Intact ATP Synthase Research Articles

An ancestral interaction module promotes oligomerization in divergent mitochondrial ATP synthases

Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo-EM structures of the intact ATP synthase dimer from Trypanosoma brucei in ten different rotational states. The model consists of 25 subunits, including nine lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit-g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit-g together with subunit-e form an ancestral oligomerization motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit-g/e module as a structural component determining ATP synthase oligomeric assemblies.

Wetting Transitions in the ATP Synthase C-Subunit Ring, a Large-Conductance Ion Channel

Apart from its known role in proton transport, the mammalian ATP synthase c-subunit ring (Fo domain) has been observed in recent electrophysiology experiments to form a high-conductance voltage-gated ion channel (∼1.5 nS). Yet, the ring's hydrophobic central lumen has been assumed to be dry, precluding a physical explanation for the observed conductance. We present an atomistic molecular dynamics simulation study of lumen wetting for mammalian and yeast c-subunit rings. Consistent with prior work, we find that the ∼12 Å diameter yeast ring remains dry on the several-hundred-nanosecond timescale of the simulations. In contrast, the narrower, ∼8 Å diameter, mammalian ring undergoes rapid (∼50 ns) liquid-vapor transitions, forming a transient but continuous water column across the lumen. Modifying the charge on the glutamate residues of the proton-transport pathway at the membrane-protein interface affects the duration and extent of lumen wetting, as do in silico mutations at various sites. The wetting variations are qualitatively consistent with electrophysiology experimental findings for wild-type and mutant forms of the mammalian c-subunit ring. We are encouraged to think that the simulations will serve as a valuable “computational microscope” for understanding how charge conductance can occur in the ATP synthase c-subunit ring. To determine the mechanism of wetting and its modulation, we are pursuing analysis of the surface hydrophobicity of the lumen lining and its response to the mutations. The simulation finding of reversible lumen wetting is supported by published work indicating c-subunit ring wetting in the intact ATP synthase. New experiments and published data additionally indicate that the Fo domain may exist at least partly dissociated from its F1 counterpart. ATP synthase c-subunit ring wetting, therefore, has implications with respect to osmotic swelling and the enigmatic mitochondrial permeability transition.

Mitochondrial (ATP Synthase) Permeability Transition Pore

ATP synthase has been suggested to contain a mitochondrial megachannel (MCC) or permeability transition pore (mPTP), which contributes the majority of the current associated with the calcium-induced mitochondrial PT. We reconstituted purified monomeric ATP synthase from porcine heart mitochondria into liposomes for patch clamp recordings. These recordings demonstrated multi-conductance, voltage and Ca2+-induced channels with sensitivity to endogenous and pharmacological mPTP and ATP synthase regulators. Pharmacological inhibitors that act by binding to the ATP synthase F1 lost their activity upon F1 removal, suggesting that regulation of channel activity occurs in response to conformational changes within ATP synthase F1. To determine the source of the channel activity, we purified and reconstituted c-subunit rings for patch recordings. These recordings demonstrated channel activity similar to that of monomeric F1FO ATP synthase except that reagents that normally bind within F1 were ineffective, presumably because of the absence of F1. In contrast, addition of purified F1 to the bath during the recordings inactivated the c-subunit channel. Based on our findings we conclude that the c-subunit forms a pore of the mPTP, the intact ATP synthase F1 binds to the c-subunit to inactivate the channel and finally, the F1 contains regulatory subunits that interact with previously known mPTP pharmacological inhibitors and novel endogenous regulators.

Isolation and Characterization of a Novel ATPase-Photosystem I Reaction Center Complex in the Chloroplast Thylakoid Membrane

A novel 1 MDa intra-membrane ‘super-complex’ dominated by the chloroplast ATP synthase and the photosystem I (PSI) reaction-center complex has been isolated from spinach thylakoid membranes. This super-complex has been characterized by assay of ATPase activity, negative stain electron microscopy, tandem mass spectrometry, and Western assay of subunits. High-resolution Orbitrap mass spectrometry was used to quantitatively analyze the presence and relative amount of the multiple major and minor polypeptide components in the super-complex. This is the first isolation of a higher order complex involving intact ATP synthase and Photosystem I to be identified and documented by negative stain electron microscopy. The importance of the ATPase-PSI super-complex in the thylakoid domain may imply efficient channeling of substrates and cofactors utilized for localized CO2 fixation. Studies supported by NIHGMS-038323 and the Henry Koffler Distinguished Professorship (WAC), and NIHDK-063491 (JPW).

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

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.

A unique conformation of the inhibitory ε subunit in a crystal structure of the Escherichia coli F1‐ATPase

We report a structure at 3.3 Åresolution for E. coli F1‐ATPase (EcF1, depleted ofδ). It was solved by combining molecular replacement and 4‐fold non‐crystallographic symmetry averaging. The sequences of α3β3γε subunits were unambiguously interpreted using selenomethionine‐labeled protein. The general architecture of the α3β3γ 'core' is like that of bovine mitochondrial F1 (MF1), but with greater asymmetry between pairs of αβ subunits. The conformation of ε (δin MF1) is the most striking feature. Unlike δ in MF1,ε's C‐terminal domain (CTD) adopts a highly extended state: helix‐1 extends up to pack on one side ofγ's 'foot' domain, the following loop packs below the 'DELSEED' motif of subunit βDP, and helix‐2 inserts into the central cavity of EcF1 to form an antiparallel coiled‐coil with part ofγ's N‐terminal helix. This interaction blocks specific contacts between γ and the CTDs of αDP/βDP subunits, which are shifted away fromγ's central rotary shaft. Remaining C‐terminal residues of ε are non‐helical and extend across the central cavity to contact βTP near the inner surface of its catalytic nucleotide‐binding site. Intrusion ofε's CTD into the central cavity is likely to block subunit motions involved in rotary catalytic coupling in F1 and in the intact ATP synthase. This structure sheds light on an inhibitory mechanism that is unique to bacterial and chloroplast ATP synthases. Support: NIH, 1R01GM083088‐01A2.

Analysis of Sequence Determinants of F1Fo-ATP Synthase in the N-terminal Region of α Subunit for Binding of δ Subunit

The stator in F(1)F(o)-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(o) and to the alpha(3)beta(3) hexagon of F(1). Previous work has shown that N-terminal residues of alpha subunit are involved in binding delta. A synthetic peptide consisting of the first 22 residues of alpha (alphaN1-22) binds specifically to isolated wild-type delta subunit with 1:1 stoichiometry and high affinity, accounting for a major portion of the binding energy between delta and F(1). Residues alpha6-18 are predicted by secondary structure algorithms and helical wheels to be alpha-helical and amphipathic, and a potential helix capping box occurs at residues alpha3-8. We introduced truncations, deletions, and mutations into alphaN1-22 peptide and examined their effects on binding to the delta subunit. The deletions and mutations were introduced also into the N-terminal region of the uncA (alpha subunit) gene to determine effects on cell growth in vivo and membrane ATP synthase activity in vitro. Effects seen in the peptides were well correlated with those seen in the uncA gene. The results show that, with the possible exception of residues close to the initial Met, all of the alphaN1-22 sequence is required for binding of delta to alpha. Within this sequence, an amphipathic helix seems important. Hydrophobic residues on the predicted nonpolar surface are important for delta binding, namely alphaIle-8, alphaLeu-11, alphaIle-12, alphaIle-16, and alphaPhe-19. Several or all of these residues probably make direct interaction with helices 1 and 5 of delta. The potential capping box sequence per se appeared less important. Impairment of alpha/delta binding brings about functional impairment due to reduced level of assembly of ATP synthase in cells.

Structure of the mitochondrial ATP synthase by electron cryomicroscopy.

We have determined the structure of intact ATP synthase from bovine heart mitochondria by electron cryomicroscopy of single particles. Docking of an atomic model of the F1-c10 subcomplex into a major segment of the map has allowed the 32 A resolution density to be interpreted as the F1-ATPase, a central and a peripheral stalk and an FO membrane region that is composed of two domains. One domain of FO corresponds to the ring of c-subunits, and the other probably contains the a-subunit, the transmembrane portion of the b-subunit and the remaining integral membrane proteins of FO. The peripheral stalk wraps around the molecule and connects the apex of F1 to the second domain of FO. The interaction of the peripheral stalk with F1-c10 implies that it binds to a non-catalytic alpha-beta interface in F1 and its inclination where it is not attached to F1 suggests that it has a flexible region that can serve as a stator during both ATP synthesis and ATP hydrolysis.

Each yeast mitochondrial F 1F 0-ATP synthase complex contains a single copy of subunit 8

The stoichiometry of subunit 8 in yeast mitochondrial F 1F 0-ATP synthase (mtATPase) has been evaluated using an immunoprecipitation approach. Single HA or FLAG epitopes were introduced at the N-terminus of subunit 8. Expression of each tagged subunit 8 variant in yeast cells lacking endogenous subunit 8 restored a respiratory phenotype and had little measurable effect on ATP hydrolase activity of the isolated enzyme. Moreover, the two epitope-tagged subunit 8 variants could be stably co-expressed in the same host cells and both of HA-Y8 and FLAG-Y8 could be detected in ATP synthase complexes isolated by native gel electrophoresis. Mitochondria isolated from each yeast strain were solubilized to release ATP synthase complexes in either the monomeric or dimeric forms. In each case, monoclonal antibodies directed against either the FLAG or HA epitope could immunoprecipitate intact ATP synthase complexes. When both HA-Y8 and FLAG-Y8 were co-expressed in cells, monomeric ATP synthases contained only a single subunit 8 variant after immunoprecipitation, corresponding to the particular antibody used (HA or FLAG). By contrast, both subunit 8 variants were recovered in samples of immunoprecipitated dimeric ATP synthase complexes, irrespective of the antibody used. We conclude that each monomeric yeast mitochondrial ATP synthase complex contains a single copy of subunit 8.

ATP Synthase from Saccharomyces cerevisiae: Location of the OSCP Subunit in the Peripheral Stalk Region

A biotinylation signal has been fused to the C terminus of the oligomycin sensitivity conferral protein (OSCP) of the ATP synthase complex from Saccharomyces cerevisiae. The signal is biotinylated in vivo and the biotinylated complex binds avidin in vitro. By electron microscopy of negatively stained particles of the ATP synthase–avidin complex, the bound avidin has been localised close to the F 1 domain. The images were subjected to multi-reference alignment and classification. Because of the presence of a flexible linker between the OSCP and the biotinylation signal, the class-averages differ in the position of the avidin relative to the F 1 domain. These positions lie on an arc, and its centre indicates the position of the C terminus of the OSCP on the surface of the F 1 domain. Since the N-terminal region of the OSCP is known to interact with the N-terminal regions of α-subunits, which are on top of the F 1 domain distal from the F o membrane domain, the OSCP extends almost 10 nm along the surface of F 1 down towards F o where it interacts with the C terminus of the b subunit, which extends up from F o. The labelling technique has also allowed a reliable 2D projection map to be developed for the intact ATP synthase from S. cerevisiae . The map reveals a marked asymmetry in the F o part of the complex that can be attributed to subunits in the F o domain.

The structure of bovine mitochondrial F1-ATPase: an example of rotary catalysis

There is now compelling evidence in support of a rotary catalytic mechanism in F1-ATPase, and, by extension, in the intact ATP synthase. Although models have been proposed to explain how protein translocation in F0 results in rotation of the gamma-subunit relative to the alpha 3/beta 3 assembly in F1 [22], these are still speculative. It seems likely that a satisfactory explanation of this mechanism will ultimately depend on structural information on the intact ATP synthase.

Determination of the structures of respiratory enzyme complexes from mammalian mitochondria

Knowledge of the atomic resolution structures of the respiratory enzymes from mammalian mitochondria is likely to be essential for understanding the molecular basis of human diseases involving their dysfunction. Because they are membrane-bound multisubunit assemblies, the determination of their high resolution structures is a rather challenging undertaking. The greatest progress has been made with the ATP synthase. The structure of its catalytic domain, F 1-ATPase, has been solved by X-ray crystallography at 2.8 Å resolution. It supports a binding change mechanism of catalysis in intact ATP synthase in which the catalytic subunits are in different states of the catalytic cycle at any instant. Interconversion of the states may be achieved by rotation of an α-helical domain of the γ-subunit relative to the α 3 β 3 sub-assembly. The membrane domain of ATP synthase has been purified, and the stalk linking the catalytic and membrane domains has been reassembled in vitro from its constituent subunits. Considerable progress has also been made in analyzing the structure of bovine complex I. It has about 43 different subunits and 42 of them have been sequenced. All of the known prosthetic groups have been localized in its extrinsic membrane arm, which has been split away from the membrane subunits and purified. The next stage is to crystallize the domain and to solve its structure.

Subunit structure of ATP synthase from chloroflexus aurantiacus

An ATP synthase has been isolated from green nonsulfur photosynthetic bacterium Chloroflexus aurantiacus, a representative of a lower branch of eubacteria. The enzyme, reconstituted with the bacterial lipids into proteoliposomes, is shown to catalyze [ 32P]P i-ATP exchange (at a rate of 180 nmol [ 32P]ATP/ min mg ). The ATP synthase is composed of nine polypeptide species (60, 50, 33, 19, 16.5, 15.5, 14.5, 13, and 8 kDa as determined by urea-SDS-PAGE). The catalytic part of the ATP synthase (which is detached by chloroform treatment) contains the first four polypeptides. In the intact ATP synthase the 14.5 and 13 kDa polypeptides are connected by disulfide bonds to form a heterodimer of 25 kDa.

Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 6.5 A resolution.

ATP synthase, the assembly which makes ATP in mitochondria, chloroplasts and bacteria, uses transmembrane proton gradients generated by respiration or photosynthesis to drive the phosphorylation of ADP. Its membrane domain is joined by a slender stalk to a peripheral catalytic domain, F1-ATPase. This domain is made of five subunits with stoichiometries of 3 alpha: 3 beta: 1 gamma: 1 delta: 1 epsilon, and in bovine mitochondria has a molecular mass of 371,000. We have determined the 3-dimensional structure of bovine mitochondrial F1-ATPase to 6.5 A resolution by X-ray crystallography. It is an approximately spherical globule 110 A in diameter, on a 40 A stem which contains two alpha-helices in a coiled-coil. This stem is presumed to be part of the stalk that connects F1 with the membrane domain in the intact ATP synthase. A pit next to the stem penetrates approximately 35 A into the F1 particle. The stem and the pit are two examples of the many asymmetric features of the structure. The central element in the asymmetry is the longer of the two alpha-helices in the stem, which extends for 90 A through the centre of the assembly and emerges on top into a dimple 15 A deep. Features with threefold and sixfold symmetry, presumed to be parts of homologous alpha and beta subunits, are arranged around the central rod and pit, but the overall structure is asymmetric. The central helix provides a possible mechanism for transmission of conformational changes induced by the proton gradient from the stalk to the catalytic sites of the enzyme.

72] Identification of essential residues in the F1-ATPases by chemical modification

Publisher Summary This chapter discusses the identification of essential residues in the F 1 -ATPases by chemical modification. The isolated F 1 -ATPases are subjected to a variety of chemical modifications that describe the molecular mechanism of ATP synthesis catalyzed by the intact ATP synthase complexes. This approach assumes that the same functional groups participate in ATP synthesis catalyzed by the intact, membrane-bound ATP synthase complex as participate in the hydrolytic reaction catalyzed by the isolated F 1 -ATPase. All the essential residues identified in the primary sequences of various F 1 -ATPases, reside in the β subunit. The reagents used to modify the F 1 -ATPases in the studies are known to inactivate a variety of enzymes; among them are other membrane-bound ATPases. Therefore, the experimental approaches and some specific procedures that were used to identify essential residues in the F 1 -ATPases are outlined in the chapter.