ATP synthase: from single molecule to human bioenergetics

Abstract

ATP synthase (F(o)F(1)) consists of an ATP-driven motor (F(1)) and a H(+)-driven motor (F(o)), which rotate in opposite directions. F(o)F(1) reconstituted into a lipid membrane is capable of ATP synthesis driven by H(+) flux. As the basic structures of F(1) (alpha(3)beta(3)gammadeltaepsilon) and F(o) (ab(2)c(10)) are ubiquitous, stable thermophilic F(o)F(1) (TF(o)F(1)) has been used to elucidate molecular mechanisms, while human F(1)F(o) (HF(1)F(o)) has been used to study biomedical significance. Among F(1)s, only thermophilic F(1) (TF(1)) can be analyzed simultaneously by reconstitution, crystallography, mutagenesis and nanotechnology for torque-driven ATP synthesis using elastic coupling mechanisms. In contrast to the single operon of TF(o)F(1), HF(o)F(1) is encoded by both nuclear DNA with introns and mitochondrial DNA. The regulatory mechanism, tissue specificity and physiopathology of HF(o)F(1) were elucidated by proteomics, RNA interference, cytoplasts and transgenic mice. The ATP synthesized daily by HF(o)F(1) is in the order of tens of kilograms, and is primarily controlled by the brain in response to fluctuations in activity.

Figure 1.

Basic structure of TF₁ (ATP-driven motor), TF_o (proton-driven motor) and TF_oF₁ (proton-driven ATP synthase). Upper left: Side view of TF₁ composed of α, β, γ, δ, and ε subunits. The γε turns counterclockwise against the α₃β₃ hexamer in the ATP hydrolysis direction when viewed from the F_o side. Middle: Top view of TF₁ with three different conformations of β subunits. Lower left: Side view of TF_o composed of a, b and c subunits. The c₁₀ ring turns clockwise against ab₂ in the proton-driven direction. Right: Side view of TF_oF₁. The central stalk (γε) turns with the c₁₀ rotor clockwise in the ATP synthesis direction when viewed from TF_o side.

Figure 2.

Side view of space filling model of HF_oF₁. As there is high homology among F_oF₁s from different species,^,–21) and quintuple yeast deletion YF₁ mutant (ΔαΔβΔγΔδΔε) is complemented by genes encoding BF₁,⁸⁾ the major structure of eukaryotic F_oF₁ is apparently universal.^,,,,–76) Thus, X-ray crystallography data for F_oF₁ subunits from different species were taken from RCSB Protein Data Bank (http://www.rcsb.org/pdb/results/results.do?outformat) and assembled according to sequence data for HF_oF₁.²¹⁾ No high-resolution structural data are available for subunit a and the hinge region of subunit b. ATP synthasome contains both PIC and ANC.³¹⁾ PIC: Phosphate carrier; ANC: Adenine nucleotide carrier.

Figure 3.

Reconstitution of F_oF₁ from subunits and F_oF₁ liposomes from phospholipids.^3,39) Isolated nucleotide-binding α and β subunits^6,56) and α₃β₃ hexamer were obtained by dissociation of TF₁ (upper left). Solubilized F_o or F_oF₁ was mixed with phospholipids and cholate, and dialyzed to reconstitute F_oF₁ liposomes (bottom right, electron micrograph) capable of proton-driven ATP synthesis.

Figure 4.

Aligned amino acid sequences^12,19) and secondary structure elements⁷¹⁾ of α and β subunits in TF₁. Solid black lines indicate folds, and these were classified into α-helices (A–H, 1–8) and β-sheets (a–f, 0–8). The labels for folds are provided only for the β subunit, except for the three C-terminal α-helices in the α subunit. Dots indicate every tenth residue. I–XI: areas of αβ contact. Red: catalytic contact areas of β. Pink: catalytic contact areas of α. Blue: non-catalytic contact areas of β. Green: non-catalytic contact areas of α. Colored bars indicate contact residues in Tβ_E. Sequences are divided by red asterisks (*) to indicate the three domains.

Figure 5.

Crystals of TF₁ and α₃β₃, and X-ray crystallography data for the catalytic center of F₁. A. Catalytic center of the β subunit of BF₁ (black text indicates residue number)⁹⁾ and TF₁ (red text indicates residue numbers).¹²⁾ Except for αR373 and αS344, all of the amino acid residues are present in β. Residues 159–164 are part of the P-loop surrounding the triphosphate residue of ATP. B. Crystals of TF₁.⁷⁰⁾ C. Crystals of α₃β₃.⁷¹⁾ D. Top view of the crystallographic structure of α₃β₃.⁷¹⁾

Figure 6.

Three-dimensional structures of Tβ_E. Three-fold axis is vertical, so that views are towards the αβ subunit interfaces. Left: non-catalytic interface (blue I–IX indicates contact areas). Right: catalytic interface (red I–XI indicates contact areas). Green letters: domain names, with domain borders being marked by red asterisks. D and R in green circles are TβD331 and TβR333, respectively, at the entrance to the crevice of the P-loop. I in red circle is TβI386 of ββ contact. H and G in yellow circles indicate hinge residues in Tβ (179–181).⁷⁴⁾

Figure 7.

Alternate splicing of human F₁γ pre-mRNA. A. Exons in the F₁ γ pre-mRNA are expressed in boxes. Exon number 8 (hatched box) is included in liver and excluded in the muscle. B. Schematic representation of wild-type and mutant F₁γ exons 8–9 (Ex8–9).²⁷⁾ Heavy underline in Ex9-wild-type indicates ESE element. Mutated nucleotides are indicated by outlined letters. In Ex9-MU2 and Ex9-MU, boxed letters indicate that these sequences are predicted to act as ESE elements. C. Selection of F₁γ exon 9 is regulated by two cis-acting regulatory elements in the same exon.⁹⁰⁾ Purine-rich ESE promotes exon 9 inclusion, which is repressed by MS-ESS under muscle-specific conditions; PPT: Polypyrimidine tract.

Figure 8.

Homoplasmy, heteroplasmy, cytoplasts and ρ^o cells.⁸⁷⁾ Small red circles indicate wild-type mtDNA and small blue circles indicate mutant mtDNA; large red circles indicate nDNA, and green ovals indicate cells. EB: ethidium bromide used to remove mtDNA; PEG: polyethylene glycol used to fuse cells or cytoplasts.

Figure 9.

Regulation of HF₁F_o biosynthesis^21,86): Transcription, splicing, translation, targeting, import, processing and assembly. PGC-1α: Peroxisome proliferator-activated receptor-γ coactivator-1α; Tfam: mitochondrial transcription factor A; NRF: nuclear respiratory factor; Circle: mitochondrial DNA.

Figure 10.

Regulation of HF₁F_o activity: Neuronal impulse from brain to human activity driven by ATP by an intricate regulation system. AMPK, AMP kinase; ecto-F₁F_o, ectopic or cell surface F₁F_o; IF, ATPase inhibitor; METs, metabolic equivalents of exercise intensity; Rs, receptors for neuro-endocrine transmitters; VDAC, voltage-dependent anion channel.