Structure and Mechanisms of F-Type ATP Synthases.

F1Fo ATP synthases produce most of the ATP in the cell. F-type ATP synthases have been investigated for more than 50 years, but a full understanding of their molecular mechanisms has become possible only with the recent structures of complete, functionally competent complexes determined by electron cryo-microscopy (cryo-EM). High-resolution cryo-EM structures offer a wealth of unexpected new insights. The catalytic F1 head rotates with the central γ-subunit for the first part of each ATP-generating power stroke. Joint rotation is enabled by subunit δ/OSCP acting as a flexible hinge between F1 and the peripheral stalk. Subunit a conducts protons to and from the c-ring rotor through two conserved aqueous channels. The channels are separated by ∼6 Šin the hydrophobic core of Fo, resulting in a strong local field that generates torque to drive rotary catalysis in F1. The structure of the chloroplast F1Fo complex explains how ATPase activity is turned off at night by a redox switch. Structures of mitochondrial ATP synthase dimers indicate how they shape the inner membrane cristae. The new cryo-EM structures complete our picture of the ATP synthases and reveal the unique mechanism by which they transform an electrochemical membrane potential into biologically useful chemical energy.

An ATP13A1-assisted topogenesis pathway for folding multi-spanning membrane proteins.

Many multi-spanning membrane proteins contain poorly hydrophobic transmembrane domains (pTMDs) protected from phospholipid in mature structure. Nascent pTMDs are difficult for translocon to recognize and insert. How pTMDs are discerned and packed into mature, muti-spanning configuration remains unclear. Here, we report that pTMD elicits a post-translational topogenesis pathway for its recognition and integration. Using six-spanning protein adenosine triphosphate-binding cassette transporter G2 (ABCG2) and cultured human cells as models, we show that ABCG2's pTMD2 can pass through translocon into the endoplasmic reticulum (ER) lumen, yielding an intermediate with inserted yet mis-oriented downstream TMDs. After translation, the intermediate recruits P5A-ATPase ATP13A1, which facilitates TMD re-orientation, allowing further folding and the integration of the remaining lumen-exposed pTMD2. Depleting ATP13A1 or disrupting pTMD-characteristic residues arrests intermediates with mis-oriented and exposed TMDs. Our results explain how a "difficult" pTMD is co-translationally skipped for insertion and post-translationally buried into the final correct structure at the late folding stage to avoid excessive lipid exposure.

Structure of the human ATP synthase.

Biological energy currency ATP is produced by F1Fo-ATP synthase. However, the molecular mechanism for human ATP synthase action remains unknown. Here, we present snapshot images for three main rotational states and one substate of human ATP synthase using cryoelectron microscopy. These structures reveal that the release of ADP occurs when the ß subunit of F1Fo-ATP synthase is in the open conformation, showing how ADP binding is coordinated during synthesis. The accommodation of the symmetry mismatch between F1 and Fo motors is resolved by the torsional flexing of the entire complex, especially the γ subunit, and the rotational substep of the c subunit. Water molecules are identified in the inlet and outlet half-channels, suggesting that the proton transfer in these two half-channels proceed via a Grotthus mechanism. Clinically relevant mutations are mapped to the structure, showing that they are mainly located at the subunit-subunit interfaces, thus causing instability of the complex.

ATP synthase.

Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms.

Cryo-EM reveals mechanistic insights into lipid-facilitated polyamine export by human ATP13A2.

The cytoplasmic polyamine maintains cellular homeostasis by chelating toxic metal cations, regulating transcriptional activity, and protecting DNA. ATP13A2 was identified as a lysosomal polyamine exporter responsible for polyamine release into the cytosol, and its dysfunction is associated with Alzheimer's disease and other neural degradation diseases. ATP13A2 belongs to the P5 subfamily of the P-type ATPase family, but its mechanisms remain unknown. Here, we report the cryoelectron microscopy (cryo-EM) structures of human ATP13A2 under four different conditions, revealing the structural coupling between the polyamine binding and the dephosphorylation. Polyamine is bound at the luminal tunnel and recognized through numerous electrostatic and π-cation interactions, explaining its broad specificity. The unique N-terminal domain is anchored to the lipid membrane to stabilize the E2P conformation, thereby accelerating the E1P-to-E2P transition. These findings reveal the distinct mechanism of P5B ATPases, thereby paving the way for neuroprotective therapy by activating ATP13A2.

Structural basis of polyamine transport by human ATP13A2 (PARK9).

Polyamines are small, organic polycations that are ubiquitous and essential to all forms of life. Currently, how polyamines are transported across membranes is not understood. Recent studies have suggested that ATP13A2 and its close homologs, collectively known as P5B-ATPases, are polyamine transporters at endo-/lysosomes. Loss-of-function mutations of ATP13A2 in humans cause hereditary early-onset Parkinson's disease. To understand the polyamine transport mechanism of ATP13A2, we determined high-resolution cryoelectron microscopy (cryo-EM) structures of human ATP13A2 in five distinct conformational intermediates, which together, represent a near-complete transport cycle of ATP13A2. The structural basis of the polyamine specificity was revealed by an endogenous polyamine molecule bound to a narrow, elongated cavity within the transmembrane domain. The structures show an atypical transport path for a water-soluble substrate, in which polyamines may exit within the cytosolic leaflet of the membrane. Our study provides important mechanistic insights into polyamine transport and a framework to understand the functions and mechanisms of P5B-ATPases.

Structural mechanisms for gating and ion selectivity of the human polyamine transporter ATP13A2.

Mutations in ATP13A2, also known as PARK9, cause a rare monogenic form of juvenile-onset Parkinson's disease named Kufor-Rakeb syndrome and other neurodegenerative diseases. ATP13A2 encodes a neuroprotective P5B P-type ATPase highly enriched in the brain that mediates selective import of spermine ions from lysosomes into the cytosol via an unknown mechanism. Here we present three structures of human ATP13A2 bound to an ATP analog or to spermine in the presence of phosphomimetics determined by cryoelectron microscopy. ATP13A2 autophosphorylation opens a lysosome luminal gate to reveal a narrow lumen access channel that holds a spermine ion in its entrance. ATP13A2's architecture suggests physical principles underlying selective polyamine transport and anticipates a "pump-channel" intermediate that could function as a counter-cation conduit to facilitate lysosome acidification. Our findings establish a firm foundation to understand ATP13A2 mutations associated with disease and bring us closer to realizing ATP13A2's potential in neuroprotective therapy.

A DNA origami rotary ratchet motor.

To impart directionality to the motions of a molecular mechanism, one must overcome the random thermal forces that are ubiquitous on such small scales and in liquid solution at ambient temperature. In equilibrium without energy supply, directional motion cannot be sustained without violating the laws of thermodynamics. Under conditions away from thermodynamic equilibrium, directional motion may be achieved within the framework of Brownian ratchets, which are diffusive mechanisms that have broken inversion symmetry1-5. Ratcheting is thought to underpin the function of many natural biological motors, such as the F1F0-ATPase6-8, and it has been demonstrated experimentally in synthetic microscale systems (for example, to our knowledge, first in ref. 3) and also in artificial molecular motors created by organic chemical synthesis9-12. DNA nanotechnology13 has yielded a variety of nanoscale mechanisms, including pivots, hinges, crank sliders and rotary systems14-17, which can adopt different configurations, for example, triggered by strand-displacement reactions18,19 or by changing environmental parameters such as pH, ionic strength, temperature, external fields and by coupling their motions to those of natural motor proteins20-26. This previous work and considering low-Reynolds-number dynamics and inherent stochasticity27,28 led us to develop a nanoscale rotary motor built from DNA origami that is driven by ratcheting and whose mechanical capabilities approach those of biological motors such as F1F0-ATPase.

Intrinsically unidirectional chemically fuelled rotary molecular motors.

Biological systems mainly utilize chemical energy to fuel autonomous molecular motors, enabling the system to be driven out of equilibrium1. Taking inspiration from rotary motors such as the bacterial flagellar motor2 and adenosine triphosphate synthase3, and building on the success of light-powered unidirectional rotary molecular motors4-6, scientists have pursued the design of synthetic molecular motors solely driven by chemical energy7-13. However, designing artificial rotary molecular motors operating autonomously using a chemical fuel and simultaneously featuring the intrinsic structural design elements to allow full 360° unidirectional rotary motion like adenosine triphosphate synthase remains challenging. Here we show that a homochiral biaryl Motor-3, with three distinct stereochemical elements, is a rotary motor that undergoes repetitive and unidirectional 360° rotation of the two aryl groups around a single-bond axle driven by a chemical fuel. It undergoes sequential ester cyclization, helix inversion and ring opening, and up to 99% unidirectionality is realized over the autonomous rotary cycle. The molecular rotary motor can be operated in two modes: synchronized motion with pulses of a chemical fuel and acid-base oscillations; and autonomous motion in the presence of a chemical fuel under slightly basic aqueous conditions. This rotary motor design with intrinsic control over the direction of rotation, simple chemical fuelling for autonomous motion and near-perfect unidirectionality illustrates the potential for future generations of multicomponent machines to perform mechanical functions.

TMK-based cell-surface auxin signalling activates cell-wall acidification.

The phytohormone auxin controls many processes in plants, at least in part through its regulation of cell expansion1. The acid growth hypothesis has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism that underlies auxin-induced cell-wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane H+-ATPase that pumps protons into the apoplast2, yet how auxin activates its phosphorylation remains unclear. Here we show that the transmembrane kinase (TMK) auxin-signalling proteins interact with plasma membrane H+-ATPases, inducing their phosphorylation, and thereby promoting cell-wall acidification and hypocotyl cell elongation in Arabidopsis. Auxin induced interactions between TMKs and H+-ATPases in the plasma membrane within seconds, as well as TMK-dependent phosphorylation of the penultimate threonine residue on the H+-ATPases. Our genetic, biochemical and molecular evidence demonstrates that TMKs directly phosphorylate plasma membrane H+-ATPase and are required for auxin-induced H+-ATPase activation, apoplastic acidification and cell expansion. Thus, our findings reveal a crucial connection between auxin and plasma membrane H+-ATPase activation in regulating apoplastic pH changes and cell expansion through TMK-based cell surface auxin signalling.

Essentials for ATP synthesis by F1F0 ATP synthases.

The majority of cellular energy in the form of adenosine triphosphate (ATP) is synthesized by the ubiquitous F(1)F(0) ATP synthase. Power for ATP synthesis derives from an electrochemical proton (or Na(+)) gradient, which drives rotation of membranous F(0) motor components. Efficient rotation not only requires a significant driving force (DeltamuH(+)), consisting of membrane potential (Deltapsi) and proton concentration gradient (DeltapH), but also a high proton concentration at the source P side. In vivo this is maintained by dynamic proton movements across and along the surface of the membrane. The torque-generating unit consists of the interface of the rotating c ring and the stator a subunit. Ion translocation through this unit involves a sophisticated interplay between the c-ring binding sites, the stator arginine, and the coupling ions on both sides of the membrane. c-ring rotation is transmitted to the eccentric shaft gamma-subunit to elicit conformational changes in the catalytic sites of F(1), leading to ATP synthesis.

FO-F1 coupling and symmetry mismatch in ATP synthase resolved in every FO rotation step.

FOF1 ATP synthase, a ubiquitous enzyme that synthesizes most ATP in living cells, is composed of two rotary motors: a membrane-embedded proton-driven FO motor and a catalytic F1 motor. These motors share both central and peripheral stalks. Although both FO and F1 have pseudo-symmetric structures, their symmetries do not match. How symmetry mismatch is solved remains elusive because of the missing intermediate structures of the rotational steps. Here, for the case of Bacillus PS3 ATP synthases with three- and 10-fold symmetries in F1 and FO, respectively, we uncovered the mechanical couplings between FO and F1 at every 36° rotation step via molecular dynamics simulations and comparative studies of cryoelectron microscopy (cryo-EM) structures from three species. We found that the mismatch could be solved using several elements: 1) the F1 head partially rotates relative to the FO a subunit via elastic distortion of the b subunits, 2) the rotor is twisted, and 3) comparisons of cryo-EM structures further suggest that the c ring rotary angles can deviate from the symmetric ones. In addition, the F1 motor may have non-canonical structures, relieving stronger frustration. Thus, we provide new insights for solving the symmetry mismatch problem.

Direct identification of the rotary angle of ATP cleavage in F1-ATPase from Bacillus PS3.

F1-ATPase is the world's smallest biological rotary motor driven by ATP hydrolysis at three catalytic ß subunits. The 120° rotational step of the central shaft γ consists of 80° substep driven by ATP binding and a subsequent 40° substep. In order to correlate timing of ATP cleavage at a specific catalytic site with a rotary angle, we designed a new F1-ATPase (F1) from thermophilic Bacillus PS3 carrying ß(E190D/F414E/F420E) mutations, which cause extremely slow rates of both ATP cleavage and ATP binding. We produced an F1 molecule that consists of one mutant ß and two wild-type ßs (hybrid F1). As a result, the new hybrid F1 showed two pausing angles that are separated by 200°. They are attributable to two slowed reaction steps in the mutated ß, thus providing the direct evidence that ATP cleavage occurs at 200° rather than 80° subsequent to ATP binding at 0°. This scenario resolves the long-standing unclarified issue in the chemomechanical coupling scheme and gives insights into the mechanism of driving unidirectional rotation.

A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps.

Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na(+),K(+)-ATPase maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps.

Rotary catalysis of bovine mitochondrial F1-ATPase studied by single-molecule experiments.

The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F1 (bMF1) were studied in single-molecule experiments. Under ATP-saturated concentrations, high-speed imaging of a single 40-nm gold bead attached to the γ subunit of bMF1 showed 2 types of intervening pauses during the rotation that were discriminated by short dwell and long dwell. Using ATPγS as a slowly hydrolyzing ATP derivative as well as using a functional mutant ßE188D with slowed ATP hydrolysis, the 2 pausing events were distinctively identified. Buffer-exchange experiments with a nonhydrolyzable analog (AMP-PNP) revealed that the long dwell corresponds to the catalytic dwell, that is, the waiting state for hydrolysis, while it remains elusive which catalytic state short pause represents. The angular position of catalytic dwell was determined to be at +80° from the ATP-binding angle, mostly consistent with other F1s. The position of short dwell was found at 50 to 60° from catalytic dwell, that is, +10 to 20° from the ATP-binding angle. This is a distinct difference from human mitochondrial F1, which also shows intervening dwell that probably corresponds to the short dwell of bMF1, at +65° from the binding pause. Furthermore, we conducted "stall-and-release" experiments with magnetic tweezers to reveal how the binding affinity and hydrolysis equilibrium are modulated by the γ rotation. Similar to thermophilic F1, bMF1 showed a strong exponential increase in ATP affinity, while the hydrolysis equilibrium did not change significantly. This indicates that the ATP binding process generates larger torque than the hydrolysis process.

The phototroph-specific ß-hairpin structure of the γ subunit of FoF1-ATP synthase is important for efficient ATP synthesis of cyanobacteria.

The FoF1 synthase produces ATP from ADP and inorganic phosphate. The γ subunit of FoF1 ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic ß-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole FoF1 complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire FoF1 ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the ß-hairpin structure of FoF1 ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the ß-hairpin structure, which provided novel insights into the regulatory mechanisms of FoF1 ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis.

Regulatory Mechanisms and Environmental Adaptation of the F-ATPase Family.

The F-type ATPase family of enzymes, including ATP synthases, are found ubiquitously in biological membranes. ATP synthesis from ADP and inorganic phosphate is driven by an electrochemical H+ gradient or H+ motive force, in which intramolecular rotation of F-type ATPase is generated with H+ transport across the membranes. Because this rotation is essential for energy coupling between catalysis and H+-transport, regulation of the rotation is important to adapt to environmental changes and maintain ATP concentration. Recently, a series of cryo-electron microscopy images provided detailed insights into the structure of the H+ pathway and the multiple subunit arrangement. However, the regulatory mechanism of the rotation has not been clarified. This review describes the inhibition mechanism of ATP hydrolysis in bacterial enzymes. In addition, properties of the F-type ATPase of Streptococcus mutans, which acts as a H+-pump in an acidic environment, are described. These findings may help in the development of novel antimicrobial agents.

A conserved, buried cysteine near the P-site is accessible to cysteine modifications and increases ROS stability in the P-type plasma membrane H+-ATPase.

Sulfur-containing amino acid residues function in antioxidative responses, which can be induced by the reactive oxygen species generated by excessive copper and hydrogen peroxide. In all Na+/K+, Ca2+, and H+ pumping P-type ATPases, a cysteine residue is present two residues upstream of the essential aspartate residue, which is obligatorily phosphorylated in each catalytic cycle. Despite its conservation, the function of this cysteine residue was hitherto unknown. In this study, we analyzed the function of the corresponding cysteine residue (Cys-327) in the autoinhibited plasma membrane H+-ATPase isoform 2 (AHA2) from Arabidopsis thaliana by mutagenesis and heterologous expression in a yeast host. Enzyme kinetics of alanine, serine, and leucine substitutions were identical with those of the wild-type pump but the sensitivity of the mutant pumps was increased towards copper and hydrogen peroxide. Peptide identification and sequencing by mass spectrometry demonstrated that Cys-327 was prone to oxidation. These data suggest that Cys-327 functions as a protective residue in the plasma membrane H+-ATPase, and possibly in other P-type ATPases as well.

Method to extract multiple states in F1-ATPase rotation experiments from jump distributions.

A method is proposed for analyzing fast (10 µs) single-molecule rotation trajectories in F1 adenosinetriphosphatase ([Formula: see text]-ATPase). This method is based on the distribution of jumps in the rotation angle that occur in the transitions during the steps between subsequent catalytic dwells. The method is complementary to the "stalling" technique devised by H. Noji et al. [Biophys. Rev. 9, 103-118, 2017], and can reveal multiple states not directly detectable as steps. A bimodal distribution of jumps is observed at certain angles, due to the system being in either of 2 states at the same rotation angle. In this method, a multistate theory is used that takes into account a viscoelastic fluctuation of the imaging probe. Using an established sequence of 3 specific states, a theoretical profile of angular jumps is predicted, without adjustable parameters, that agrees with experiment for most of the angular range. Agreement can be achieved at all angles by assuming a fourth state with an ∼10 µs lifetime and a dwell angle about 40° after the adenosine 5'-triphosphate (ATP) binding dwell. The latter result suggests that the ATP binding in one ß subunit and the adenosine 5'-diphosphate (ADP) release from another ß subunit occur via a transient whose lifetime is ∼10 µs and is about 6 orders of magnitude smaller than the lifetime for ADP release from a singly occupied [Formula: see text]-ATPase. An internal consistency test is given by comparing 2 independent ways of obtaining the relaxation time of the probe. They agree and are ∼15 µs.

Insights into the origin of the high energy-conversion efficiency of F1-ATPase.

Our understanding of the rotary-coupling mechanism of F1-ATPase has been greatly enhanced in the last decade by advances in X-ray crystallography, single-molecular imaging, and theoretical models. Recently, Volkán-Kacsó and Marcus [S. Volkán-Kacsó, R. A. Marcus, Proc. Natl. Acad. Sci. U.S.A. 112, 14230 (2015)] presented an insightful thermodynamic model based on the Marcus reaction theory coupled with an elastic structural deformation term to explain the observed γ-rotation angle dependence of the adenosine triphosphate (ATP)/adenosine diphosphate (ADP) exchange rates of F1-ATPase. Although the model is successful in correlating single-molecule data, it is not in agreement with the available theoretical results. We describe a revision of the model, which leads to consistency with the simulation results and other experimental data on the F1-ATPase rotor compliance. Although the free energy liberated on ATP hydrolysis by F1-ATPase is rapidly dissipated as heat and so cannot contribute directly to the rotation, we show how, nevertheless, F1-ATPase functions near the maximum possible efficiency. This surprising result is a consequence of the differential binding of ATP and its hydrolysis products ADP and Pi along a well-defined pathway.