36 results found
Blum TB, Hahn A, Meier T, et al., 2019, Dimers of mitochondrial ATP synthase induce membrane curvature and self-assemble into rows, Proceedings of the National Academy of Sciences of the United States of America, Vol: 116, Pages: 4250-4255, ISSN: 0027-8424
Mitochondrial ATP synthases form dimers, which assemble intolong ribbons at the rims of the inner membrane cristae. We re-constituted detergent-purified mitochondrial ATP synthase dimersfrom the green algaPolytomellasp. and the yeastYarrowialipolyticainto liposomes and examined them by electron cryo-tomography. Tomographic volumes revealed that ATP synthasedimers from both species self-assemble into rows and bend thelipid bilayer locally. The dimer rows and the induced degree ofmembrane curvature closely resemble those in the inner mem-brane cristae. Monomers of mitochondrial ATP synthase reconsti-tuted into liposomes do not bend membrane visibly and do notform rows. No specific lipids or proteins other than ATP synthasedimers are required for row formation and membrane remodelling.Long rows of ATP synthase dimers are a conserved feature ofmitochondrial inner membranes. They are required for cristaeformation and a main factor in mitochondrial morphogenesis.
Barringer R, 2019, Illuminating the secrets of crystals: microcrystal electron diffraction in structural biology, Bioscience Horizons, Vol: 11, Pages: 1-12, ISSN: 1754-7431
X-ray crystallography (XRC) has visualised biological macromolecules in exquisite detail for over 50 years, relying on a combination of mathematical principles to offer insight into atomic structures. Crystals can diffract various elec-tromagnetic waves aside from the conventional X-ray, offering an alternative approach to crystallographic structural analysis. Microcrystal Electron Diffraction (MicroED) illuminates crystals with electron waves instead of X-rays. Two specialised groups have demonstrated that MicroED can give high resolution (often atomic) data, and now appears to be developing into a powerful alternative method to XRC or electron microscopy of macromolecules. How Mi-croED compares to XRC will be key to assessing it as a stand-alone crystallographic technique. This review presents a critical analysis of MicroED, with comments on theoretical and practical aspects and suggestions of further work and development.
Hahn A, Vonck J, Mills D, et al., 2018, Structure, mechanism, and regulation of the chloroplast ATP synthase, Science, Vol: 360, ISSN: 0036-8075
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 F1Fo ATP synthase (cF1Fo), 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 α3β3 head (cF1) is powered by the cFo rotary motor in the membrane. cFo 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 ε transmits the torque from the Fo motor to the catalytic cF1 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 cF1 with cFo.All rotary ATP synthases are, in principle, fully reversible. To prevent wasteful ATP hydrolysis, cF1Fo 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 cF1Fo 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 cF1Fo ATP synthase in lipid nanodiscs a
Dautant A, Meier TK, Hahn A, et al., 2018, ATP synthase diseases of mitochondrial genetic origin, Frontiers in Physiology, Vol: 9, ISSN: 1664-042X
Devastating human neuromuscular disorders have been associated to defects in the ATP synthase. This enzyme is found in the inner mitochondrial membrane and catalyzes the last step in oxidative phosphorylation, which provides aerobic eukaryotes with ATP. With the advent of structures of complete ATP synthases, and the availability of genetically approachable systems such as the yeast Saccharomyces cerevisiae, we can begin to understand these molecular machines and their associated defects at the molecular level. In this review, we describe what is known about the clinical syndromes induced by 58 different mutations found in the mitochondrial genes encoding membrane subunits 8 and a of ATP synthase, and evaluate their functional consequences with respect to recently described cryo-EM structures.
Eisel B, Hartrampf F, Meier TK, et al., 2018, Reversible optical control of F1Fo-ATP synthase using photoswitchable inhibitors, FEBS Letters, Vol: 592, Pages: 343-355, ISSN: 0014-5793
F1Fo-ATP synthase is one of the best studied macromolecular machines in nature. It can be inhibited by a range of small molecules, which include the polyphenols resveratrol and piceatannol. Here, we introduce Photoswitchable Inhibitors of ATP Synthase, termed PIAS, which were synthetically derived from these polyphenols. They can be used to reversibly control the enzymatic activity of purified yeast Yarrowia lipolytica ATP synthase by light. Our experiments indicate that the PIAS described here bind to the same site in the ATP synthase F1 complex as the polyphenols in their trans form but they do not bind in their cis form. The new PIAS compounds could be useful tools for the optical precision control of ATP synthase in a variety of biochemical and biotechnological applications.
Schulz S, Wilkes M, Mills DJ, et al., 2017, Molecular architecture of the N type ATPase rotor ring from Burkholderia pseudomallei, Embo Reports, Vol: 18, Pages: 526-535, ISSN: 1469-3178
The genome of the highly infectious bacterium Burkholderia pseudomallei harbours an atp-operon that encodes an N-type rotary ATPase, in addition to an operon for a regular F-type rotary ATPase. The molecular architecture of N-type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N1No-type ATPase and investigated the structure and ion specificity of its membrane-embedded c-ring rotor by single-particle electron cryo-microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low-density, low-CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c-ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c17 ring is H+ specific, demonstrating that the ATPase is proton-coupled. The c17 ring stoichiometry results in a very high ion-to-ATP ratio of 5.7. We propose that this N-ATPase is a highly efficient proton pump that helps these melioidosis-causing bacteria to survive in the hostile, acidic environment of phagosomes.
He CH, Preiss LP, Wang BW, et al., 2016, Structural Simplification of Bedaquiline: the Discovery of 3-(4-(N,N-dimethylaminomethyl)phenyl)quinoline Derived Antitubercular Lead Compounds, Chemmedchem, Vol: 12, Pages: 106-119, ISSN: 1860-7187
Bedaquiline (BDQ) is a novel and highly potent last-line antituberculosis drug that was approved by the US FDA in 2013. Owing to its stereo-structural complexity, chemical synthesis and compound optimization are rather difficult and expensive. This study describes the structural simplification of bedaquiline while preserving antitubercular activity. The compound's structure was split into fragments and reassembled in various combinations while replacing the two chiral carbon atoms with an achiral linkage instead. Four series of analogues were designed; these candidates retained their potent antitubercular activity at sub-microgram per mL concentrations against both sensitive and multidrug-resistant (MDR) Mycobacterium tuberculosis strains. Six out of the top nine MIC-ranked candidates were found to inhibit mycobacterial ATP synthesis activity with IC50 values between 20 and 40 μm, one had IC50>66 μm, and two showed no inhibition, despite their antitubercular activity. These results provide a basis for the development of chemically less complex, lower-cost bedaquiline derivatives and describe the identification of two derivatives with antitubercular activity against non-ATP synthase related targets.
Hahn A, Parey K, Bublitz M, et al., 2016, Structure of a Complete ATP Synthase Dimer Reveals the Molecular Basis of Inner Mitochondrial Membrane Morphology, Molecular Cell, Vol: 63, Pages: 1-12, ISSN: 1097-4164
We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing a strong membrane curvature of ∼100°. Our structure explains the structural basis of cristae formation in mitochondria, a landmark signature of eukaryotic cell morphology.
Preiss L, Eisel B, Grell E, et al., 2016, Structural characterization of the mycobacterial ATP synthase c-ring and its interaction with the novel anti-tuberculosis drug Bedaquiline, Publisher: INT UNION CRYSTALLOGRAPHY, Pages: S210-S210, ISSN: 2053-2733
Preiss L, Hicks DB, Suzuki S, et al., 2015, Alkaliphilic bacteria with impact on industrial applications, concepts of early life forms, and bioenergetics of ATP synthesis, Frontiers in Bioengineering and Biotechnology, Vol: 3, ISSN: 2296-4185
Alkaliphilic bacteria typically grow well at pH 9, with the most extremophilic strains growing up to pH values as high as pH 12–13. Interest in extreme alkaliphiles arises because they are sources of useful, stable enzymes, and the cells themselves can be used for biotechnological and other applications at high pH. In addition, alkaline hydrothermal vents represent an early evolutionary niche for alkaliphiles and novel extreme alkaliphiles have also recently been found in alkaline serpentinizing sites. A third focus of interest in alkaliphiles is the challenge raised by the use of proton-coupled ATP synthases for oxidative phosphorylation by non-fermentative alkaliphiles. This creates a problem with respect to tenets of the chemiosmotic model that remains the core model for the bioenergetics of oxidative phosphorylation. Each of these facets of alkaliphilic bacteria will be discussed with a focus on extremely alkaliphilic Bacillus strains. These alkaliphilic bacteria have provided a cogent experimental system to probe adaptations that enable their growth and oxidative phosphorylation at high pH. Adaptations are clearly needed to enable secreted or partially exposed enzymes or protein complexes to function at the high external pH. Also, alkaliphiles must maintain a cytoplasmic pH that is significantly lower than the pH of the outside medium. This protects cytoplasmic components from an external pH that is alkaline enough to impair their stability or function. However, the pH gradient across the cytoplasmic membrane, with its orientation of more acidic inside than outside, is in the reverse of the productive orientation for bioenergetic work. The reversed gradient reduces the trans-membrane proton-motive force available to energize ATP synthesis. Multiple strategies are hypothesized to be involved in enabling alkaliphiles to circumvent the challenge of a low bulk proton-motive force energizing proton-coupled ATP synthesis at high pH.
Preiss L, Langer JD, Yildiz O, et al., 2015, Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline., Science Advances, Vol: 1, Pages: e1500106-e1500106, ISSN: 2375-2548
Multidrug-resistant tuberculosis (MDR-TB) is more prevalent today than at any other time in human history. Bedaquiline (BDQ), a novel Mycobacterium-specific adenosine triphosphate (ATP) synthase inhibitor, is the first drug in the last 40 years to be approved for the treatment of MDR-TB. This bactericidal compound targets the membrane-embedded rotor (c-ring) of the mycobacterial ATP synthase, a key metabolic enzyme required for ATP generation. We report the x-ray crystal structures of a mycobacterial c9 ring without and with BDQ bound at 1.55- and 1.7-Å resolution, respectively. The structures and supporting functional assays reveal how BDQ specifically interacts with the rotor ring via numerous interactions and thereby completely covers the c-ring’s ion-binding sites. This prevents the rotor ring from acting as an ion shuttle and stalls ATP synthase operation. The structures explain how diarylquinoline chemicals specifically inhibit the mycobacterial ATP synthase and thus enable structure-based drug design of next-generation ATP synthase inhibitors against Mycobacterium tuberculosis and other bacterial pathogens.
Leone V, Pogoryelov D, Meier T, et al., 2015, On the principle of ion selectivity in Na+/H+-coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor, Proceedings of the National Academy of Sciences, Vol: 112, Pages: E1057-E1066, ISSN: 0027-8424
<jats:p>Numerous membrane transporters and enzymes couple their mechanisms to the permeation of Na<jats:sup>+</jats:sup>or H<jats:sup>+</jats:sup>, thereby harnessing the energy stored in the form of transmembrane electrochemical potential gradients to sustain their activities. The molecular and environmental factors that control and modulate the ion specificity of most of these systems are, however, poorly understood. Here, we use isothermal titration calorimetry to determine the Na<jats:sup>+</jats:sup>/H<jats:sup>+</jats:sup>selectivity of the ion-driven membrane rotor of an F-type ATP synthase. Consistent with earlier theoretical predictions, we find that this rotor is significantly H<jats:sup>+</jats:sup>selective, although not sufficiently to be functionally coupled to H<jats:sup>+</jats:sup>, owing to the large excess of Na<jats:sup>+</jats:sup>in physiological settings. The functional Na<jats:sup>+</jats:sup>specificity of this ATP synthase thus results from two opposing factors, namely its inherent chemical selectivity and the relative availability of the coupling ion. Further theoretical studies of this membrane rotor, and of two others with a much stronger and a slightly weaker H<jats:sup>+</jats:sup>selectivity, indicate that, although the inherent selectivity of their ion-binding sites is largely set by the balance of polar and hydrophobic groups flanking a conserved carboxylic side chain, subtle variations in their structure and conformational dynamics, for a similar chemical makeup, can also have a significant contribution. We propose that the principle of ion selectivity outlined here may provide a rationale for the differentiation of Na<jats:sup>+</jats:sup>- and H<jats:sup>+</jats:sup>-coupled systems in other families of membrane transporters and enzymes.</jats:p>
Matthies D, Zhou W, Klyszejko AL, et al., 2014, High-resolution structure and mechanism of an F/V-hybrid rotor ring in a Na+-coupled ATP synthase, Nature Communications, Vol: 5
Preiss L, Langer JD, Hicks DB, et al., 2014, The c-ring ion binding site of the ATP synthase fromBacillus pseudofirmus OF4 is adapted to alkaliphilic lifestyle, Molecular Microbiology, Vol: 92, Pages: 973-984, ISSN: 0950-382X
Leone V, Pogoryelov D, Meier T, et al., 2014, Experimental Determination of the Ion Selectivity of an ATP-Synthase Membrane Rotor by Isothermal Titration Calorimetry, Biophysical Journal, Vol: 106, Pages: 372a-372a, ISSN: 0006-3495
Schulz S, Iglesias-Cans M, Krah A, et al., 2013, A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif, PLOS Biology, Vol: 11, ISSN: 1545-7885
The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na+. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1Fo-ATP synthase with a novel Na+ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na+ specificity in physiological settings. Consistently, activity measurements showed Na+ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na+ ionophore monensin. Furthermore, Na+ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na+ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na+ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na+ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.
Preiss L, Klyszejko AL, Hicks DB, et al., 2013, The c-ring stoichiometry of ATP synthase is adapted to cell physiological requirements of alkaliphilic Bacillus pseudofirmus OF4, Proceedings of the National Academy of Sciences, Vol: 110, Pages: 7874-7879, ISSN: 0027-8424
Preiss L, Eckhardt-Strelau L, Langer J, et al., 2012, A novel TB drug targets the ATP synthase rotor ring of mycobacteria, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol: 1817, Pages: S22-S22, ISSN: 0005-2728
Pogoryelov D, Klyszejko AL, Krasnoselska GO, et al., 2012, Engineering rotor ring stoichiometries in the ATP synthase, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol: 1817, Pages: S21-S21, ISSN: 0005-2728
Krasnoselska GO, Pogoryelov D, Krah A, et al., 2012, Tuning the ion specificity of the ATP synthase rotor, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol: 1817, Pages: S17-S17, ISSN: 0005-2728
Hakulinen JK, Klyszejko AL, Hoffmann J, et al., 2012, Structural study on the architecture of the bacterial ATP synthase Fomotor, Proceedings of the National Academy of Sciences, Vol: 109, Pages: E2050-E2056, ISSN: 0027-8424
Pogoryelov D, Klyszejko AL, Krasnoselska GO, et al., 2012, Engineering rotor ring stoichiometries in the ATP synthase, Proceedings of the National Academy of Sciences, Vol: 109, Pages: E1599-E1608, ISSN: 0027-8424
Symersky J, Pagadala V, Osowski D, et al., 2012, Structure of the c10 ring of the yeast mitochondrial ATP synthase in the open conformation, Nature Structural & Molecular Biology, Vol: 19, Pages: 485-491, ISSN: 1545-9993
Pogoryelov D, Krah A, Langer JD, et al., 2010, Microscopic rotary mechanism of ion translocation in the Fo complex of ATP synthases, Nature Chemical Biology, Vol: 6, Pages: 891-899, ISSN: 1552-4450
Preiss L, Yildiz Ö, Hicks DB, et al., 2010, A New Type of Proton Coordination in an F1Fo-ATP Synthase Rotor Ring, PLoS Biology, Vol: 8, Pages: e1000443-e1000443
Krah A, Pogoryelov D, Langer J, et al., 2010, Structural basis for the ion selectivity of F-ATP-synthase c-ring rotors, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol: 1797, Pages: 35-35, ISSN: 0005-2728
Pogoryelov D, Yildiz Ö, Faraldo-Gómez JD, et al., 2009, High-resolution structure of the rotor ring of a proton-dependent ATP synthase, Nature Structural & Molecular Biology, Vol: 16, Pages: 1068-1073, ISSN: 1545-9993
Meier T, Krah A, Bond PJ, et al., 2009, Complete Ion-Coordination Structure in the Rotor Ring of Na+-Dependent F-ATP Synthases, Journal of Molecular Biology, Vol: 391, Pages: 498-507, ISSN: 0022-2836
Pogoryelov D, Schlattner U, Meier T, et al., 2008, S1.35 The rotor subunit interface of the ATP synthase from Ilyobacter tartaricus, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol: 1777, Pages: S17-S17, ISSN: 0005-2728
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