Rotary ATPases Research Articles

The distribution of different types of rotary ATPases among prokaryotes

The distribution of different types of rotary ATPases among prokaryotes

Visualizing Single V-ATPase Rotation Using Janus Nanoparticles.

Understanding the function of rotary molecular motors, such as the rotary ATPases, relies on our ability to visualize the single-molecule rotation. Traditional imaging methods often involve tagging those motors with nanoparticles (NPs) and inferring their rotation from translational motion of NPs. Here, we report an approach using "two-faced" Janus NPs to directly image the rotation of single V-ATPase from Enterococcus hirae, an ATP-driven rotary ion pump. By employing a 500-nm silica/gold Janus NP, we exploit its asymmetric optical contrast - silica core with a gold cap on one hemisphere - to achieve precise imaging of the unidirectional counter-clockwise rotation of single V-ATPase motors immobilized on surfaces. Despite the added viscous load from the relatively large Janus NP probe, our approach provides accurate torque measurements of single V-ATPase. This study underscores the advantages of Janus NPs over conventional probes, establishing them as powerful tools for single-molecule analysis of rotary molecular motors.

Eukaryotic yeast V1-ATPase rotary mechanism insights revealed by high-resolution single-molecule studies.

Vacuolar ATP-dependent proton pumps (V-ATPases) belong to a super-family of rotary ATPases and ATP synthases. The V1 complex consumes ATP to drive rotation of a central rotor that pumps protons across membranes via the Vo complex. Eukaryotic V-ATPases are regulated by reversible disassembly of subunit C, V1 without C, and VO. ATP hydrolysis is thought to generate an unknown rotary state that initiates regulated disassembly. Dissociated V1 is inhibited by subunit H that traps it in a specific rotational position. Here, we report the first single-molecule studies with high resolution of time and rotational position of Saccharomyces cerevisiae V1-ATPase lacking subunits H and C (V1ΔHC), which resolves previously elusive dwells and angular velocity changes. Rotation occurred in 120° power strokes separated by dwells comparable to catalytic dwells observed in other rotary ATPases. However, unique V1ΔHC rotational features included: 1) faltering power stroke rotation during the first 60°; 2) a dwell often occurring ∼45° after the catalytic dwell, which did not increase in duration at limiting MgATP; 3) a second dwell, ∼2-fold longer occurring 112° that increased in duration and occurrence at limiting MgATP; 4) limiting MgATP-dependent decreases in power stroke angular velocity where dwells were not observed. The results presented here are consistent with MgATP binding to the empty catalytic site at 112° and MgADP released at ∼45°, and provide important new insight concerning the molecular basis for the differences in rotary positions of substrate binding and product release between V-type and F-type ATPases.

Transforming yeast into a facultative photoheterotroph via expression of vacuolar rhodopsin

Phototrophic metabolism, the capture of light for energy, was a pivotal biological innovation that greatly increased the total energy available to the biosphere. Chlorophyll-based photosynthesis is the most familiar phototrophic metabolism, but retinal-based microbial rhodopsins transduce nearly as much light energy as chlorophyll does,1 via a simpler mechanism, and are found in far more taxonomic groups. Although this system has apparently spread widely via horizontal gene transfer,2,3,4 little is known about how rhodopsin genes (with phylogenetic origins within prokaryotes5,6) are horizontally acquired by eukaryotic cells with complex internal membrane architectures or the conditions under which they provide a fitness advantage. To address this knowledge gap, we sought to determine whether Saccharomyces cerevisiae, a heterotrophic yeast with no known evolutionary history of phototrophy, can function as a facultative photoheterotroph after acquiring a single rhodopsin gene. We inserted a rhodopsin gene from Ustilago maydis,7 which encodes a proton pump localized to the vacuole, an organelle normally acidified via a V-type rotary ATPase, allowing the rhodopsin to supplement heterotrophic metabolism. Probes of the physiology of modified cells show that they can deacidify the cytoplasm using light energy, demonstrating the ability of rhodopsins to ameliorate the effects of starvation and quiescence. Further, we show that yeast-bearing rhodopsins gain a selective advantage when illuminated, proliferating more rapidly than their non-phototrophic ancestor or rhodopsin-bearing yeast cultured in the dark. These results underscore the ease with which rhodopsins may be horizontally transferred even in eukaryotes, providing novel biological function without first requiring evolutionary optimization.

Mechanism of ATP hydrolysis dependent rotation of bacterial ATP synthase

F1 domain of ATP synthase is a rotary ATPase complex in which rotation of central γ-subunit proceeds in 120° steps against a surrounding α3β3 fueled by ATP hydrolysis. How the ATP hydrolysis reactions occurring in three catalytic αβ dimers are coupled to mechanical rotation is a key outstanding question. Here we describe catalytic intermediates of the F1 domain in FoF1 synthase from Bacillus PS3 sp. during ATP mediated rotation captured using cryo-EM. The structures reveal that three catalytic events and the first 80° rotation occur simultaneously in F1 domain when nucleotides are bound at all the three catalytic αβ dimers. The remaining 40° rotation of the complete 120° step is driven by completion of ATP hydrolysis at αDβD, and proceeds through three sub-steps (83°, 91°, 101°, and 120°) with three associated conformational intermediates. All sub-steps except for one between 91° and 101° associated with phosphate release, occur independently of the chemical cycle, suggesting that the 40° rotation is largely driven by release of intramolecular strain accumulated by the 80° rotation. Together with our previous results, these findings provide the molecular basis of ATP driven rotation of ATP synthases.

Rotary mechanism of V/A-ATPases-how is ATP hydrolysis converted into a mechanical step rotation in rotary ATPases?

V/A-ATPase is a rotary molecular motor protein that produces ATP through the rotation of its central rotor. The soluble part of this protein, the V1 domain, rotates upon ATP hydrolysis. However, the mechanism by which ATP hydrolysis in the V1 domain couples with the mechanical rotation of the rotor is still unclear. Cryo-EM snapshot analysis of V/A-ATPase indicated that three independent and simultaneous catalytic events occurred at the three catalytic dimers (ABopen, ABsemi, and ABclosed), leading to a 120° rotation of the central rotor. Besides the closing motion caused by ATP bound to ABopen, the hydrolysis of ATP bound to ABsemi drives the 120° step. Our recent time-resolved cryo-EM snapshot analysis provides further evidence for this model. This review aimed to provide a comprehensive overview of the structure and function of V/A-ATPase from a thermophilic bacterium, one of the most well-studied rotary ATPases to date.

Molecular simulations reveal chemomechanical forces driving bacterial gliding motility.

The cell distributes energy between two main branches: locomotion, and reproduction. This partitioning scheme is dynamic, and dependent on the cell's environment. In view of deconvoluting this energy balance, we present an investigation of the costs associated with cellular locomotion in Bacteroidetes. This exemplary organism displays a unique gliding motility. Such translocation is independent of well-known propulsive structures like flagella, pili, or fimbriae; rather, the cell uniquely glides on a surface. I will present a ‘rotary rack and pinion’ model engaging the Type IX Secretion System rotary motor to describe the gliding mechanism. (1) Using a Bayesian model of maximum entropy, we investigate the torque-guided, conformational landscape of the Secretion System in the periplasmic space. After 2 microseconds of simulation, an allosteric network is discovered that couples the power-strokes in the peripheral domains with the rotatory proton-pumping in the transmembrane part of the motor. (2) In analogy with our past computations of rotary ATPases, a combination of pH-simulations and Anisotropic Network Model are separately employed to decipher the chemo-mechanical forces that drive proton gradient across the motor. These coupling forces are monitored using light microscopy and tuned biochemically using mutations to control the gliding movements of the bacteria. (3) Finally, guided by recent cryo-EM experiments, both the periplasmic and membrane domains of the secretion system are integrated to drive a kinetic description of the energy cost associated with gliding motility, comparing with the growth necessities.

Cryo-EM analysis of V/A-ATPase intermediates reveals the transition of the ground-state structure to steady-state structures by sequential ATP binding

Vacuolar/archaeal-type ATPase (V/A-ATPase) is a rotary ATPase that shares a common rotary catalytic mechanism with FoF1 ATP synthase. Structural images of V/A-ATPase obtained by single-particle cryo-electron microscopy during ATP hydrolysis identified several intermediates, revealing the rotary mechanism under steady-state conditions. However, further characterization is needed to understand the transition from the ground state to the steady state. Here, we identified the cryo-electron microscopy structures of V/A-ATPase corresponding to short-lived initial intermediates during the activation of the ground state structure by time-resolving snapshot analysis. These intermediate structures provide insights into how the ground-state structure changes to the active, steady state through the sequential binding of ATP to its three catalytic sites. All the intermediate structures of V/A-ATPase adopt the same asymmetric structure, whereas the three catalytic dimers adopt different conformations. This is significantly different from the initial activation process of FoF1, where the overall structure of the F1 domain changes during the transition from a pseudo-symmetric to a canonical asymmetric structure (PNAS NEXUS, pgac116, 2022). In conclusion, our findings provide dynamical information that will enhance the future prospects for studying the initial activation processes of the enzymes, which have unknown intermediate structures in their functional pathway.

Structural snapshots of V/A-ATPase reveal the rotary catalytic mechanism of rotary ATPases

V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FoF1 ATP synthase. When powered by ATP hydrolysis, the V1 domain rotates the central rotor against the A3B3 hexamer, composed of three catalytic AB dimers adopting different conformations (ABopen, ABsemi, and ABclosed). Here, we report the atomic models of 18 catalytic intermediates of the V1 domain of V/A-ATPase under different reaction conditions, determined by single particle cryo-EM. The models reveal that the rotor does not rotate immediately after binding of ATP to the V1. Instead, three events proceed simultaneously with the 120˚ rotation of the shaft: hydrolysis of ATP in ABsemi, zipper movement in ABopen by the binding ATP, and unzipper movement in ABclosed with release of both ADP and Pi. This indicates the unidirectional rotation of V/A-ATPase by a ratchet-like mechanism owing to ATP hydrolysis in ABsemi, rather than the power stroke model proposed previously for F1-ATPase.

Cryo-EM Structural Snapshots of V/A-ATPase Reveal the Rotay Mechanism of Rotary ATPases

Cryo-EM Structural Snapshots of V/A-ATPase Reveal the Rotay Mechanism of Rotary ATPases

Chained Structure of Dimeric F1-like ATPase in Mycoplasma mobile Gliding Machinery.

ABSTRACTMycoplasma mobile, a fish pathogen, exhibits gliding motility using ATP hydrolysis on solid surfaces, including animal cells. The gliding machinery can be divided into surface and internal structures. The internal structure of the motor is composed of 28 so-called “chains” that are each composed of 17 repeating protein units called “particles.” These proteins include homologs of the catalytic α and β subunits of F1-ATPase. In this study, we isolated the particles and determined their structures using negative-staining electron microscopy and high-speed atomic force microscopy. The isolated particles were composed of five proteins, MMOB1660 (α-subunit homolog), -1670 (β-subunit homolog), -1630, -1620, and -4530, and showed ATP hydrolyzing activity. The two-dimensional (2D) structure, with dimensions of 35 and 26 nm, showed a dimer of hexameric ring approximately 12 nm in diameter, resembling F1-ATPase catalytic (αβ)3. We isolated the F1-like ATPase unit, which is composed of MMOB1660, -1670, and -1630. Furthermore, we isolated the chain and analyzed the three-dimensional (3D) structure, showing that dimers of mushroom-like structures resembling F1-ATPase were connected and aligned along the dimer axis at 31-nm intervals. An atomic model of F1-ATPase catalytic (αβ)3 from Bacillus PS3 was successfully fitted to each hexameric ring of the mushroom-like structure. These results suggest that the motor for M. mobile gliding shares an evolutionary origin with F1-ATPase. Based on the obtained structure, we propose possible force transmission processes in the gliding mechanism.

Rotary Ion-Translocating ATPases/ATP Synthases: Diversity, Similarities, and Differences.

Ion-translocating ATPases and ATP synthases (F-, V-, A-type ATPases, and several P-type ATPases and ABC-transporters) catalyze ATP hydrolysis or ATP synthesis coupled with the ion transport across the membrane. F-, V-, and A-ATPases are protein nanomachines that combine transmembrane transport of protons or sodium ions with ATP synthesis/hydrolysis by means of a rotary mechanism. These enzymes are composed of two multisubunit subcomplexes that rotate relative to each other during catalysis. Rotary ATPases phosphorylate/dephosphorylate nucleotides directly, without the generation of phosphorylated protein intermediates. F-type ATPases are found in chloroplasts, mitochondria, most eubacteria, and in few archaea. V-type ATPases are eukaryotic enzymes present in a variety of cellular membranes, including the plasma membrane, vacuoles, late endosomes, and trans-Golgi cisternae. A-type ATPases are found in archaea and some eubacteria. F- and A-ATPases have two main functions: ATP synthesis powered by the proton motive force (pmf) or, in some prokaryotes, sodium-motive force (smf) and generation of the pmf or smf at the expense of ATP hydrolysis. In prokaryotes, both functions may be vitally important, depending on the environment and the presence of other enzymes capable of pmf or smf generation. In eukaryotes, the primary and the most crucial function of F-ATPases is ATP synthesis. Eukaryotic V-ATPases function exclusively as ATP-dependent proton pumps that generate pmf necessary for the transmembrane transport of ions and metabolites and are vitally important for pH regulation. This review describes the diversity of rotary ion-translocating ATPases from different organisms and compares the structural, functional, and regulatory features of these enzymes.

Cryo-EM studies of the rotary H<sup>+</sup>-ATPase/synthase from <i>Thermus thermophilus</i>

Proton-translocating rotary ATPases couple proton influx across the membrane domain and ATP hydrolysis/synthesis in the soluble domain through rotation of the central rotor axis against the surrounding peripheral stator apparatus. It is a significant challenge to determine the structure of rotary ATPases due to their intrinsic conformational heterogeneity and instability. Recent progress of single particle analysis of protein complexes using cryogenic electron microscopy (cryo-EM) has enabled the determination of whole rotary ATPase structures and made it possible to classify different rotational states of the enzymes at a near atomic resolution. Three cryo-EM maps corresponding to different rotational states of the V/A type H+-rotary ATPase from a bacterium Thermus thermophilus provide insights into the rotation of the whole complex, which allow us to determine the movement of each subunit during rotation. In addition, this review describes methodological developments to determine higher resolution cryo-EM structures, such as specimen preparation, to improve the image contrast of membrane proteins.

The Peripheral Stalk of Rotary ATPases.

Rotary ATPases are a family of enzymes that are thought of as molecular nanomotors and are classified in three types: F, A, and V-type ATPases. Two members (F and A-type) can synthesize and hydrolyze ATP, depending on the energetic needs of the cell, while the V-type enzyme exhibits only a hydrolytic activity. The overall architecture of all these enzymes is conserved and three main sectors are distinguished: a catalytic core, a rotor and a stator or peripheral stalk. The peripheral stalks of the A and V-types are highly conserved in both structure and function, however, the F-type peripheral stalks have divergent structures. Furthermore, the peripheral stalk has other roles beyond its stator function, as evidenced by several biochemical and recent structural studies. This review describes the information regarding the organization of the peripheral stalk components of F, A, and V-ATPases, highlighting the key differences between the studied enzymes, as well as the different processes in which the structure is involved.

Cryo EM structure of intact rotary H+-ATPase/synthase from Thermus thermophilus

Proton translocating rotary ATPases couple ATP hydrolysis/synthesis, which occurs in the soluble domain, with proton flow through the membrane domain via a rotation of the common central rotor complex against the surrounding peripheral stator apparatus. Here, we present a large data set of single particle cryo-electron micrograph images of the V/A type H+-rotary ATPase from the bacterium Thermus thermophilus, enabling the identification of three rotational states based on the orientation of the rotor subunit. Using masked refinement and classification with signal subtractions, we obtain homogeneous reconstructions for the whole complexes and soluble V1 domains. These reconstructions are of higher resolution than any EM map of intact rotary ATPase reported previously, providing a detailed molecular basis for how the rotary ATPase maintains structural integrity of the peripheral stator apparatus, and confirming the existence of a clear proton translocation path from both sides of the membrane.

Structure, mechanism, and regulation of the chloroplast ATP synthase

INTRODUCTION Green plant chloroplasts convert light into chemical energy, and adenosine triphosphate (ATP) generated by photosynthesis is the prime source of biologically useful energy on the planet. Plants produce ATP by the chloroplast F 1 F o ATP synthase (cF 1 F o ), a macromolecular machine par excellence, driven by the electrochemical proton gradient across the photosynthetic membrane. It consists of 26 protein subunits, 17 of them wholly or partly membrane-embedded. ATP synthesis in the hydrophilic α 3 β 3 head (cF 1 ) is powered by the cF o rotary motor in the membrane. cF o contains a rotor ring of 14 c subunits, each with a conserved protonatable glutamate. Subunit a conducts the protons to and from the c-ring protonation sites. The central stalk of subunits γ and e transmits the torque from the F o motor to the catalytic cF 1 head, resulting in the synthesis of three ATP per revolution. The peripheral stalk subunits b, b′, and δ act as a stator to prevent unproductive rotation of cF 1 with cF o . All rotary ATP synthases are, in principle, fully reversible. To prevent wasteful ATP hydrolysis, cF 1 F o has a redox switch that inhibits adenosine triphosphatase (ATPase) activity in the dark. RATIONALE Understanding the molecular mechanisms of this elaborate nanomachine requires detailed structures of the whole complex, ideally at atomic resolution. Because of the dynamic nature of this membrane protein complex, crystallization has been difficult and no high-resolution structure of an entire, functional ATP synthase is available. We reconstituted cF 1 F o from spinach chloroplasts into lipid nanodiscs and determined its structure by cryo–electron microscopy (cryo-EM). Cryo-EM is the ideal technique for this study because it can deliver high-resolution structures of large, dynamic macromolecular assemblies that adopt a mixture of conformational states. RESULTS We present the cryo-EM structure of the intact cF 1 F o ATP synthase in lipid nanodiscs at a resolution of 2.9 A (cF 1 ) to 3.4 A (cF o ). In the cF 1 ATPase head, we observe nucleotides with their coordinating Mg ions and water molecules, allowing assignment to the three well-characterized functional states involved in rotary ATP synthesis. Subunit δ on top of the ATPase head binds to all three α subunits, ensuring that only one peripheral stalk can attach. The loosely entwined, long α helices of the peripheral stalk subunits b and b′ clamp the integral membrane subunit a in its position next to the c-ring rotor, thus connecting cF 1 to cF o . Subunit γ has an L-shaped double hairpin with a redox sensor that can form a disulfide bond and a chock that blocks rotation to avoid wasteful ATP hydrolysis at night. Protons are translocated through access routes in subunit a in all rotary ATPases. We observe a hydrophilic channel on the lumenal surface that connects to the glutamate residues on the c-ring rotor that carry protons for an almost full rotation before releasing them into the stroma through another hydrophilic channel. A strictly conserved arginine separates the access and exit channels, preventing leakage of protons through the membrane. CONCLUSION We observe three cF 1 F o conformations, each with the central rotor stalled in a different position. Ring rotation is unexpectedly divided into three unequal steps. The peripheral stalk may thus act like an elastic spring, evening out the different energy contributions of each step. The features of ATP synthase nanomachines are remarkably similar in chloroplasts and mitochondria, considering their evolutionary distance of a billion years or more.

Cryo EM structure of intact rotary H+-ATPase/synthase from Thermus thermophilus

Proton translocating rotary ATPases couple ATP hydrolysis/synthesis, which occurs in the soluble domain, with proton flow through the membrane domain via a rotation of the common central rotor complex against the surrounding peripheral stator apparatus. Here, we present a large data set of single particle cryo-electron micrograph images of the V/A type H+-rotary ATPase from the bacterium Thermus thermophilus, enabling the identification of three rotational states based on the orientation of the rotor subunit. Using masked refinement and classification with signal subtractions, we obtain homogeneous reconstructions for the whole complexes and soluble V1 domains. These reconstructions are of higher resolution than any EM map of intact rotary ATPase reported previously, providing a detailed molecular basis for how the rotary ATPase maintains structural integrity of the peripheral stator apparatus, and confirming the existence of a clear proton translocation path from both sides of the membrane.

Electron cryomicroscopy of rotary ATPases

Ion‐translocating rotary ATPases serve either as adenosine triphosphate (ATP) synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. Our group uses electron cryomicroscopy (cryo‐EM) of single protein particles to study the structures of rotary ATPases. We also work to develop new methods for cryo‐EM to facilitate these studies. Our recent studies have not only illuminated the structures of these fascinating molecular motors at unprecedented resolution, but have also started to uncover their dynamics through computational isolation of the different conformations of the enzymes that exist simultaneously in solution. This lecture will describe the latest structures we have determined for the mitochondrial ATP synthase and proton pumping V‐ATPase, and what we have learned about how these enzymes function and how they interact with molecules that affect their activities.Support or Funding InformationCanadian Institutes of Health Research, Canada Research Chairs

Cryo-EM analysis of a domain antibody bound rotary ATPase complex.

The bacterial A/V-type ATPase/synthase rotary motor couples ATP hydrolysis/synthesis with proton translocation across biological membranes. The A/V-type ATPase/synthase from Thermus thermophilus has been extensively studied both structurally and functionally for many years. Here we provide an 8.7Å resolution cryo-electron microscopy 3D reconstruction of this complex bound to single-domain antibody fragments, small monomeric antibodies containing just the variable heavy domain. Docking of known structures into the density revealed the molecular orientation of the domain antibodies, suggesting that structure determination of co-domain antibody:protein complexes could be a useful avenue for unstable or smaller proteins. Although previous studies suggested that the presence of fluoroaluminate in this complex could change the rotary state of this enzyme, we observed no gross structural rearrangements under these conditions.

In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits

We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis.