Thomas Meier
Imperial College London, Life Sciences, Faculty Member
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The genome of the highly infectious bacterium Burkholderia pseudomallei harbors 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... more
The genome of the highly infectious bacterium Burkholderia pseudomallei harbors 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...
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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... more
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.
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Acinetobacter baumannii is a clinically relevant pathogen which causes multi-drug resistant, hospital-acquired infections and is a top priority target for antibiotic development. Cryo-EM structures of the A. baumannii F1Fo-ATP synthase in... more
Acinetobacter baumannii is a clinically relevant pathogen which causes multi-drug resistant, hospital-acquired infections and is a top priority target for antibiotic development. Cryo-EM structures of the A. baumannii F1Fo-ATP synthase in three conformational states reveal unique features, which represent attractive sites for the development of novel therapeutics.One sentence summaryStructure of Acinetobacter baumannii ATP synthase
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Research Interests: Chemistry, Molecular Dynamics Simulation, Biology, Medicine, Macromolecular X-Ray Crystallography, and 10 moreBiological Sciences, Saccharomyces cerevisiae, ATP synthase, CHEMICAL SCIENCES, Protein Conformation, Protons, Molecular Dynamic Simulation, Binding Site, binding sites, and Medical and Health Sciences
The Na+-translocating F-ATPase of the thermoalkaliphilic bacterium Clostridium paradoxum harbors an oligomeric ring of c subunits that resists dissociation by sodium dodecyl sulfate. The c ring has been isolated and crystallized in two... more
The Na+-translocating F-ATPase of the thermoalkaliphilic bacterium Clostridium paradoxum harbors an oligomeric ring of c subunits that resists dissociation by sodium dodecyl sulfate. The c ring has been isolated and crystallized in two dimensions. From electron microscopy of these c-ring crystals, a projection map was calculated to 7 Å resolution. In the projection map, each c ring consists of two concentric, slightly staggered, packed rings, each composed of 11 densities representing the α-helices. On the basis of these results, it was determined that the F-ATPase from C. paradoxum contains an undecameric c ring.
Research Interests: Electron Microscopy, Structural Biology, Bacteriology, Crystallization, Biological Sciences, and 11 moreSequence alignment, ATP synthase, Sodium, Amino Acid Sequence, Two Dimensions, Protons, Clostridium, Molecular Sequence Data, Sodium Dodecyl Sulfate, binding sites, and Medical and Health Sciences
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Research Interests: Thermodynamics, Chemistry, Molecular Dynamics Simulation, Structural Dynamics, Mass Spectrometry, and 15 moreBiological Sciences, Computer Simulation, Membrane transport, Physical sciences, ATP synthase, Enzyme, Protein Conformation, Spirulina, Proton Motive Force, Amino Acid Substitution Rates, Site-directed Mutagenesis, Molecular Dynamic Simulation, Binding Site, Biochemistry and cell biology, and binding sites
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FF-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... more
FF-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 lipolyticaATP synthase by light. Our experiments indicate that the PIAS bind to the same site in the ATP synthase Fcomplex as the polyphenols in their trans form, but they do not bind in their cis form. The PIAS could be useful tools for the optical precision control of ATP synthase in a variety of biochemical and biotechnological applications.
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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... more
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 ...
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Driven by transmembrane electrochemical ion gradients, F-type ATP synthases are the primary source of the universal energy currency, adenosine triphosphate (ATP), throughout all domains of life. The ATP synthase found in the thylakoid... more
Driven by transmembrane electrochemical ion gradients, F-type ATP synthases are the primary source of the universal energy currency, adenosine triphosphate (ATP), throughout all domains of life. The ATP synthase found in the thylakoid membranes of photosynthetic organisms has some unique features not present in other bacterial or mitochondrial systems. Among these is a larger-than-average transmembrane rotor ring and a redox-regulated switch capable of inhibiting ATP hydrolysis activity in the dark by uniquely adapted rotor subunit modifications. Here, we review recent insights into the structure and mechanism of ATP synthases specifically involved in photosynthesis and explore the cellular physiological consequences of these adaptations at short and long time scales.
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F-type adenosine triphosphate (ATP) synthase is a membrane-bound macromolecular complex, which is responsible for the synthesis of ATP, the universal energy source in living cells. This enzyme uses the proton- or sodium-motive force to... more
F-type adenosine triphosphate (ATP) synthase is a membrane-bound macromolecular complex, which is responsible for the synthesis of ATP, the universal energy source in living cells. This enzyme uses the proton- or sodium-motive force to power ATP synthesis by a unique rotary mechanism and can also operate in reverse, ATP hydrolysis, to generate ion gradients across membranes. The FF-ATP synthases from bacteria consist of eight different structural subunits, forming a complex of ∼550 kDa in size. In the bacterium Ilyobacter tartaricus the ATP synthase has the stoichiometry αβγδεabc. This chapter describes a wet-lab working protocol for the purification of several tens of milligrams of pure, heterologously (E. coli-)produced I. tartaricus Na-driven FF-ATP synthase and its subsequent efficient reconstitution into proteoliposomes. The methods are useful for a broad range of subsequent biochemical and biotechnological applications.
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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... more
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 , 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 and of ATP synthase, and evaluate their functional consequences with respect to recently described cryo-EM structures.
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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... more
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 enabl...
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Numerous membrane transporters and enzymes couple their mechanisms to the permeation of Na(+) or H(+), thereby harnessing the energy stored in the form of transmembrane electrochemical potential gradients to sustain their activities. The... more
Numerous membrane transporters and enzymes couple their mechanisms to the permeation of Na(+) or H(+), 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(+)/H(+) 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(+) selective, although not sufficiently to be functionally coupled to H(+), owing to the large excess of Na(+) in physiological settings. The functional Na(+) 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 o...
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All rotary ATPases catalyse the interconversion of ATP and ADP-Pi through a mechanism that is coupled to the transmembrane flow of H(+) or Na(+). Physiologically, however, F/A-type enzymes specialize in ATP synthesis driven by downhill... more
All rotary ATPases catalyse the interconversion of ATP and ADP-Pi through a mechanism that is coupled to the transmembrane flow of H(+) or Na(+). Physiologically, however, F/A-type enzymes specialize in ATP synthesis driven by downhill ion diffusion, while eukaryotic V-type ATPases function as ion pumps. To begin to rationalize the molecular basis for this functional differentiation, we solved the crystal structure of the Na(+)-driven membrane rotor of the Acetobacterium woodii ATP synthase, at 2.1 Å resolution. Unlike known structures, this rotor ring is a 9:1 heteromer of F- and V-type c-subunits and therefore features a hybrid configuration of ion-binding sites along its circumference. Molecular and kinetic simulations are used to dissect the mechanisms of Na(+) recognition and rotation of this c-ring, and to explain the functional implications of the V-type c-subunit. These structural and mechanistic insights indicate an evolutionary path between synthases and pumps involving ad...
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Research Interests: Electron Microscopy, Molecular Evolution, Atomic Force Microscopy, ATP synthase, Molecular motor proteins, and 6 moreMolecular Phylogenetics and Evolution, Protein Secondary Structure Prediction, Protein Binding, Biochemistry and cell biology, binding sites, and Medical biochemistry and metabolomics
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Research Interests: Science, Multidisciplinary, Macromolecular X-Ray Crystallography, Crystal structure, ATP synthase, and 11 moreMolecular motor proteins, Protein Secondary Structure Prediction, Sodium, Protein Conformation, Amino Acid Sequence, Ion Transport, Cytoplasm, Binding Site, Glutamic Acid, Molecular Sequence Data, and binding sites
Research Interests: Molecular Dynamics Simulation, Kinetics, Lithium, Macromolecular X-Ray Crystallography, Biocatalysis, and 12 moreBiological Sciences, Humans, Sodium, Substrate Specificity, Detergents, Hydrogen-Ion Concentration, Protons, Ions, Cell Membrane, binding sites, Fusobacterium nucleatum, and Medical and Health Sciences
Research Interests: Biochemistry, Genetics, Biophysics, Cell Cycle, DNA replication, and 15 moreCell Biology, DNA repair, Apoptosis, Gene expression, Biological Sciences, Crystal structure, Chromatin remodeling, ATP synthase, Chromatin, Chromatin structure, CHEMICAL SCIENCES, Amino Acid Sequence, Cations, DNA recombination, and binding sites
Research Interests: Thermodynamics, Crystallization, Energy Metabolism, Lipid Membranes, ATP synthase, and 15 moreMolecular motor proteins, Membrane Lipids, Free Energy, X ray diffraction, Protein Conformation, Spirulina, Atomic Structure, Thermodynamic Stability, Hydrogen-Ion Concentration, Protons, Molecular Dynamic Simulation, Binding Site, Ions, Biochemistry and cell biology, and binding sites
Research Interests: Molecular Biology, Phospholipids, Atomic Force Microscopy, Molecular, Bacteria, and 15 moreATP synthase, Protein Secondary Structure Prediction, Sodium, Cryo Electron Microscopy, Amino Acid Profile, Amino Acid Sequence, Protein Quaternary Structure, Two Dimensions, Cytoplasm, Binding Site, Ions, Biochemistry and cell biology, Electron Crystallography, Molecular Sequence Data, and binding sites
Research Interests: Electron Microscopy, Molecular Biology, EM, Western blotting, Protein synthesis, and 13 moreEscherichia coli, Molecular Machine, ATP synthase, Liposomes, FM, Membrane Integrity, Enzyme activity, Single Particle Analysis, Open Reading Frame, Membrane Protein, Detergents, Biochemistry and cell biology, and Bacillaceae
Research Interests: Molecular Biology, Molecular Dynamics Simulation, Carbon, Molecular, Crystal structure, and 15 moreGlutamate, Membrane transport, ATP synthase, Enzyme, Hydrogen Bond, Atomic Structure, Lipid bilayers, Amino Acid Sequence, Molecular Replacement, Molecular Dynamic Simulation, Binding Site, Biochemistry and cell biology, Interaction Network, Molecular Sequence Data, and binding sites
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ABSTRACT F-type ATP synthases typically harbor c-subunits of approximately 8-9 kDa that form oligomeric rings of 8-15 monomers with one ion-binding site per subunit. Each c-subunit in the ring forms a hairpin-like structure in the... more
ABSTRACT F-type ATP synthases typically harbor c-subunits of approximately 8-9 kDa that form oligomeric rings of 8-15 monomers with one ion-binding site per subunit. Each c-subunit in the ring forms a hairpin-like structure in the membrane with an inner α-helix, a cytoplasmatic loop and an outer α-helix. The membrane rings in eukaryotic V-type ATPases and archaeal A-type ATP synthases often have analogous but duplicated c-subunits with four instead of two transmembrane α-helices, harboring either one or two ion-binding sites per subunit. The anaerobic, acetogenic bacterium Acetobacterium woodii has a Na+-driven F-type ATP synthase, which contains a unique rotor composed of c-subunits of both types and transmembrane topologies. The enzyme is encoded by an atp-operon comprising 11 genes (atpIBE1E2E3FHAGDC) with three genes coding for two different c-subunits: atpE1 codes for a duplicated c-subunit, c1, whereas atpE2 and atpE3 code for single hairpin c-subunits, c2 and c3. We solved the structure of the A. woodii c-ring by X-ray crystallography at 2.4 Å resolution. This high resolution structure confirms earlier indications that this c-ring consists of c1:c2/3 subunits in a 1:9 stoichiometry. All c2/3-subunits (single hairpin) exhibit a Na+-binding site. A conserved glutamate residue in the locked conformation, halfway along the outer helix, three other carbonyl/hydroxyl groups and a water molecule are involved in Na+-coordination. Interestingly, the c1-subunit (double hairpin) contains only one Na+-binding site, of identical make-up. In the second site, a glutamine replaces the conserved glutamate, and prevents Na+-binding. Hence, the c-ring from the A. woodii ATP synthase carries 10 sodium ions across the membrane per 360-degree rotation. Similar to some of the V-type c-subunits, the c1-subunit has an N-terminal extension, which is also visible by atomic force microscopy. In summary, we present the first atomic-resolution structure of a heteromeric ATP synthase ring with intriguing mechanistic implications.
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Mass spectrometry of large macromolecules is still a methodological challenge. We here report on the application of the recently developed LILBID (laser induced liquid bead ion desorption) mass spectrometry by which the biomolecules... more
Mass spectrometry of large macromolecules is still a methodological challenge. We here report on the application of the recently developed LILBID (laser induced liquid bead ion desorption) mass spectrometry by which the biomolecules dissolved in microdroplets are desorbed/ablated by a mid-IR laser into vacuum. Two modes of desorption are possible: an ultrasoft mode at low laser intensity in which a
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Mass spectrometry of large macromolecules is still a methodological challenge. We here report on the application of the recently developed LILBID (laser induced liquid bead ion desorption) mass spectrometry by which the biomolecules... more
Mass spectrometry of large macromolecules is still a methodological challenge. We here report on the application of the recently developed LILBID (laser induced liquid bead ion desorption) mass spectrometry by which the biomolecules dissolved in microdroplets are desorbed/ablated by a mid-IR laser into vacuum. Two modes of desorption are possible: an ultrasoft mode at low laser intensity in which a