Fo Domain Research Articles

SQ31f is a potent non-tuberculous mycobacteria antibiotic by specifically targeting the mycobacterial F-ATP synthase.

Non-tuberculous mycobacteria (NTM) infection presents a growing global health problem and requires new antibiotics targeting enzymes that are essential for the pathogens under various metabolic conditions, with high target specificity, good solubility and with attractive combinatory potency. SQ31f was synthesized by a simplified synthesis protocol, and its effect on growth inhibition of fast- and slow-growing NTM and clinical isolates, whole-cell ATP depletion, ex vivo macrophages and its potency in combination with other antibiotics were evaluated. Molecular docking studies were employed to assess SQ31f's binding mode. We present- squaramide SQ31f as a novel anti-NTM inhibitor targeting the NTM F1FO-ATP synthase, essential for ATP formation, regulation of ATP homeostasis and proton motive force under multiple growth conditions. The potency of SQ31f in growth inhibition of fast- and slow-growing NTM and clinical isolates correlates with whole-cell ATP depletion, which is not caused by altered oxygen consumption. SQ31f's high aqueous solubility enables binding to the waterfilled cytosolic proton half channel in the subunits a-c interface of the FO domain. As presented for the fast-growing Mycobacterium abscessus, the compound is active against intracellular-residing M. abscessus. Importantly, SQ31f shows an additive effect of the anti-M. abscessus drugs clofazimine, rifabutin or amikacin, and an attractive potentiation of linezolid, clarithromycin, or the oral pair tebipenem and avibactam. SQ31f represents an attractive inhibitor to tackle the issues associated with NTM drug tolerance and toxicity. Its combinatory potency with anti-M. abscessus drugs holds potential for overcoming resistance, while also reducing intensive compound synthesis and associated costs.

H+-slip correlated to rotor free-wheeling as cause of F1FO-ATPase dysfunction in primary mitochondrial disorders.

Inborn errors of metabolism are related to mitochondrial disorders caused by dysfunction of the oxidative phosphorylation (OXPHOS) system. Congenital hypermetabolism in the infant is a rare disease belonging to Luft syndrome, nonthyroidal hypermetabolism, arising from a singular example of a defect in OXPHOS. The mitochondria lose coupling of mitochondrial substrates oxidation from the ADP phosphorylation. Since Luft syndrome is due to uncoupled cell respiration responsible for deficient in ATP production that originates in the respiratory complexes, a de novo heterozygous variant in the catalytic subunit of mitochondrial F1FO-ATPase arises as the main cause of an autosomal dominant syndrome of hypermetabolism associated with dysfunction in ATP production, which does not involve the respiratory complexes. The F1FO-ATPase works as an embedded molecular machine with a rotary action using two different motor engines. The FO, which is an integral domain in the membrane, dissipates the chemical potential difference for H+, a proton motive force (Δp), across the inner membrane to generate a torsion. The F1 domain-the hydrophilic portion responsible for ATP turnover-is powered by the molecular rotary action to synthesize ATP. The structural and functional coupling of F1 and FO domains support the energy transduction for ATP synthesis. The dissipation of Δp by means of an H+ slip correlated to rotor free-wheeling of the F1FO-ATPase has been discovered to cause enzyme dysfunction in primary mitochondrial disorders. In this insight, we try to offer commentary and analysis of the molecular mechanism in these impaired mitochondria.

Activity modulation of the Escherichia coli F1FO ATP synthase by a designed antimicrobial peptide via cardiolipin sequestering

Most antimicrobial peptides (AMPs) exert their microbicidal activity through membrane permeabilization. The designed AMP EcDBS1R4 has a cryptic mechanism of action involving the membrane hyperpolarization of Escherichia coli, suggesting that EcDBS1R4 may hinder processes involved in membrane potential dissipation. We show that EcDBS1R4 can sequester cardiolipin, a phospholipid that interacts with several respiratory complexes of E.coli. Among these, F1FO ATP synthase uses membrane potential to fuel ATP synthesis. We found that EcDBS1R4 can modulate the activity of ATP synthase upon partition to membranes containing cardiolipin. Molecular dynamics simulations suggest that EcDBS1R4 alters the membrane environment of the transmembrane FO motor, impairing cardiolipin interactions with the cytoplasmic face of the peripheral stalk that binds the catalytic F1 domain to the FO domain. The proposed mechanism of action, targeting membrane protein function through lipid reorganization may open new venues of research on the mode of action and design of other AMPs.

PHB3 Is Required for the Assembly and Activity of Mitochondrial ATP Synthase in Arabidopsis.

Mitochondrial ATP synthase is a multiprotein complex, which consists of a matrix-localized F1 domain (F1-ATPase) and an inner membrane-embedded Fo domain (Fo-ATPase). The assembly process of mitochondrial ATP synthase is complex and requires the function of many assembly factors. Although extensive studies on mitochondrial ATP synthase assembly have been conducted on yeast, much less study has been performed on plants. Here, we revealed the function of Arabidopsis prohibitin 3 (PHB3) in mitochondrial ATP synthase assembly by characterizing the phb3 mutant. The blue native PAGE (BN-PAGE) and in-gel activity staining assays showed that the activities of ATP synthase and F1-ATPase were significantly decreased in the phb3 mutant. The absence of PHB3 resulted in the accumulation of the Fo-ATPase and F1-ATPase intermediates, whereas the abundance of the Fo-ATPase subunit a was decreased in the ATP synthase monomer. Furthermore, we showed that PHB3 could interact with the F1-ATPase subunits β and δ in the yeast two-hybrid system (Y2H) and luciferase complementation imaging (LCI) assay and with Fo-ATPase subunit c in the LCI assay. These results indicate that PHB3 acts as an assembly factor required for the assembly and activity of mitochondrial ATP synthase.

The mitochondrial Hsp70 controls the assembly of the F1FO-ATP synthase

The mitochondrial F1FO-ATP synthase produces the bulk of cellular ATP. The soluble F1 domain contains the catalytic head that is linked via the central stalk and the peripheral stalk to the membrane embedded rotor of the FO domain. The assembly of the F1 domain and its linkage to the peripheral stalk is poorly understood. Here we show a dual function of the mitochondrial Hsp70 (mtHsp70) in the formation of the ATP synthase. First, it cooperates with the assembly factors Atp11 and Atp12 to form the F1 domain of the ATP synthase. Second, the chaperone transfers Atp5 into the assembly line to link the catalytic head with the peripheral stalk. Inactivation of mtHsp70 leads to integration of assembly-defective Atp5 variants into the mature complex, reflecting a quality control function of the chaperone. Thus, mtHsp70 acts as an assembly and quality control factor in the biogenesis of the F1FO-ATP synthase.

Binding properties of the anti-TB drugs bedaquiline and TBAJ-876 to a mycobacterial F-ATP synthase.

Tuberculosis (TB), the deadly disease caused by Mycobacterium tuberculosis (Mtb), kills more people worldwide than any other bacterial infectious disease. There has been a recent resurgence of TB drug discovery activities, resulting in the identification of a number of novel enzyme inhibitors. Many of these inhibitors target the electron transport chain complexes and the F1FO-ATP synthase; these enzymes represent new target spaces for drug discovery, since the generation of ATP is essential for the bacterial pathogen's physiology, persistence, and pathogenicity. The anti-TB drug bedaquiline (BDQ) targets the Mtb F-ATP synthase and is used as salvage therapy against this disease. Medicinal chemistry efforts to improve the physio-chemical properties of BDQ resulted in the discovery of 3,5-dialkoxypyridine (DARQ) analogs to which TBAJ-876 belongs. TBAJ-876, a clinical development candidate, shows attractive in vitro and in vivo antitubercular activity. Both BDQ and TBAJ-876 inhibit the mycobacterial F1FO-ATP synthase by stopping rotation of the c-ring turbine within the FO domain, thereby preventing proton translocation and ATP synthesis to occur. While structural data for the BDQ bound state are available, no structural information about TBAJ-876 binding have been described. In this study, we show how TBAJ-876 binds to the FO domain of the M. smegmatis F1FO-ATP synthase. We further calculate the binding free energy of both drugs bound to their target and predict an increased affinity of TBAJ-876 for the FO domain. This approach will be useful in future efforts to design new and highly potent DARQ analogs targeting F-ATP synthases of Mtb, nontuberculosis mycobacteria (NTM) as well as the M. leprosis complex.

Dissociation Strategies to Maximize Coverage of α-Helical Domains in Top-Down Mass Spectrometry of Integral Membrane Proteins.

Transmembrane α-helical domains of membrane proteins tend to remain structured in the gas phase, presenting a challenge for efficient electron capture/transfer dissociation during top-down dissociation mass spectrometry (MS) experiments. In this study, we compare results from different dissociation modes on a modern Orbitrap platform applied to a model integral membrane protein containing two transmembrane helices, the c-subunit of the Fo domain of the chloroplast ATP synthase. Using commercially available options, we compare collisionally activated dissociation (CAD) with the related variant higher-energy collisional dissociation (HCD) and with electron transfer dissociation (ETD). HCD performed better than CAD and ETD. A combined method utilizing both ETD and HCD (EThcD) demonstrates significant synergy over HCD or ETD alone, representing a robust option analogous to activated ion electron capture dissociation, whereby an infrared laser was used to heat the protein ion alongside electron bombardment. Ultraviolet photodissociation at 213 nm displays at least three backbone dissociation mechanisms and covered nearly 100% of backbone bonds, suggesting significant potential for this technique.

Complexome profiling reveals novel insights into the composition and assembly of the mitochondrial ATP synthase of Arabidopsis thaliana

The mitochondrial ATP synthase is producing most of the energy required to support eucaryotic life. It is located in the mitochondrial inner-membrane and couples the dissipation of the proton gradient produced by the electron transfer chain with ATP production. It is composed of two domains, the F1 domain located in the matrix and the FO domain embedded in the inner membrane. The mitochondrial ATP synthase belongs to the F-type ATP synthase family together with bacterial and chloroplastic enzymes. The composition of the mitochondrial ATP synthase is well conserved across species, except in plants where several subunits found in opisthokonts were not identified and additional, plant-specific, subunits were found. The assembly of the F-type ATP synthase has been extensively studied in bacteria, yeast and mammals. The overall assembly pattern is conserved but species-specific steps have been identified. In plant, little is known about the assembly of the mitochondrial ATP synthase. We have mined our previously published complexome profiling dataset in order to identity assembly steps of the ATP synthase in the reference plant Arabidopsis thaliana. Several assembly intermediates were identified and we propose a model for the assembly pathway of the ATP synthase of plant mitochondria. In addition, combining complexome profiling with homology searches, we found that the previously described plant-specific subunits are actually present in other organisms. Overall, our work show that the subunit composition and the assembly pathway of the plant mitochondria ATP synthase are mostly conserved with other mitochondrial enzymes.

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.

Functional production of an archaeal ATP synthase with a V-type c subunit in Escherichia coli

ATP synthases are the key elements of cellular bioenergetics and present in any life form and the overall structure and function of this rotary energy converter is conserved in all domains of life. However, ancestral microbes, the archaea, have a unique and huge diversity in the size and number of ion-binding sites in their membrane-embedded rotor subunit c. Due to the harsh conditions for ATP synthesis in these life forms it has never been possible to address the consequences of these unusual c subunits for ATP synthesis. Recently, we have found a Na+-dependent archaeal ATP synthase with a V-type c subunit in a mesophilic bacterium and here, we have cloned and expressed the genes in the ATP synthase-negative strain Escherichia coli DK8. The enzyme was present in membranes of E. coli DK8 and catalyzed ATP hydrolysis with a rate of 35 nmol·min−1·mg protein−1. Inverted membrane vesicles of this strain were then checked for their ability to synthesize ATP. Indeed, ATP was synthesized driven by NADH oxidation despite the V-type c subunit. ATP synthesis was dependent on Na+ and inhibited by ionophores. Most importantly, ATPase activity was inhibited by DCCD and this inhibition was relieved by addition of Na+, indicating a functional coupling of the F1 and FO domains, a prerequisite for studies on structure-function relationship. A first step in this direction was the exchange of a conserved arginine (Arg530) in the FO motor subunit a which led to loss of ATP synthesis whereas ATP hydrolysis was retained.

The 3 × 120° rotary mechanism of Paracoccus denitrificans F1-ATPase is different from that of the bacterial and mitochondrial F1-ATPases

The rotation of Paracoccus denitrificans F1-ATPase (PdF1) was studied using single-molecule microscopy. At all concentrations of adenosine triphosphate (ATP) or a slowly hydrolyzable ATP analog (ATPγS), above or below Km, PdF1 showed three dwells per turn, each separated by 120°. Analysis of dwell time between steps showed that PdF1 executes binding, hydrolysis, and probably product release at the same dwell. The comparison of ATP binding and catalytic pauses in single PdF1 molecules suggested that PdF1 executes both elementary events at the same rotary position. This point was confirmed in an inhibition experiment with a nonhydrolyzable ATP analog (AMP-PNP). Rotation assays in the presence of adenosine diphosphate (ADP) or inorganic phosphate at physiological concentrations did not reveal any obvious substeps. Although the possibility of the existence of substeps remains, all of the datasets show that PdF1 is principally a three-stepping motor similar to bacterial vacuolar (V1)-ATPase from Thermus thermophilus This contrasts with all other known F1-ATPases that show six or nine dwells per turn, conducting ATP binding and hydrolysis at different dwells. Pauses by persistent Mg-ADP inhibition or the inhibitory ζ-subunit were also found at the same angular position of the rotation dwell, supporting the simplified chemomechanical scheme of PdF1 Comprehensive analysis of rotary catalysis of F1 from different species, including PdF1, suggests a clear trend in the correlation between the numbers of rotary steps of F1 and Fo domains of F-ATP synthase. F1 motors with more distinctive steps are coupled with proton-conducting Fo rings with fewer proteolipid subunits, giving insight into the design principle the F1Fo of ATP synthase.

A decidable and expressive fragment of Many-Sorted First-Order Linear Temporal Logic

First-Order Linear Temporal Logic (FOLTL) and its Many-Sorted variant (MSFOLTL) are well-suited to specify infinite-state systems. However, the satisfiability of (MS)FOLTL is not even semi-decidable, thus preventing automated verification. In this paper, we exhibit various fragments of increasing scope that provide a pertinent basis for the abstract specification of infinite-state systems. We show that these fragments enjoy the Bounded Domain Property (any satisfiable (MS)FOLTL formula has a model with a finite, bounded FO domains), which provides a basis for complete, automated verification by reduction to LTL satisfiability. Finally, we present a simple case study illustrating the applicability and limitations of our results.

Cryo-EM structure of the entire mammalian F-type ATP synthase.

The majority of adenosine triphosphate (ATP) powering cellular processes in eukaryotes is produced by the mitochondrial F1Fo ATP synthase. Here, we present the atomic models of the membrane Fo domain and the entire mammalian (ovine) F1Fo, determined by cryo-electron microscopy. Subunits in the membrane domain are arranged in the 'proton translocation cluster' attached to the c-ring and a more distant 'hook apparatus' holding subunit e. Unexpectedly, this subunit is anchored to a lipid 'plug' capping the c-ring. We present a detailed proton translocation pathway in mammalian Fo and key inter-monomer contacts in F1Fo multimers. Cryo-EM maps of F1Fo exposed to calcium reveal a retracted subunit e and a disassembled c-ring, suggesting permeability transition pore opening. We propose a model for the permeability transition pore opening, whereby subunit e pulls the lipid plug out of the c-ring. Our structure will allow the design of drugs for many emerging applications in medicine.

3D reconstruction and flexibility of the hybrid engine Acetobacterium woodii F-ATP synthase

The Na+-translocating F1FO ATP synthase from Acetobacterium woodii (AwF-ATP synthase) with a subunit stoichiometry of α3:β3:γ:δ:ε:a:b2:(c2/3)9:c1 represents an evolutionary path between ATP-synthases and vacuolar ATPases, by containing a heteromeric rotor c-ring, composed of subunits c1, c2 and c3, and an extra loop (γ195-211) within the rotary γ subunit. Here, the recombinant AwF-ATP synthase was subjected to negative stain electron microscopy and single particle analysis. The reference free 2D class averages revealed high flexibility of the enzyme, wherein the F1 and FO domains distinctively bended to adopt multiple conformations. Moreover, both the F1 and FO domains tilted relative to each other to a maximum extent of 28° and 30°, respectively. The first 3D reconstruction of the AwF-ATP synthase was determined which accommodates well the modelled structure of the AwF-ATP synthase as well as the γ195-211-loop. Molecular simulations of the enzyme underlined the bending features and flexibility observed in the electron micrographs, and enabled assessment of the dynamics of the extra γ195-211-loop.

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases.

F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Escherichia coli, the enzyme consists of rotor subunits γεc10 and stator subunits (αβ)3δab2. Subunits c10 or (αβ)3 alone are rotationally symmetric. However, symmetry is broken by the b2 homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded FO domain with the soluble F1 domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)3 catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b2δ in F1 and with b2a in FO. We monitored the enzyme’s rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling.

Molecular basis of diseases caused by the mtDNA mutation m.8969G>A in the subunit a of ATP synthase

The ATP synthase which provides aerobic eukaryotes with ATP, organizes into a membrane-extrinsic catalytic domain, where ATP is generated, and a membrane-embedded FO domain that shuttles protons across the membrane. We previously identified a mutation in the mitochondrial MT-ATP6 gene (m.8969G>A) in a 14-year-old Chinese female who developed an isolated nephropathy followed by brain and muscle problems. This mutation replaces a highly conserved serine residue into asparagine at amino acid position 148 of the membrane-embedded subunit a of ATP synthase. We showed that an equivalent of this mutation in yeast (aS175N) prevents FO-mediated proton translocation. Herein we identified four first-site intragenic suppressors (aN175D, aN175K, aN175I, and aN175T), which, in light of a recently published atomic structure of yeast FO indicates that the detrimental consequences of the original mutation result from the establishment of hydrogen bonds between aN175 and a nearby glutamate residue (aE172) that was proposed to be critical for the exit of protons from the ATP synthase towards the mitochondrial matrix. Interestingly also, we found that the aS175N mutation can be suppressed by second-site suppressors (aP12S, aI171F, aI171N, aI239F, and aI200M), of which some are very distantly located (by 20–30 Å) from the original mutation. The possibility to compensate through long-range effects the aS175N mutation is an interesting observation that holds promise for the development of therapeutic molecules.

Near-neighbor interactions of the membrane-embedded subunits of the mitochondrial ATP synthase of a chlorophycean alga

Mitochondrial F1FO-ATP synthase of the chlorophycean algae Polytomella sp. can be isolated as a highly stable dimeric complex of 1600kDa. It is composed of eight highly conserved orthodox subunits (α, β, γ, δ, ε, OSCP, a, and c) and nine subunits (Asa1-9) that are exclusive of chlorophycean algae. The Asa subunits replace those that build up the peripheral stalk and the dimerization domains of the ATP synthase in other organisms. Little is known about the disposition of subunits Asa6, Asa8 and Asa9, that are predicted to have transmembrane stretches and that along with subunit a and a ring of c-subunits, seem to constitute the membrane-embedded Fo domain of the algal ATP synthase. Here, we over-expressed and purified the three Asa hydrophobic subunits and explored their interactions in vitro using a combination of immunochemical techniques, affinity chromatography, and an in vivo yeast-two hybrid assays. The results obtained suggest the following interactions Asa6–Asa6, Asa6–Asa8, Asa6–Asa9, Asa8–Asa8 and Asa8–Asa9. Cross-linking experiments carried out with the intact enzyme corroborated some of these interactions. Based on these results, we propose a model of the disposition of these hydrophobic subunits in the membrane-embedded sector of the algal ATP synthase. We also propose based on sequence analysis and hydrophobicity plots, that the algal subunit a is atypical in as much it lacks the first transmembrane stretch, exhibiting only four hydrophobic, tilted alpha helices.

Perfect chemomechanical coupling of FoF1-ATP synthase

FoF1-ATP synthase (FoF1) couples H+ flow in Fo domain and ATP synthesis/hydrolysis in F1 domain through rotation of the central rotor shaft, and the H+/ATP ratio is crucial to understand the coupling mechanism and energy yield in cells. Although H+/ATP ratio of the perfectly coupling enzyme can be predicted from the copy number of catalytic β subunits and that of H+ binding c subunits as c/β, the actual H+/ATP ratio can vary depending on coupling efficiency. Here, we report actual H+/ATP ratio of thermophilic Bacillus FoF1, whose c/β is 10/3. Proteoliposomes reconstituted with the FoF1 were energized with ΔpH and Δψ by the acid-base transition and by valinomycin-mediated diffusion potential of K+ under various [ATP]/([ADP]⋅[Pi]) conditions, and the initial rate of ATP synthesis/hydrolysis was measured. Analyses of thermodynamically equilibrated states, where net ATP synthesis/hydrolysis is zero, show linear correlation between the chemical potential of ATP synthesis/hydrolysis and the proton motive force, giving the slope of the linear function, that is, H+/ATP ratio, 3.3 ± 0.1. This value agrees well with the c/β ratio. Thus, chemomechanical coupling between Fo and F1 is perfect.

Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues

F1Fo-ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H+ flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H+ within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F1 and FO domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo.

On the structural possibility of pore-forming mitochondrial FoF1 ATP synthase

The mitochondrial permeability transition is an inner mitochondrial membrane event involving the opening of the permeability transition pore concomitant with a sudden efflux of matrix solutes and breakdown of membrane potential. The mitochondrial FoF1 ATP synthase has been proposed as the molecular identity of the permeability transition pore. The likeliness of potential pore-forming sites in the mitochondrial FoF1 ATP synthase is discussed and a new model, the death finger model, is described. In this model, movement of a p-side density that connects the lipid-plug of the c-ring with the distal membrane bending Fo domain allows reversible opening of the c-ring and structural cross-talk with OSCP and the catalytic (αβ)3 hexamer. This article is part of a Special Issue entitled ‘EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2–6, 2016’, edited by Prof. Paolo Bernardi.