Regulation of ATP synthases

ATP synthases have various mechanisms of inactivation and reactivation of ATP synthesis under conditions of low and increasing available energy. In mitochondria and chloroplasts, when Δp is low, ADP-Mg (without inorganic phosphate) remains bound to one of the three catalytic sites of the enzyme forming an inactive complex. In chloroplasts, it is thought that this inactive ADP inhibited state is stabilized during the hours of darkness by formation of an intramolecular disulfide linkage in the γ-subunit of the ATP synthase. When daylight is restored, the enzyme is reactivated by thioredoxin regulated reduction of the disulfide, and energy dependent dissociation of the ADP-Mg [1][2].

Mitochondrial enzymes

Mitochondria contain an inhibitor protein known as IF1 [3]. In vitro, at a pH of about 6.7 or below, this protein forms a 1:1 complex with either F1-ATPase or with the intact enzyme complex, the formation of this inhibited complex requiring the hydrolysis of two ATP molecules. In the presence of Δp, the direction of rotation of the rotor reverses, the bound IF1 is released and ATP synthesis resumes. Hence, it has been assumed that the physiological role of IF1 is to prevent the wasteful hydrolysis of ATP in mitochondria under anaerobic conditions.

Figure Inhibition of bovine F1-ATPase by IF1.

The mode of inhibition of F1-ATPase by IF1 has been studied in detail. The active bovine protein is a homo-dimer, where the C-terminal regions of the largely α-helical proteins forman antiparallel coiled-coil (Part A) [4]. The protruding N-terminal regions provide the inhibitory part of the protein, and each active dimer can bind to two F1-ATPase moieties [5]. At pH values around 8.0 and higher, the dimers of bovine IF1 form dimers of dimers and higher aggregates, occluding the inhibitory portion of the protein and rendering the inhibitor inactive. Disaggregation of these oligomers into dimers appears to be controlled by the ionic state of a histidine residue, H49, which is thought to provide a pH sensitive switch between inactive and active states [6]. Removal of the capacity to dimerise by deletion of the C-terminal region from residues 61-84 produces a potent monomeric inhibitor that binds to a single F1-ATPase moiety [7]. In some species, for example S. cerevisiae, IF1 lacks the C-terminal dimerization region, and the protein is naturally monomeric [8].

The structures of the monomeric inhibited bovine [7] and yeast F1-IF1 [8] complexes show that IF1 is bound in a complex binding site at a catalytic interface between the αDP- and βDP-subunits (Part B). In the bovine complex, the inhibitor protein occupies a deep groove lined with α-helices in the C-terminal domains of the αDP- and βDP-subunits (Part C), and its N-terminal region interacts with the coiled-coil region of the γ-subunit via a short α-helix (Part D), and extends into the central aqueous cavity of F1-ATPase. Yeast IF1 binds to yeast F1-ATPase in a similar manner. However, the yeast inhibitor arrests the catalytic cycle at an earlier stage than in the bovine complex, where the magnesium ion and phosphate have been released following hydrolysis of ATP, but ADP still remains bound to the βE-subunit [8]. In the bovine inhibited complex, the ADP molecule has been released from the βE-subunit. The residues in bovine IF1 that provide its binding energy have been identified by systematic mutational analysis and measurement of kinetic constants [9]. Most of the residues are involved in hydrophobic interactions with the C-terminal domain of the βDP-and βTP-subunits. Additional binding energy is provided by an ionic interaction involving glutamate residue E30 of IF1 and arginine residue R408 in the βDP-subunit. This information is being used to construct more potent inhibitor proteins to be used, for example, to probe the catalytic pathway of the enzyme, and for investigation of the pathway of formation of the inhibited F1-IF1 complex.

One striking feature of the inhibitory region of IF1 is that, when it is free in solution and not bound to the enzyme, it is intrinsically unfolded, and it folds progressively into the α-helical structure observed in the inhibited complex as it becomes bound to the enzyme [10]. Two ATP molecules have to be hydrolysed for F1-IF1 complex to achieve the fully inhibited state. By systematic mutagenesis, we have identified the amino acid residues in IF1 that are required for it to bind to F1-ATPase [9].

Eubacterial enzymes

The eubacterial ATP synthase from Paracoccus denitrificans and other α-proteobacteria have their own inhibitor protein, known as the ξ-subunit, that is thought to prevent ATP hydrolysis [11]. The structure of the free protein in solution has a bundle of 4 α-helices, and its N-terminal region from residues 1-18 is unfolded. Conversely, in the inhibited ATP synthase complex, and very similar to IF1, the N-terminal region from residues 1-32 is folded into an α-helix, which is bound in catalytic interface of the F1-domain, very similar to the mode of binding of IF1 to mitochondrial enzymes. The C-terminal domain was not detected in the crystal structure, presumably because it is mobile [12]. The sequence of the ζ-subunit is unrelated to that of IF1.

Figure Binding of the ζ-subunit to the ATP synthase from Paracoccus denitrificans. Part A, the ξ-subunit.  The brown helices are those resolved in the crystal structure of the ATP synthase from P. denitrificans and the cyan helices are from an NMR structure and are unresolved in the crystal structure. Parts B and C, the ξ-subunit binds in a similar position to IF1. Part D, similarity of the ξ-subunit N-terminal helix (brown) with the bovine and yeast IF1 (light blue and pink, respectively).

The ATP synthases from E. coli, G. stearothermophilus and Thermosynechococcus elongatus can synthesize ATP by oxidative phosphorylation, and, under anoxic conditions, hydrolyze ATP made by glycolysis so as to maintain a pmf required for driving other essential cellular functions, such as transport of nutrients and cell motility. Nonetheless, it has been proposed that in these organisms, when cellular ATP concentration and pmf are low, another inhibitory mechanism may operate to prevent wastage of ATP [13][14][15]. It involves the ε-subunits of these enzymes. This subunit, a component of the rotor at the interface between the central stalk and the membrane bound c-ring, is folded into an N-terminal nine-stranded β-sandwich and a C-terminal α-helical hair-pin. The β-sandwich binds the subunit to the γ-subunit and to the c-ring, and the α-helices adopt two conformations “up” and “down”. In the down state, the α-helices bind an ATP molecule, and are associated with the β-sandwich; in the absence of bound ATP the α-helices assume the up position, interact with the α3β3-catalytic domain and inhibit ATP hydrolysis.

Figure Regulation of F1-ATPase from E. coli and C. thermarum. Parts A and B, comparisson of the positions of the ε-subunit in E. coli and C. thermarum, respectively.  The β-subunits, the γ-subunit, and the ε-subunit are cloured yellow, blue and green respectively.  The three α-subunits and one of the β-subunits have been removed for clarity.  Part C, the nucleotide binding site of βEmpty showing the tightly bound phosphate ion and weakly bound ADP molecule. 

In the structures of F1-ATPase from C. thermarum [16], the ε-subunit is in the down position. An ATP molecule with an accompanying magnesium ion, is bound to the ε-subunit. However, when the capacity to bind an ATP molecule was removed, the ε-subunit remained down. Thus, it appears that the ε-subunit does not inhibit ATP hydrolysis in this organism. In the enzyme from M. smegmatis, the ε-subunit, has a truncated C-terminus. It also lacks the arginine residue of the ATP binding motif (I/LDXXRA) suggesting that it is unlikely to bind nucleotide. From their structures, it seems much more likely that the ATP hydrolytic activities of the C. thermarum and M. smegmatis enzymes are inhibited by the failure to release phosphate from one of their three catalytic sites [16]. In the F. nucleatum enzyme, the ε-subunit lacks both the arginine and the alanine from the ATP binding motif and the basis for the inhibition of ATP hydrolysis is not yet clear.


References

  1. Nalin CM & McCarty RE (1984) Role of a disulfide bond in the gamma subunit in activation of the ATPase of chloroplast coupling factor 1. J Biol Chem 259, 7275-80
  2. Walker JE (1994) The regulation of catalysis in ATP synthase. Curr Opin Struct Biol 4, 912-8
  3. PULLMAN ME & MONROY GC (1963) A NATURALLY OCCURRING INHIBITOR OF MITOCHONDRIAL ADENOSINE TRIPHOSPHATASE. J Biol Chem 238, 3762-9
  4. Cabezón E, Runswick MJ, Leslie AG & Walker JE (2001) The structure of bovine IF(1), the regulatory subunit of mitochondrial F-ATPase. EMBO J 20, 6990-6
  5. Cabezón E, Arechaga I, Jonathan P, Butler G & Walker JE (2000) Dimerization of bovine F1-ATPase by binding the inhibitor protein, IF1. J Biol Chem 275, 28353-5
  6. Cabezón E, Butler PJ, Runswick MJ & Walker JE (2000) Modulation of the oligomerization state of the bovine F1-ATPase inhibitor protein, IF1, by pH. J Biol Chem 275, 25460-4
  7. Gledhill JR, Montgomery MG, Leslie AGW & Walker JE (2007) How the regulatory protein, IF(1), inhibits F(1)-ATPase from bovine mitochondria. Proc Natl Acad Sci U S A 104, 15671-6
  8. Robinson GC, Bason JV, Montgomery MG, Fearnley IM, Mueller DM, Leslie AGW & Walker JE (2013) The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF₁. Open Biol 3, 120164
  9. Bason JV, Runswick MJ, Fearnley IM & Walker JE (2011) Binding of the inhibitor protein IF(1) to bovine F(1)-ATPase. J Mol Biol 406, 443-53
  10. Bason JV, Montgomery MG, Leslie AGW & Walker JE (2015) How release of phosphate from mammalian F1-ATPase generates a rotary substep. Proc Natl Acad Sci U S A 112, 6009-14
  11. Morales-Rios E, de la Rosa-Morales F, Mendoza-Hernández G, Rodríguez-Zavala JS, Celis H, Zarco-Zavala M & García-Trejo JJ (2010) A novel 11-kDa inhibitory subunit in the F1FO ATP synthase of Paracoccus denitrificans and related alpha-proteobacteria. FASEB J 24, 599-608
  12. Morales-Rios E, Montgomery MG, Leslie AGW & Walker JE (2015) Structure of ATP synthase from Paracoccus denitrificans determined by X-ray crystallography at 4.0 Å resolution. Proc Natl Acad Sci U S A 112, 13231-6
  13. Laget PP & Smith JB (1979) Inhibitory properties of endogenous subunit epsilon in the Escherichia coli F1 ATPase. Arch Biochem Biophys 197, 83-9
  14. Kato-Yamada Y, Bald D, Koike M, Motohashi K, Hisabori T & Yoshida M (1999) Epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, also inhibits F(0)F(1)-ATPase. J Biol Chem 274, 33991-4
  15. Yagi H, Konno H, Murakami-Fuse T, Isu A, Oroguchi T, Akutsu H, Ikeguchi M & Hisabori T (2009) Structural and functional analysis of the intrinsic inhibitor subunit epsilon of F1-ATPase from photosynthetic organisms. Biochem J 425, 85-94
  16. Ferguson SA, Cook GM, Montgomery MG, Leslie AGW & Walker JE (2016) Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum. Proc Natl Acad Sci U S A 113, 10860-5