The results imply that if FoF1 is resting in the ϵ-inhibited state, Fo motor must transmit to γ a torque larger than expected from thermodynamic equilibrium to initiate ATP synthesis.

Conformational Transitions of Subunit ɛ in ATP Synthase from Thermophilic Bacillus PS3

Subunit epsilon of bacterial and chloroplast F(O)F(1)-ATP synthase is responsible for inhibition of ATPase activity. In Bacillus PS3 enzyme, subunit epsilon can adopt two conformations. In the extended, inhibitory conformation, its two C-terminal alpha-helices are stretched along subunit gamma. In the contracted, noninhibitory conformation, these helices form a hairpin. The transition of subunit epsilon from an extended to a contracted state was studied in ATP synthase incorporated in Bacillus PS3 membranes at 59 degrees C. Fluorescence energy resonance transfer between fluorophores introduced in the C-terminus of subunit epsilon and in the N-terminus of subunit gamma was used to follow the conformational transition in real time. It was found that ATP induced the conformational transition from the extended to the contracted state (half-maximum transition extent at 140 microM ATP). ADP could neither prevent nor reverse the ATP-induced conformational change, but it did slow it down. Acid residues in the DELSEED region of subunit beta were found to stabilize the extended conformation of epsilon. Binding of ATP directly to epsilon was not essential for the ATP-induced conformational change. The ATP concentration necessary for the half-maximal transition (140 microM) suggests that subunit epsilon probably adopts the extended state and strongly inhibits ATP hydrolysis only when the intracellular ATP level drops significantly below the normal value.

IF1, a natural inhibitor of mitochondrial ATP synthase, is not essential for the normal growth and breeding of mice.

IF1 is an endogenous inhibitor protein of mitochondrial ATP synthase. It is evolutionarily conserved throughout all eukaryotes and it has been proposed to play crucial roles in prevention of the wasteful reverse reaction of ATP synthase, in the metabolic shift from oxidative phosphorylation to glycolysis, in the suppression of ROS (reactive oxygen species) generation, in mitochondria morphology and in haem biosynthesis in mitochondria, which leads to anaemia. Here, we report the phenotype of a mouse strain in which IF1 gene was destroyed. Unexpectedly, individuals of this IF1-KO (knockout) mouse strain grew and bred without defect. The general behaviours, blood test results and responses to starvation of the IF1-KO mice were apparently normal. There were no abnormalities in the tissue anatomy or the autophagy. Mitochondria of the IF1-KO mice were normal in morphology, in the content of ATP synthase molecules and in ATP synthesis activity. Thus, IF1 is not an essential protein for mice despite its ubiquitous presence in eukaryotes.

Polypeptide in the chaperonin cage partly protrudes out and then folds inside or escapes outside

The current mechanistic model of chaperonin‐assisted protein folding assumes that the substrate protein in the cage, formed by GroEL central cavity capped with GroES, is isolated from outside and exists as a free polypeptide. However, using ATPase‐deficient GroEL mutants that keep GroES bound, we found that, in the rate‐limiting intermediate of a chaperonin reaction, the unfolded polypeptide in the cage partly protrudes through a narrow space near the GroEL/GroES interface. Then, the entire polypeptide is released either into the cage or to the outside medium. The former adopts a native structure very rapidly and the latter undergoes spontaneous folding. Partition of the in‐cage folding and the escape varies among substrate proteins and is affected by hydrophobic interaction between the polypeptide and GroEL cavity wall. The ATPase‐active GroEL with decreased in‐cage folding produced less of a native model substrate protein in Escherichia coli cells. Thus, the polypeptide in the critical GroEL–GroES complex is neither free nor completely confined in the cage, but it is interacting with GroELs apical region, partly protruding to outside.

Kinetic equivalence of transmembrane pH and electrical potential differences in ATP synthesis

Background: ATP synthesis is driven by the combination of transmembrane electrical potential and pH difference. Results: Either electrical potential or pH difference can drive synthesis even when the other opposes. Conclusion: The synthesis rate depends on the algebraic sum of the two, irrespective of the individual magnitudes and signs. Significance: Comprehensive data sets directly attest to kinetic equivalence of the two. ATP synthase is the key player of Mitchells 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.