Martin G. Montgomery

The structure of the central stalk in bovine F 1 -ATPase at 2.4 Å resolution

The central stalk in ATP synthase, made of γ, δ and ɛ subunits in the mitochondrial enzyme, is the key rotary element in the enzymes catalytic mechanism. The γ subunit penetrates the catalytic (αβ) 3 domain and protrudes beneath it, interacting with a ring of c subunits in the membrane that drives rotation of the stalk during ATP synthesis. In other crystals of F1-ATPase, the protrusion was disordered, but with crystals of F1-ATPase inhibited with dicyclohexylcarbodiimide, the complete structure was revealed. The δ and ɛ subunits interact with a Rossmann fold in the γ subunit, forming a foot. In ATP synthase, this foot interacts with the c-ring and couples the transmembrane proton motive force to catalysis in the (αβ)3 domain.

Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria

The catalytic domain of the F-ATPase in mitochondria protrudes into the matrix of the organelle, and is attached to the membrane domain by central and peripheral stalks. Energy for the synthesis of ATP from ADP and phosphate is provided by the transmembrane proton-motive-force across the inner membrane, generated by respiration. The proton-motive force is coupled mechanically to ATP synthesis by the rotation at about 100 times per second of the central stalk and an attached ring of c-subunits in the membrane domain. Each c-subunit carries a glutamate exposed around the midpoint of the membrane on the external surface of the ring. The rotation is generated by protonation and deprotonation successively of each glutamate. Each 360° rotation produces three ATP molecules, and requires the translocation of one proton per glutamate by each c-subunit in the ring. In fungi, eubacteria, and plant chloroplasts, ring sizes of c10–c15 subunits have been observed, implying that these enzymes need 3.3–5 protons to make each ATP, but until now no higher eukaryote has been examined. As shown here in the structure of the bovine F1-c-ring complex, the c-ring has eight c-subunits. As the sequences of c-subunits are identical throughout almost all vertebrates and are highly conserved in invertebrates, their F-ATPases probably contain c8-rings also. Therefore, in about 50,000 vertebrate species, and probably in many or all of the two million invertebrate species, 2.7 protons are required by the F-ATPase to make each ATP molecule.

Mechanism of Inhibition of Bovine F1-ATPase by Resveratrol and Related Polyphenols.

The structures of F1-ATPase from bovine heart mitochondria inhibited with the dietary phytopolyphenol, resveratrol, and with the related polyphenols quercetin and piceatannol have been determined at 2.3-, 2.4- and 2.7-Å resolution, respectively. The inhibitors bind to a common site in the inside surface of an annulus made from loops in the three α- and three β-subunits beneath the “crown” of β-strands in their N-terminal domains. This region of F1-ATPase forms a bearing to allow the rotation of the tip of the γ-subunit inside the annulus during catalysis. The binding site is a hydrophobic pocket between the C-terminal tip of the γ-subunit and the βTP subunit, and the inhibitors are bound via H-bonds mostly to their hydroxyl moieties mediated by bound water molecules and by hydrophobic interactions. There are no equivalent sites between the γ-subunit and either the βDP or the βE subunit. The inhibitors probably prevent both the synthetic and hydrolytic activities of the enzyme by blocking both senses of rotation of the γ-subunit. The beneficial effects of dietary resveratrol may derive in part by preventing mitochondrial ATP synthesis in tumor cells, thereby inducing apoptosis.

Ground State Structure of F1-ATPase from Bovine Heart Mitochondria at 1.9 A Resolution

The structure of bovine F1-ATPase, crystallized in the presence of AMP-PNP and ADP, but in the absence of azide, has been determined at 1.9Å resolution. This structure has been compared with the previously described structure of bovine F1-ATPase determined at 1.95Å resolution with crystals grown under the same conditions but in the presence of azide. The two structures are extremely similar, but they differ in the nucleotides that are bound to the catalytic site in the βDP-subunit. In the present structure, the nucleotide binding sites in the βDP- and βTP-subunits are both occupied by AMP-PNP, whereas in the earlier structure, the βTP site was occupied by AMP-PNP and the βDP site by ADP, where its binding is enhanced by a bound azide ion. Also, the conformation of the side chain of the catalytically important residue, αArg-373 differs in the βDP- and βTP-subunits. Thus, the structure with bound azide represents the ADP inhibited state of the enzyme, and the new structure represents a ground state intermediate in the active catalytic cycle of ATP hydrolysis.

The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride

The structure of bovine F1‐ATPase inhibited with ADP and beryllium fluoride at 2.0 Å resolution contains two ADP.BeF3− complexes mimicking ATP, bound in the catalytic sites of the βTP and βDP subunits. Except for a 1 Å shift in the guanidinium of αArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the γ‐phosphate of ATP during hydrolysis is 2.6 Å from the beryllium in the βDP subunit and 3.8 Å away in the βTP subunit, strongly indicating that the βDP subunit is the catalytically active conformation. In the structure of F1‐ATPase with five bound ADP molecules (three in α‐subunits, one each in the βTP and βDP subunits), which has also been determined, the conformation of αArg373 suggests that it senses the presence (or absence) of the γ‐phosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F1‐ATPase.

The structure of bovine F1-ATPase in complex with its regulatory protein IF1

In mitochondria, the hydrolytic activity of ATP synthase is prevented by an inhibitor protein, IF1. The active bovine protein (84 amino acids) is an α-helical dimer with monomers associated via an antiparallel α-helical coiled coil composed of residues 49–81. The N-terminal inhibitory sequences in the active dimer bind to two F1-ATPases in the presence of ATP. In the crystal structure of the F1−IF1 complex at 2.8 Å resolution, residues 1–37 of IF1 bind in the αDP-βDP interface of F1-ATPase, and also contact the central γ subunit. The inhibitor opens the catalytic interface between the αDP and βDP subunits relative to previous structures. The presence of ATP in the catalytic site of the βDP subunit implies that the inhibited state represents a pre-hydrolysis step on the catalytic pathway of the enzyme.

Structure of Bovine Mitochondrial F1-ATPase Inhibited by Mg2+Adp and Aluminium Fluoride

BACKGROUND The globular domain of the membrane-associated F(1)F(o)-ATP synthase complex can be detached intact as a water-soluble fragment known as F(1)-ATPase. It consists of five different subunits, alpha, beta, gamma, delta and epsilon, assembled with the stoichiometry 3:3:1:1:1. In the crystal structure of bovine F(1)-ATPase determined previously at 2.8 A resolution, the three catalytic beta subunits and the three noncatalytic alpha subunits are arranged alternately around a central alpha-helical coiled coil in the gamma subunit. In the crystals, the catalytic sites have different nucleotide occupancies. One contains the triphosphate form of the nucleotide, the second contains the diphosphate, and the third is unoccupied. Fluoroaluminate complexes have been shown to mimic the transition state in several ATP and GTP hydrolases. In order to understand more about its catalytic mechanism, F(1)-ATPase was inhibited with Mg(2+)ADP and aluminium fluoride and the structure of the inhibited complex was determined by X-ray crystallography. RESULTS The structure of bovine F(1)-ATPase inhibited with Mg(2+)ADP and aluminium fluoride determined at 2.5 A resolution differs little from the original structure with bound AMP-PNP and ADP. The nucleotide occupancies of the alpha and beta subunits are unchanged except that both aluminium trifluoride and Mg(2+)ADP are bound in the nucleotide-binding site of the beta(DP) subunit. The presence of aluminium fluoride is accompanied by only minor adjustments in the surrounding protein. CONCLUSIONS The structure appears to mimic a possible transition state. The coordination of the aluminofluoride group has many features in common with other aluminofluoride-NTP hydrolase complexes. Apparently, once nucleotide is bound to the catalytic beta subunit, no additional major structural changes are required for catalysis to occur.

How the regulatory protein, IF1, inhibits F1-ATPase from bovine mitochondria

The structure of bovine F1-ATPase inhibited by a monomeric form of the inhibitor protein, IF1, known as I1–60His, lacking most of the dimerization region, has been determined at 2.1-Å resolution. The resolved region of the inhibitor from residues 8–50 consists of an extended structure from residues 8–13, followed by two α-helices from residues 14–18 and residues 21–50 linked by a turn. The binding site in the βDP-αDP catalytic interface is complex with contributions from five different subunits of F1-ATPase. The longer helix extends from the external surface of F1 via a deep groove made from helices and loops in the C-terminal domains of subunits βDP, αDP, βTP, and αTP to the internal cavity surrounding the central stalk. The linker and shorter helix interact with the γ-subunit in the central stalk, and the N-terminal region extends across the central cavity to interact with the nucleotide binding domain of the αE subunit. To form these complex interactions and penetrate into the core of the enzyme, it is likely that the initial interaction of the inhibitor with F1 forms via the open conformation of the βE subunit. Then, as two ATP molecules are hydrolyzed, the βE-αE interface converts to the βDP-αDP interface via the βTP-αTP interface, trapping the inhibitor progressively in its binding site and a nucleotide in the catalytic site of subunit βDP. The inhibition probably arises by IF1 imposing the structure and properties of the βTP-αTP interface on the βDP-αDP interface, thereby preventing it from hydrolyzing the bound ATP.

Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM.

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases. DOI: http://dx.doi.org/10.7554/eLife.10180.001

Structure of ATP synthase from Paracoccus denitrificans determined by X-ray crystallography at 4.0 Å resolution.

Significance ATP, the fuel of life, is produced in living cells by a complex molecular machine consisting of two motors linked by a rotor. One motor generates rotation by consuming energy derived from oxidative metabolism or photosynthesis; the other uses energy transmitted by the rotor to put ATP molecules together from their building blocks, ADP and phosphate. One such intact machine from the α-proteobacterium Paracoccus denitrificans has been induced to form crystals, providing the means of deducing a blueprint of the machine, giving details of how its components are organized, and providing insights into how it works. The mechanistic principles deduced from the bacterial machine apply to similar molecular machines found in all living organisms. The structure of the intact ATP synthase from the α-proteobacterium Paracoccus denitrificans, inhibited by its natural regulatory ζ-protein, has been solved by X-ray crystallography at 4.0 Å resolution. The ζ-protein is bound via its N-terminal α-helix in a catalytic interface in the F1 domain. The bacterial F1 domain is attached to the membrane domain by peripheral and central stalks. The δ-subunit component of the peripheral stalk binds to the N-terminal regions of two α-subunits. The stalk extends via two parallel long α-helices, one in each of the related b and b′ subunits, down a noncatalytic interface of the F1 domain and interacts in an unspecified way with the a-subunit in the membrane domain. The a-subunit lies close to a ring of 12 c-subunits attached to the central stalk in the F1 domain, and, together, the central stalk and c-ring form the enzyme’s rotor. Rotation is driven by the transmembrane proton-motive force, by a mechanism where protons pass through the interface between the a-subunit and c-ring via two half-channels in the a-subunit. These half-channels are probably located in a bundle of four α-helices in the a-subunit that are tilted at ∼30° to the plane of the membrane. Conserved polar residues in the two α-helices closest to the c-ring probably line the proton inlet path to an essential carboxyl group in the c-subunit in the proton uptake site and a proton exit path from the proton release site. The structure has provided deep insights into the workings of this extraordinary molecular machine.