Alexander Hahn

Structure of a Complete ATP Synthase Dimer Reveals the Molecular Basis of Inner Mitochondrial Membrane Morphology

Summary 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.

The interplay between siderophore secretion and coupled iron and copper transport in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120.

Iron uptake is essential for Gram-negative bacteria including cyanobacteria. In cyanobacteria, however, the iron demand is higher than in proteobacteria due to the function of iron as a cofactor in photosynthesis and nitrogen fixation, but our understanding of iron uptake by cyanobacteria stands behind the knowledge in proteobacteria. Here, two genes involved in this process in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were identified. ORF all4025 encodes SchE, a putative cytoplasmic membrane-localized transporter involved in TolC-dependent siderophore secretion. Inactivation of schE resulted in an enhanced sensitivity to high metal concentrations and decreased secretion of hydroxamate-type siderophores. ORF all4026 encodes a predicted outer membrane-localized TonB-dependent iron transporter, IacT. Inactivation of iacT resulted in decreased sensitivity to elevated iron and copper levels. Expression of iacT from the artificial trc promoter (P(trc)) resulted in sensitization against tested metals. Further analysis showed that iron and copper effects are synergistic because a decreased supply of iron induced a significant decrease of copper levels in the iacT insertion mutant but an increase of those levels in the strain carrying P(trc)-iacT. Our results unravel a link between iron and copper homeostasis in Anabaena sp. PCC 7120.

The components of the putative iron transport system in the cyanobacterium Anabaena sp. PCC 7120.

Iron uptake in Gram-negative bacteria involves four distinct steps: (i) siderophore synthesis, (ii) siderophore secretion into the extracellular space, (iii) iron chelation by the siderophores, and (iv) siderophore/iron uptake via complexes in the outer membrane and the intermembrane space as well as in the plasma membrane. This process is well characterized for some proteobacterial systems, but largely unexplored and scarcely investigated in cyanobacteria such as the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Two putative siderophore synthesis clusters have been recently identified in this cyanobacterium. In addition, the export system for the main siderophore, schizokinen, secreted by Anabaena sp. PCC 7120 was described as well as the outer membrane transporter for its import from the extracellular space. We present the identification of components of three additional systems involved in siderophore-mediated iron uptake under iron-limiting conditions, namely TonB3, the ExbB3/ExbD3 and the Fhu systems. The transcription level of these genes is elevated under iron limitations and decreased under excess iron, while the expression levels of other members of these gene families and systems are impacted in distinct ways by other environmental conditions. Mutants of the tonB3, exbB3/exbD3 and fhu genes show an iron starvation phenotype. Thus, Anabaena sp. has a similar, yet distinct system for siderophore-dependent iron uptake compared with other proteobacteria.

Structure and Conservation of the Periplasmic Targeting Factor Tic22 Protein from Plants and Cyanobacteria

Background: Although Tic22 is involved in protein import into chloroplasts, the function in cyanobacteria is unknown. Results: Cyanobacterial Tic22 is required for OM biogenesis, shares structural features with chaperones, and can be substituted by plant Tic22. Conclusion: Tic22, involved in outer membrane biogenesis, is functionally conserved in cyanobacteria and plants. Significance: The findings are important for the understanding of periplasmic protein transport. Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.

The outer membrane TolC-like channel HgdD is part of tripartite resistance-nodulation-cell division (RND) efflux systems conferring multiple-drug resistance in the Cyanobacterium Anabaena sp. PCC7120.

Background: The RND efflux transporters of cyanobacteria are largely unknown. Results: Six RNDs with different functionality and mutant phenotypes exist in Anabaena sp. Conclusion: Antibiotic export involves a single AcrB-like RND protein. Significance: The diversity of the RND function in cyanobacteria is initially dissected. The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component “AB-D” systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox− phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.

Structure, mechanism, and regulation of the chloroplast ATP synthase

Protons find a path Adenosine triphosphate (ATP) synthases are dynamos that interconvert rotational and chemical energy. Capturing the complete structure of these multisubunit membrane-bound complexes has been hindered by their inherent ability to adopt multiple conformations. Srivastava et al. used protein engineering to freeze mitochondrial ATP synthase from yeast in a single conformation and obtained a structure with the inhibitor oligomycin, which binds to the rotating c-ring within the membrane. Hahn et al. show that chloroplast ATP synthase contains a built-in inhibitor triggered by oxidizing conditions in the dark chloroplast. The mechanisms by which these machines are powered are remarkably similar: Protons are shuttled through a channel to the membrane-embedded c-ring, where they drive nearly a full rotation of the rotor before exiting through another channel on the opposite side of the membrane (see the Perspective by Kane). Science, this issue p. eaas9699, p. eaat4318; see also p. 600 The mechanism by which protons find a path through the key enzyme involved in plant energy generation is elucidated. 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 at a resolution of 2.9 Å (cF1) to 3.4 Å (cFo). In the cF1 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 cF1 to cFo. 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 cF1Fo 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 the chloroplast ATP synthase. Subunits α and β contain the nucleotide-binding sites with resolved nucleotides, Mg2+, and water molecules. Subunit δ joins cF1 to the membrane-embedded motor via the peripheral stalk (b, b′) that positions subunit a against the rotor ring. The electrochemical proton gradient drives ring rotation (arrow). The central stalk (γε) transmits torque to cF1. The redox regulator blocks rotation in the dark. The chloroplast adenosine triphosphate (ATP) synthase uses the electrochemical proton gradient generated by photosynthesis to produce ATP, the energy currency of all cells. Protons conducted through the membrane-embedded Fo motor drive ATP synthesis in the F1 head by rotary catalysis. We determined the high-resolution structure of the complete cF1Fo complex by cryo–electron microscopy, resolving side chains of all 26 protein subunits, the five nucleotides in the F1 head, and the proton pathway to and from the rotor ring. The flexible peripheral stalk redistributes differences in torsional energy across three unequal steps in the rotation cycle. Plant ATP synthase is autoinhibited by a β-hairpin redox switch in subunit γ that blocks rotation in the dark.

Manganese, iron and sulfur K edge XAFS of promoted sulfated zirconia catalysts

Promoted sulfated zirconia samples were prepared by the incipient wetness technique to produce isomerization catalysts which were active for the conversion of n-butane to isobutane at 338K (up to 10% conversion of 1% n-butane, 1atm., 0.25 h(-1) WHSV). The local structure of Fe and Mn in promoted sulfated zirconia was investigated using fluorescence yield XAS. Spectra were taken of calcined samples, activated samples, and samples after reaction with n-butane (maximum activity and deactivated). Factor analysis reveals that the Mn K edge XANES can be described by a linear combination of the spectra of two separate components, and that the ratio of these components changes with activation of the catalyst, and during use in the n-butane isomerization reaction. The change in ratio of the Mn species during activation and reaction results in a reduction of the average Mn valence from 2.4 to 2.2. The Fe K edge XANES was not similarly affected by activation and reaction with n-butane.

Outer Membrane Proteins

The outer membrane is considered to be a barrier in Gram-negative bacteria. However, bacteria are not autonomous and have to communicate in many ways with their surroundings. This interaction ranges from their association to surfaces, uptake of solutes, and sensing of environmental conditions to export of solutes into attacked cells. To perform these various functions, the outer membrane hosts many proteins. The protein family initially discovered was named porins, as they form pores for the uptake of solutes. They were considered to be unspecific for small solutes and thereby gave an impression of a molecular sieve behavior of the outer membrane. Research in recent decades, however, challenged this opinion, as several solute-specific porins were discovered. As these proteins are unique with respect to their structure, we first will introduce the structural concept of the outer membrane proteome. Subsequently, the classical porins and solute-specific porins will be described. Within this chapter, we aim to explain their importance for cell function and consequently will focus in an separate chapter on their regulation by small noncoding RNAs. Recently, the outer membrane proteins involved in outer membrane biogenesis have moved into the focus of investigations. Here, proteins involved in the biogenesis of the lipid and lipopolysaccharide layer and of the outer membrane proteins are studied. Finally, the export (secretion) systems will be compared. As the range of outer membrane proteins is too large to be covered fully, the reader will find general concepts of the function of outer membrane proteins, which are discussed in selected examples.

ATP Synthase Diseases of Mitochondrial Genetic Origin

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.

Silicon Photomultiplier Research and Development Studies for the Large Size Telescope of the Cherenkov Telescope Array

Riccardo Rando∗ab, Daniele Cortia, Francesco Dazzic, Alessandro De Angelisd, Antonios Dettlaffc, Daniela Dornere, David Finkc, Nadia Fouque f , Felix Grundnerc, Werner Habererc, Alexander Hahnc, Richard Hermel f , Samo Korparg, Gasper Kukec Mezekh, Ronald Maierc, Christian Maneaa, Mose Mariottiab, Daniel Mazinci, Fatima Mehrez f , Razmik Mirzoyanc, Sergey Podkladkinc, Ignasi Reichardta, Wolfgang Rhode j, Sylvie Rosier f , Cornelia Schultzab, Carlo Stellak, Masahiro Teshimaci, Holger Wetteskindc and Marko Zavrtanikgh for the CTA Consortium† E-mail: riccardo.rando@pd.infn.it a INFN Section of Padova, Italy; b University of Padova, Italy; c Max Planck Institute for Physics, Munich, Germany; d LIP/IST Lisboa, Portugal; e University of Wurzburg, Germany; f LAPP, CNRS-IN2P3, Annecy, France; g Jožef Stefan Institute, Ljubljana, Slovenia; h University of Nova Gorica, Slovenia; i University of Tokyo and ICRR, Tokyo, Japan; j Technische Universitat, Dortmund, Germany; k University of Udine and INFN, Italy. The Cherenkov Telescope Array (CTA) is the the next generation facility of imaging atmospheric Cherenkov telescopes; two sites will cover both hemispheres. CTA will reach unprecedented sensitivity, energy and angular resolution in very-high-energy gamma-ray astronomy. Each CTA array will include four Large Size Telescopes (LSTs), designed to cover the low-energy range of the CTA sensitivity (∼20 GeV to 200 GeV). In the baseline LST design, the focal-plane camera will be instrumented with 265 photodetector clusters; each will include seven photomultiplier tubes (PMTs), with an entrance window of 1.5 inches in diameter. The PMT design is based on mature and reliable technology. Recently, silicon photomultipliers (SiPMs) are emerging as a competitor. Currently, SiPMs have advantages (e.g. lower operating voltage and tolerance to high illumination levels) and disadvantages (e.g. higher capacitance and cross talk rates), but this technology is still young and rapidly evolving. SiPM technology has a strong potential to become superior to the PMT one in terms of photon detection efficiency and price per square mm of detector area. While the advantage of SiPMs has been proven for high-density, small size cameras, it is yet to be demonstrated for large area cameras such as the one of the LST. We are working to develop a SiPM-based module for the LST camera, in view of a possible camera upgrade. We will describe the solutions we are exploring in order to balance a competitive performance with a minimal impact on the overall LST camera design. The 34th International Cosmic Ray Conference, 30 July6 August, 2015 The Hague, The Netherlands