9 research outputs found
Strukturelle und funktionelle Untersuchungen am Cytochrom-bc1-Komplex zur Klärung des molekularen Wirkungsmechanismus
The cytochrome bc1 complex is a cornerstone in bioenergetic electron transfer chains, where it carries out tasks as diverse as respiration, photosynthesis, and nitrogen fixation. This homodimeric multisubunit membrane protein has been studied extensively for several decades and the enzyme mechanism is described with the modified protonmotive Q cycle. Still, the molecular and kinetic description of the catalytic cycle is not complete and questions remain regarding the bifurcation of electron transfer at the quinol oxidation (Qo) site, substrate occupancy, pathways of proton conduction, and the nature of the Rieske protein domain movement. We used competitive inhibitors to study the molecular architecture at the Qo site with X-ray crystallography. The structure of the enzyme with the substrate analog 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole (HHDBT) bound at the Qo site was determined at 2.5 Å resolution. Spectroscopic studies showed that HHDBT is negatively charged when bound at the active site. Mechanistic interpretations from inhibitor binding are in line with single occupancy model for quinol oxidation and structural analysis supports the proposed proton transfer pathway. For functional insight into the enzyme mechanism, redox-sensitive protonation changes were studied by Fourier transform infrared spectroscopy. The protein purification procedure was optimized for less delipidation and the isolated enzyme was more active. Furthermore, two new phospholipids were identified in the X-ray structures, including a cardiolipin. Strikingly, conserved lipid binding cavities were observed in structural comparison with homologous enzymes. The functional role of tightly bound phospholipids will be discussed. Finally, the Qo site is a target for various compounds of agricultural and pharmaceutical importance. Importantly, the X-ray structures permit detailed analysis of the molecular reasons for acquired resistance to and treatment failure of Qo site inhibitors, such as atovaquone, that is used to treat malaria and pneumonia, as discussed herein.Der Cytochrom-bc1-Komplex ist eine essentielle Komponente von Elektronen-transportketten in Eukaryoten und Prokaryoten, wo er an verschiedenen bioenergetischen Prozessen wie der Atmungskette, der Photosynthese und der Stickstoffixierung beteiligt ist. Der Mechanismus dieses Enzyms ist in seinen Grundzügen charakterisiert und mit dem Model des modifizierten Q-Zyklus beschrieben. Jedoch sind Regulationsprozesse und insbesondere der molekulare Mechanismus der Ubichinoloxidation immer noch unverstanden. Um den molekularen Mechanismus der Katalyse in der Qo-Bindungsstelle zu untersuchen, wurde der Bindungsmodus des Qo-Inhibitors HHDBT mittels der Röntgenkristallographie bestimmt. Diese 2.5 Å Kristallstruktur erlaubte wichtige Rückschlüsse auf den Katalysemechanismus. Vorherigen kristallographischen Arbeiten am UHDBT-Inhibitorkomplex waren nicht erfolgreich und führten nicht zu einer hochaufgelösten Struktur. Unter Verwendung von Heptyl-HDBT, das eine kürzere Alkyl-Kettenlänge aufweist, wurden erfolgreich dreidimensionale Kristalle des Komplexes gezüchtet. Dieses Hydroxychinon bindet in seiner ionisierten Form, entgegen vorherigen Behauptungen, dass eine ionisierte Verbindung nicht in der katalytischen Substrattasche binden könne. Demnach weisen unsere Studien darauf hin, dass die pH-Abhängigkeit der alkyl-HDBT Inhibierung durch eine dissoziierbare Gruppe im Komplex, vermutlich den Liganden des [Fe-2S]-Zentrums His181, bewirkt wird und nicht durch eine Verringerung der Inhibitoreffizienz durch Deprotonierung der Hydroxylgruppe. Die strukturelle Ähnlichkeit zu Ubichinol und die kompetitive Hemmung der Qo-Bindungsstelle erlauben es, HHDBT als Substratanalogon zu betrachten. Die Konformationsänderungen an der Bindungsstelle unterstützen den zuvor postulierten Protonentransferweg und offenbaren die Plastizität der katalytischen Bindungsstelle. Aus dem beobachteten Bindungsmodus des Hydroxychinon-Anions und dem Protonentransferweg wurde ein katalytischer Mechanismus abgeleitet, der sich mit dem Modell einer einfachen Besetzung der Ubichinol-Oxidationstasche in Einklang bringen lässt. Um das vorgeschlagene mechanistische Modell zu überprüfen, wurde die FTIR- Spektroskopie eingesetzt. Diese Studie zeigte zum ersten Mal, dass die Bindung der Inhibitoren HHDBT und Stigmatellin den Protonierungszustand und/oder die Orientierung von sauren Seitenketten beeinflusst. Des weiteren wurde die Interaktion der Stigmatellin Carbonylgruppe mit dem Enzym nachgewiesen. Die Röntgenkristallstrukturen der Hefe Cytochrom-bc1-Komplexe mit gebundenen HHDBT oder Stigmatellin in der Qo-Bindungsstelle sind wertvoll um die molekularen Ursachen menschlicher Krankheiten zu verstehen, die auf Mutationen im Cytochrom b zurückzuführen sind. Ebenfalls von großer Bedeutung sind diese Strukturen für die gezielte Entwicklung von Pestiziden oder pharmazeutischen Wirkstoffen, die gegen das aktive Zentrum der Komplexe in Parasiten oder Pilzen gerichtet sind. Zielorganismen entwickeln oft eine Resistenz gegenüber diesen Wirkstoffen. Bei einem Vergleich von Cytochrom-bc1-Komplex Strukturen von Hefe und Rind, mit verschiedenem Besetzungsgrad der Chinon-Bindungsstellen wurde die Plastizität der Qo-Bindungsstelle beobachtet. Basierend auf dem HHDBT Bindungsmodus in der Qo-Bindungsstelle wurde die Interaktion mit z.b. dem Antimalariawirkstoff Atovaquone modelliert. Die strukturellen Grundlagen von dessen Spezies-spezifischer Wirkung können so diskutiert werden. Ein weiteres Ergebnis der Doktorarbeit war die Optimierung der Proteinreinigung im Hinblick auf eine geringere Delipidierung der Proteinprobe und eine vergrößerte Kapazität. Dabei wurde die Ausbeute verbessert sowie die spezifische Enzymaktivität des gereinigten Proteins erhöht. So konnten zwei neue Phospholipidmoleküle reproduzierbar in unterschiedlichen Datensätzen identifiziert werden. Eine Analyse des Bindungsmodus von fest gebundenen Lipiden in Membranproteinstrukturen wurde durchgeführt. Es wurden hierbei spezifische Bindungsmuster für die Stabilisierung der Interaktionen zwischen den Phosphodiestergruppen und den Seitenketten der Aminosäurereste identifiziert. Eine bevorzugte Stabilisierung der Phospholipide auf der negativen Seite der Membranen wurde beobachtet. Die in der Kristallstruktur identifizierten, spezifisch gebundenen Phospholipide, die an Interaktionsflächen von Proteinuntereinheiten auftreten, weisen auf eine wichtige Rolle für die strukturelle Integrität des Komplexes hin. Eine vergleichende Analyse der Lipidbindungsstellen in Strukturen homologer Proteine wurde durchgeführt und zeigte, dass sie Spezies-übergreifend konserviert sind. Die Röntgenstrukturen des Cytochrom-bc1-Komplexes bieten die Grundlage für detaillierte Struktur-Funktions-Studien zur Beschreibung der molekularen katalytischen Vorgänge. Sie fördern aufregende Entdeckungen für das tiefere Verständnis der fundamentalen Prozesse des Lebens, welche durch diesen Typ der Enzyme katalysiert werden
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3D Ultrastructure of the Cochlear Outer Hair Cell Lateral Wall Revealed By Electron Tomography.
Outer Hair Cells (OHCs) in the mammalian cochlea display a unique type of voltage-induced mechanical movement termed electromotility, which amplifies auditory signals and contributes to the sensitivity and frequency selectivity of mammalian hearing. Electromotility occurs in the OHC lateral wall, but it is not fully understood how the supramolecular architecture of the lateral wall enables this unique form of cellular motility. Employing electron tomography of high-pressure frozen and freeze-substituted OHCs, we visualized the 3D structure and organization of the membrane and cytoskeletal components of the OHC lateral wall. The subsurface cisterna (SSC) is a highly prominent feature, and we report that the SSC membranes and lumen possess hexagonally ordered arrays of particles. We also find the SSC is tightly connected to adjacent actin filaments by short filamentous protein connections. Pillar proteins that join the plasma membrane to the cytoskeleton appear as variable structures considerably thinner than actin filaments and significantly more flexible than actin-SSC links. The structurally rich organization and rigidity of the SSC coupled with apparently weaker mechanical connections between the plasma membrane (PM) and cytoskeleton reveal that the membrane-cytoskeletal architecture of the OHC lateral wall is more complex than previously appreciated. These observations are important for our understanding of OHC mechanics and need to be considered in computational models of OHC electromotility that incorporate subcellular features
Structure-function relationships in the cytochrome bc1 complex from Saccharomyces cerevisiae
Lipids in Membrane Protein Structures
AbstractThis review describes the recent knowledge about tightly bound lipids in membrane protein structures and deduces general principles of the binding interactions. Bound lipids are grouped in annular, nonannular, and integral protein lipids. The importance of lipid binding for vertical positioning and tight integration of proteins in the membrane, for assembly and stabilization of oligomeric and multisubunit complexes, for supercomplexes, as well as their functional roles are pointed out. Lipid binding is stabilized by multiple noncovalent interactions from protein residues to lipid head groups and hydrophobic tails. Based on analysis of lipids with refined head groups in membrane protein structures, distinct motifs were identified for stabilizing interactions between the phosphodiester moieties and side chains of amino acid residues. Differences between binding at the electropositive and electronegative membrane side, as well as a preferential binding to the latter, are observed. A first attempt to identify lipid head group specific binding motifs is made. A newly identified cardiolipin binding site in the yeast cytochrome bc1 complex is described. Assignment of unsaturated lipid chains and evolutionary aspects of lipid binding are discussed
Protonmotive pathways and mechanisms in the cytochrome bc1 complex
AbstractThe cytochrome bc1 complex catalyzes electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane. In the Q cycle mechanism proton translocation is the net result of topographically segregated reduction of quinone and reoxidation of quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the quinol. The linkage of proton chemistry to electron transfer during quinol oxidation and quinone reduction requires pathways for moving protons to and from the aqueous phase and the hydrophobic environment in which the quinol and quinone redox reactions occur. Crystal structures of the mitochondrial cytochrome bc1 complexes in various conformations allow insight into possible proton conduction pathways. In this review we discuss pathways for proton conduction linked to ubiquinone redox reactions with particular reference to recently determined structures of the yeast bc1 complex
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3D Ultrastructure of the Cochlear Outer Hair Cell Lateral Wall Revealed By Electron Tomography.
Outer Hair Cells (OHCs) in the mammalian cochlea display a unique type of voltage-induced mechanical movement termed electromotility, which amplifies auditory signals and contributes to the sensitivity and frequency selectivity of mammalian hearing. Electromotility occurs in the OHC lateral wall, but it is not fully understood how the supramolecular architecture of the lateral wall enables this unique form of cellular motility. Employing electron tomography of high-pressure frozen and freeze-substituted OHCs, we visualized the 3D structure and organization of the membrane and cytoskeletal components of the OHC lateral wall. The subsurface cisterna (SSC) is a highly prominent feature, and we report that the SSC membranes and lumen possess hexagonally ordered arrays of particles. We also find the SSC is tightly connected to adjacent actin filaments by short filamentous protein connections. Pillar proteins that join the plasma membrane to the cytoskeleton appear as variable structures considerably thinner than actin filaments and significantly more flexible than actin-SSC links. The structurally rich organization and rigidity of the SSC coupled with apparently weaker mechanical connections between the plasma membrane (PM) and cytoskeleton reveal that the membrane-cytoskeletal architecture of the OHC lateral wall is more complex than previously appreciated. These observations are important for our understanding of OHC mechanics and need to be considered in computational models of OHC electromotility that incorporate subcellular features
Three-Dimensional Macromolecular Organization of Cryofixed Myxococcus xanthus Biofilms as Revealed by Electron Microscopic Tomography ▿ †
Despite the fact that most bacteria grow in biofilms in natural and pathogenic ecosystems, very little is known about the ultrastructure of their component cells or about the details of their community architecture. We used high-pressure freezing and freeze-substitution to minimize the artifacts of chemical fixation, sample aggregation, and sample extraction. As a further innovation we have, for the first time in biofilm research, used electron tomography and three-dimensional (3D) visualization to better resolve the macromolecular 3D ultrastructure of a biofilm. This combination of superb specimen preparation and greatly improved resolution in the z axis has opened a window in studies of Myxococcus xanthus cell ultrastructure and biofilm community architecture. New structural information on the chromatin body, cytoplasmic organization, membrane apposition between adjacent cells, and structure and distribution of pili and vesicles in the biofilm matrix is presented
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Deep nuclear invaginations are linked to cytoskeletal filaments - integrated bioimaging of epithelial cells in 3D culture.
The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growth-arrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect to the outer nuclear envelope. The cytoskeleton is also connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues