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Mitochondria perform essential energetic, metabolic and signalling functions within the cell. To fulfil these, the integrity of the mitochondrial proteome has to be preserved. Therefore, each mitochondrial subcompartment harbours its own system for protein quality control. However, if the capacity of mitochondrial chaperones and proteases is overloaded, mitochondrial misfolding stress (MMS) occurs. Upon this stress condition, mitochondria communicate with the nucleus to increase the transcription of nuclear encoded mitochondrial chaperones and proteases. This proteotoxic stress pathway was termed the mitochondrial unfolded protein response (UPRmt) aiming at restoring protein homeostasis. Despite being discovered over 25 years ago, the signalling molecules released by stressed mitochondria as well as the corresponding receptor and transcription factor remain poorly understood. With this study, we aimed at characterising the underlying signalling events and mechanisms of how mitochondria react to misfolded proteins. First, we aimed to establish different methods to induce MMS that triggers the transcriptional induction of mitochondrial chaperones and proteases detected by quantitative polymerase chain reaction. We were able to induce UPRmt signalling by overexpression of an aggregation-prone protein and by knock-down or inhibition of mitochondrial protein quality control components. To study the signalling in a time-resolved manner, we focused on the usage of the mitochondrial HSP90 inhibitor GTPP and the mitochondrial LONP1 protease inhibitor CDDO.
Early time point RNA sequencing analysis of cells stressed with GTPP or CDDO revealed upregulated genes in response to oxidative stress. Indeed, measurements of mitochondrial superoxide with the fluorescent dye MitoSOX showed increased levels of reactive oxygen species (ROS) upon MMS induction. In contrast, there was no induction of mitochondrial chaperones and proteases when combining MMS with antioxidants. Compartment-specific targeting of the hydrogen peroxide sensor HyPer7 revealed increased ROS levels in the intermembrane space and matrix of mitochondria, followed by elevated ROS levels in the cytosol at later time points. The importance of cytosolic ROS for the signalling was supported by preventing UPRmt induction with an inhibitor blocking the outer mitochondrial membrane pore. Thus, ROS were identified as an essential UPRmt signal.
To understand which cytosolic factor is modified by ROS, redox proteomics was performed. Here, reversible changes on cysteine residues of the HSP40 co-chaperone DNAJA1 were observed upon MMS. Consequently, transcriptional induction of UPRmt genes was abolished by DNAJA1 knock-down. To understand the function of DNAJA1 during UPRmt signalling, quantitative interaction proteomics upon MMS revealed an increased binding to mitochondrial proteins and its interaction partner HSP70. Immunoprecipitation confirmed a ROS-dependent interaction between HSP40 and HSP70. Increased binding to mitochondrial proteins represented a cytosolic interaction of DNAJA1 with mitochondrial precursor proteins, whose accumulation was confirmed by western blot. Moreover, a fluorescent protein targeted to mitochondria accumulated in the cytosol during GTPP treatment, confirming a reduced import efficiency upon MMS. Preventing the accumulation of precursors by a translation inhibitor or depletion of a general mitochondrial transcription factor resulted in reduced UPRmt activation. Thus, DNAJA1 is essential for UPRmt signalling, since its oxidation by mitochondrial ROS and its enhanced recruitment to mitochondrial precursors allows the integration of both MMS-induced signals.
To link these findings to an increased transcription of mitochondrial chaperones and proteases, we screened for transcription factors accumulating in the nucleus upon MMS by cellular fractionation mass spectrometry. We demonstrated that specifically HSF1 accumulates in nuclei of cells stressed with GTPP or CDDO. Depletion of HSF1 by knock-down or knock-out resulted in the abrogation of the UPRmt-specific transcriptional response. HSF1 activation was visualised by nuclear accumulation on western blot, a process inhibited by ROS and precursor suppression. Moreover, DNAJA1 depletion prevented HSF1 activation. Ultimately, we proved by immunoprecipitation that the inhibitory interaction between HSF1 and HSP70 is reduced upon MMS.
Thus, we conclude that MMS increases mitochondrial ROS that are released into the cytosol. In addition, the import efficiency is reduced upon MMS, resulting in the accumulation of non-imported mitochondrial precursor proteins in the cytosol. Both signals are recognised via DNAJA1 oxidation and substrate binding. The concurrent recruitment of HSP70 to DNAJA1 results in the loss of the inhibitory HSP70-HSF1 interaction. Thus, active HSF1 can migrate to the nucleus to initiate transcription of mitochondrial chaperones and proteases. These findings are in accordance with observations in yeast, where mistargeted mitochondrial proteins activate cellular stress responses. Our results highlight a surprising interconnection and dependence of the mitochondrial and the cytosolic proteostasis network, in which the UPRmt is activated by a combination of two mitochondria-specific proteotoxic stress signals.
In the course of systematic investigations on sila-substituted parasympatholytics the diphenyl(2-aminoethoxymethyl)silanols 3b and 4b (and its carbon analogue 4a) were synthesized and characterized by their physical and chemical properties. In the solid state 4a and 4b form strong O-H---N hydrogen bonds, which are intramolecular (4a) and intermolecular (4b), respectively. 4a and 4b were found to be weak antimuscarinic agents (4b >4a) and strong papaverine-like spasmolytics (4a ≈4b).
Receptor tyrosine kinases (RTKs) orchestrate cell motility and differentiation. Deregulated RTKs may promote cancer and are prime targets for specific inhibitors. Increasing evidence indicates that resistance to inhibitor treatment involves receptor cross-interactions circumventing inhibition of one RTK by activating alternative signaling pathways. Here, we used single-molecule super-resolution microscopy to simultaneously visualize single MET and epidermal growth factor receptor (EGFR) clusters in two cancer cell lines, HeLa and BT-20, in fixed and living cells. We found heteromeric receptor clusters of EGFR and MET in both cell types, promoted by ligand activation. Single-protein tracking experiments in living cells revealed that both MET and EGFR respond to their cognate as well as non-cognate ligands by slower diffusion. In summary, for the first time, we present static as well as dynamic evidence of the presence of heteromeric clusters of MET and EGFR on the cell membrane that correlates with the relative surface expression levels of the two receptors
The transient receptor potential (TRP) ankyrin type 1 (TRPA1) channel is highly expressed in a subset of sensory neurons where it acts as an essential detector of painful stimuli. However, the mechanisms that control the activity of sensory neurons upon TRPA1 activation remain poorly understood. Here, using in situ hybridization and immunostaining, we found TRPA1 to be extensively co-localized with the potassium channel Slack (KNa1.1, Slo2.2, or Kcnt1) in sensory neurons. Mice lacking Slack globally (Slack−/−) or conditionally in sensory neurons (SNS-Slack−/−) demonstrated increased pain behavior after intraplantar injection of the TRPA1 activator allyl isothiocyanate. By contrast, pain behavior induced by the TRP vanilloid 1 (TRPV1) activator capsaicin was normal in Slack-deficient mice. Patch-clamp recordings in sensory neurons and in a HEK cell line transfected with TRPA1 and Slack revealed that Slack-dependent potassium currents (IKS) are modulated in a TRPA1-dependent manner. Taken together, our findings highlight Slack as a modulator of TRPA1-mediated, but not TRPV1-mediated, activation of sensory neurons.
Keywords: TRPA1; slack; dorsal root ganglia; pain; mice
Natural science is only just beginning to understand the complex processes surrounding transcription. Epitranscriptional regulation is in large parts conveyed by transcription factors (TFs) and two recently discovered small RNA (smRNA) species: microRNAs (miRNAs) and transfer RNA fragments (tRFs). As opposed to the fairly well-characterised function of TFs in shaping the phenotype of the cell, the effects and mechanism of action of smRNA species is less well understood. In particular, the multi-levelled combinatorial interaction (many-to-many) of smRNAs presents new challenges to molecular biology. This dissertation contributes to the study of smRNA dynamics in mammalian cells in several ways, which are presented in three main chapters.
I) The exhaustive analysis of the many-to-many network of smRNA regulation is reliant on bioinformatic support. Here, I describe the development of an integrative database capable of fast and efficient computation of complex multi-levelled transcriptional interactions, named miRNeo. This infrastructure is then applied to two use cases. II) To elucidate smRNA dynamics of cholinergic systems and their relevance to psychiatric disease, an integrative transcriptomics analysis is performed on patient brain sample data, single-cell sequencing data, and two closely related in vitro human cholinergic cellular models reflecting male and female phenotypes. III) The dynamics between small and large RNA transcripts in the blood of stroke victims are analysed via a combination of sequencing, analysis of sorted blood cell populations, and bioinformatic methods based on the miRNeo infrastructure. Particularly, importance and practicality of smRNA:TF:gene feedforward loops are assessed.
In both analytic scenarios, I identify the most pertinent regulators of disease-relevant processes and biological pathways implicated in either pathogenesis or responses to the disease. While the examples described in chapters three and four of this dissertation are disease-specific applications of miRNeo, the database and methods described have been developed to be applicable to the whole genome and all known smRNAs.
Herein, we present a multi-cycle chemoenzymatic synthesis of modified RNA with simplified solid-phase handling to overcome size limitations of RNA synthesis. It combines the advantages of classical chemical solid-phase synthesis and enzymatic synthesis using magnetic streptavidin beads and biotinylated RNA. Successful introduction of light-controllable RNA nucleotides into the tRNAMet sequence was confirmed by gel electrophoresis and mass spectrometry. The methods tolerate modifications in the RNA phosphodiester backbone and allow introductions of photocaged and photoswitchable nucleotides as well as photocleavable strand breaks and fluorophores.
The potential of a protein-engineered His tag to immobilize macromolecules in a predictable orientation at metal-chelating lipid interfaces was investigated using recombinant 20 S proteasomes His-tagged in various positions. Electron micrographs demonstrated that the orientation of proteasomes bound to chelating lipid films could be controlled via the location of their His tags: proteasomes His-tagged at their sides displayed exclusively side-on views, while proteasomes His-tagged at their ends displayed exclusively end-on views. The activity of proteasomes immobilized at chelating lipid interfaces was well preserved. In solution, His-tagged proteasomes hydrolyzed casein at rates comparable with wild-type proteasomes, unless the His tags were located in the vicinity of the N termini of α-subunits. The N termini of α-subunits might partly occlude the entrance channel in α-rings through which substrates enter the proteasome for subsequent degradation. A combination of electron micrographs and atomic force microscope topographs revealed a propensity of vertically oriented proteasomes to crystallize in two dimensions on fluid lipid films. The oriented immobilization of His-tagged proteins at biocompatible lipid interfaces will assist structural studies as well as the investigation of biomolecular interaction via a wide variety of surface-sensitive techniques including single-molecule analysis.
Ein Hauptziel dieser Arbeit war die spektroskopische Charakterisierung einer neuartigen photolabilen Schutzgruppe (Photocage). Diese besteht aus dem weitverbreiteten (7-Diethylaminocumarin)methyl (DEACM), welches zusätzlich mit einer Art Antenne (ATTO 390) ausgestattet ist. Letztere soll die Zwei-Photonen-Absorption (2PA) erleichtern, was neben dem Energietransfer von der Antenne zur photolabilen Schutzgruppe sowie die Freisetzungsreaktion eines gebundenen Effektormoleküls untersucht wurde. Der Nachweis der erhöhten 2PA wurde durch Zwei-Photonen-induzierte Fluoreszenz erbracht, welche die Bestimmung des Zwei-Photonen-Einfangquerschnitts ermöglicht. Die 2PA wurde durch Messungen mit variierender Anregungsenergie an Rhodamin B und dem neuartigen Antennen-Photocage-System bestätigt, welche eine fast perfekte quadratische Abhängigkeit der Fluoreszenzintensität nach vorangegangener 2PA widerspiegelten. Die Werte des Zwei-Photonen-Einfangquerschnitts der neuartigen photolabilen Schutzgruppe sind über alle Wellenlängen hinweg größer als die von DEACM-OH. Der Beweis eines intramolekularen Energietransfers von der Antenne zu DEACM erfolgte durch transiente Absorptionsspektroskopie. Hierfür wurde der Photocage mit 365nm angeregt, was überwiegend die Antenne adressiert. Ein intramolekularer Energietransfer konnte mit einer Zeitkonstante von 20 ps beobachtet werden, welcher wahrscheinlich von einem nachgelagerten Ladungstransfer von DEACM auf ATTO 390 begleitet wurde. Die Funktionalität des neuartigen Photocages wurde durch Aufnahme von Absorptionsspektren im IR-Bereich während kontinuierlicher Belichtung bei 365 nm untersucht. Hierbei konnte die Entstehung der intensiven Absorption von Kohlendioxid aufgrund der Photodecarboxylierung detektiert werden. Absorptionsänderungen während kontinuierlicher Belichtung wurden ebenfalls im UV/Vis-Bereich detektiert, in welchen eine hypsochrome Verschiebung der langwelligen Absorptionsbande sowie ein Anstieg der Absorption festgestellt wurden. Hieraus konnte eine Quantenausbeute der Freisetzungsreaktion von 1,5% ermittelt werden. Die Ergebnisse zum Antennen-Photocage-System zeigen auf, dass durch Anbringen einer Antenne die 2PA verbessert werden kann, ohne die Funktionalität des Freisetzungsprozesses negativ zu beeinflussen. In einem nächsten Schritt zielen Verbesserungen des untersuchten Photocages darauf ab, den Ladungstransfer zu unterdrücken. Die Validierung dieses Ansatzes sollte die Einführung anderer Antennen mit erhöhten Zwei-Photonen-Einfangquerschnitten, wie z.B. Quantenpunkte, weiter motivieren. Der zweite Ergebnisteil dieser Arbeit konzentriert sich auf drei verschiedene Photosysteme, die sich durch eine sehr kurzlebige Fluoreszenz auszeichnen, welche mit einem Kerrschalter aufgenommen wurde. Das erste der drei untersuchten Systeme umfasst eine kooperative BODIPY-DTE-Dyade(Bordipyrromethen-Dithienylethen), die einen hocheffizienten photochromen Förster-Resonanzenergietransfer aufweist. Dieser wurde durch verkürzte Lebenszeiten der Differenzsignale im transienten Absorptionsspektrum der Dyade im photostationären Zustand abgeleitet. In diesem stellt BODIPY-DTE eine hochkonjugierte Einheit dar, welches durch die geschlossene Form des photochromen DTEs einen Energietransfer vom photoangeregten BODIPY zum DTE ermöglicht. Bei diesem Prozess wird die Fluoreszenz des Donors um einige Größenordnungen reduziert. Die Ergebnisse der transienten Absorptionsmessung wurde durch ein zeitaufgelöstes Fluoreszenzexperimentbestätigt. Die detektierte Fluoreszenztransiente zerfällt mit einer Zeitkonstante von etwa 15 ps und weist somit sehr hohe Ähnlichkeit mit dem Signal des Grundzustandsbleichens (GSB) aus dem transienten Absorptionsexperiment auf. Des Weiteren wurde die photochrome Ringschlussreaktion eines wasserlöslichen Indolylfulgimids spektroskopisch charakterisiert. Transiente Absorptionsmessungen geben einen direkten Einblick in den Mechanismus der Reaktion, in welcher, nach Photoanregung, die Relaxation aus dem Franck-Condon Bereich und die schnelle biphasische Relaxation des Moleküls über die konische Durchschneidung abgeleitet werden kann. Zusätzlich wurden zeitaufgelöste Fluoreszenzmessungen mit Hilfe des Kerrschalters durchgeführt, da die stimulierte Emission (SE) in transienten Absorptionsmessungen durch die Überlagerung mehrerer Signale nicht vollständig zu erkennen war. Die globale Lebensdaueranalyse der mit dem Kerrschalter aufgenommenen Breitband-Fluoreszenz lieferte drei Zeitkonstanten, welche wesentliche Übereinstimmung mit den Zeitkonstanten aus der globalen Lebensdaueranalyse der transienten Absorptionsmessungen aufweisen. Schlussendlich wurde die Deaktivierung des elektronisch angeregten Zustands des flavinbindenden Dodecins aus Mycobacterium tuberculosis mit Hilfe von unterschiedlichen spektroskopischen Methoden charakterisiert. Stationäre Fluoreszenzmessungen bei unterschiedlichen pH-Werten zeigten bei pH 5 eine im Vergleich zu nahezu physiologischen Bedingungen (pH 7,5)reduzierte Fluoreszenz auf. Auffällig ist, dass diese Beobachtungen durch transiente Absorptionsmessungen nicht bestätigt werden konnten, da diese eine große Ähnlichkeit bezüglich der Dynamik und der spektralen Signatur zueinander besaßen. Ein negatives Signal, hervorgerufen durch die SE, wurde hierbei nicht gefunden. Allerdings konnte in den zerfallsassoziierten Spektren eine spektrale Signatur beobachtet werden, die auf eine SE hindeutete, welche allerdings mit größeren positiven Signalen überlagert ist. Dieser Aspekt wurde in einer Kerrschalter-Messung untersucht, in der eine schwache Emission bei pH 7,5 festgestellt werden konnte. Zusätzlich wies die Zerfallsdynamik der Emission Übereinstimmung mit dem GSB-Signal aus den transienten Absorptionsmessungen auf.
Die Steuerung biochemischer Prozesse oder die Verbesserung von Materialien erfordert zunächst ein tiefgründiges Verständnis über die zugrundeliegenden Systeme. Zur Untersuchung eignet sich Licht als ideales Werkzeug, da hiermit nützliche Informationen über die chemische Struktur, ihre Eigenschaften sowie den zusammenhängenden, schnellen Reaktionsabläufen erhalten werden können. Um die Aufklärung zu erleichtern können kleine, chemische Verbindungen eingeführt werden, welche beispielsweise ein Fluoreszenzmarker, eine photolabile Schutzgruppe oder eine photoschaltbare Verbindung sein können. Von jeweils einem Vertreter dieser Moleküle wurden unterschiedliche Studien durchgeführt, dessen Ergebnisse in dieser Arbeit in insgesamt drei Projekten zusammengefasst werden.
Zunächst wurde die Funktionalität der Helikase RhlB untersucht, die der Familie der DEAD-Box Proteine zugeordnet wird, und RNA-Duplexe in ihre Einzelstränge entwindet. Als RNA-Modellduplex diente JM2h, an dem ein RNA-Einzelstrang fluoreszenzmarkiert war (M2AP6). Die Einführung dieses Markers ermöglichte die Durchführung von statischen Fluoreszenzmessungen sowie von Mischexperimenten, die mit Hilfe der stopped-flow-Technik durchgeführt wurden. In den einleitenden Studien wurde die Helikase weggelassen, wodurch der Fokus auf den Fluoreszenzeigenschaften der RNA gelegt wurde. Die Ergebnisse hierzu zeigten, dass die Fluoreszenzintensität des Einzelstrangs durch Zugabe des komplementären Strangs deutlich abnimmt, wobei das Minimum bei einem äquimolaren Verhältnis erreicht wird. Die dazugehörigen stopped-flow-Messungen zeigten eine Beschleunigung der Hybridisierungsreaktion, wenn höhere Konzentrationen des Gegenstrangs in der Lösung vorhanden waren. Nach anschließender Zugabe der Helikase zur Lösung wurde ein Anstieg der Fluoreszenzintensität erwartet, der vom separierten Einzelstrang M2AP6 herrühren sollte. Dieser Anstieg wurde jedoch erst nach weiterer Zugabe von ATP beobachtet, der auf eine ATP-Abhängigkeit der Entwindungsreaktion von RhlB hindeutet. Diese Abhängigkeit wurde auch bereits für andere Helikasen der DEAD-Box Familie entdeckt. Die korrekte Funktionalität sowie die ATP-Abhängigkeit wurden in stopped-flow-Messungen verfiziert, bei denen der Fluoreszenzanstieg auch zeitaufgelöst betrachtet werden konnte. Für die spektralen Korrekturen der Fluoreszenzspektren wurde ein selbstgeschriebenes MATLAB-Programm namens FluCY verwendet (engl.: Fluorescence Correction & Quantum yield), welches eine schnelle und fehlerfreie Verarbeitung des Datensatzes ermöglichte.
Die zwei im folgenden beschriebenen Projekte handeln von photoaktivierbaren Molekülen. Zum einen photolabile Verbindungen, welche die Funktion z.B. eines Biomoleküls durch eine chemische Modifikation deaktivieren können. Durch eine lichtinduzierte Reaktion kommt es zur Abspaltung der Modifikation und die Funktion ist wiederhergestellt. In dieser Arbeit wurden verschiedene photolabile Schutzgruppen untersucht, die denselben Chromophor BIST (BIsStyryl-Thiophen) tragen. Durch die Einführung dieses Chromophors absorbierten sämtliche untersuchte Verbindungen sehr effizient sichtbares Licht (epsilon(445)=55.700 M^(-1) cm^(-1)), wodurch der photoinduzierte Bindungsbruch mit Wellenlängen durchgeführt werden, die bei einer biologischen Anwendungen keinen Schaden an der Zelle anrichten würden. Hieraufhin wurden in statischen und zeitaufgelösten Absorptionsmessungen Teilschritte der Freisetzungsreaktion untersucht, indem nach Photoanregung die Absorptionsänderungen auf verschiedenen Zeitskalen analysiert wurden. Die ultraschnelle Dynamik im Piko- bis Nanosekundenbereich (10^(-12)-10^(-9) s) wird durch eine spektral breite, positive Absorptionsänderng dominiert. Diese impliziert, dass die Deaktivierung über den Triplettpfad abläuft, der die vergleichsweise niedrigen Freisetzungsausbeuten erklärt (phi(u) < 5). Aufgrund des hohen Extinktionskoeffizienten reichen dennoch bereits niedrige Strahlungsdosen aus, um eine Freisetzung zu initiieren. Der geschwindigkeitsbestimmende Schritt dieser Reaktion ist dem Zerfall des aci-nitro Intermediats zugeordnet. Für ein sekundäres Amin, welches mit BIST geschützt wurde, ist eine Lebensdauer des Intermediats von 71 µs gefunden worden.
In einigen Fällen ist es erwünscht, eine vorliegende Aktivität nicht nur ein-, sondern auch ausschalten zu können, wofür photochrome Verbindungen (oder Photoschalter) verwendet werden. Die in dieser Arbeit untersuchte Verbindung ceCAM ist ein Alken-Photoschalter und vollführt bei Bestrahlung mit Licht eine cis/trans-Isomerisierung. ceCAM ist das Cyanoester-Derivat (ce) von Cumarin-substituierten Allylidenmalonat, von denen beide Konformere sehr effizient sichtbares Licht absorbieren trans: epsilon(489)=50.300 M^(-1) cm^(-1); cis: epsilon(437)=18.600 M^(-1) cm^(-1)). Andere photophysikalische Eigenschaften umfassen u.a. hohe thermische und photochemische Stabilität. Letztere wurde über ein Experiment nachgewiesen, bei dem die lichtinduzierte Isomerisierung alternierend durchgeführt wurde und selbst bei über 250 Zyklen keine signifikate Abnahme der Absorption beobachtet werden konnte. Des Weiteren konnte die Reaktion mit Quantenausbeuten von 39% (trans) und 42% (cis) induziert werden, wobei im photostationären Gleichgewicht auch hohe Isomerenverhältnisse mit bis zu 80% (trans) und 96% (cis) akkumuliert werden konnten. Die Geschwindigkeit der Reaktion wurde mit Hilfe der Ultakurzzeit-Spektroskopie untersucht. Die Dynamik im Zeitbereich von ps-ns zeigte, dass die trans/cis-Isomerisierung unterhalb von 0,5 ns und die umgekehrte Reaktion noch viel schneller (wenige ps) abgeschlossen ist. Durch die Untersuchungen in dieser Arbeit an den BIST-Verbindungen und ceCAM sind viele vorteilhafte, photophysikalische Eigenschaften charakterisiert worden, wodurch sie als verbesserte Alternative zu den bisher bekannten photolabilen Schutzgruppen oder Photoschaltern anzusehen sind.
Im Rahmen dieser vorliegenden Thesis wurden verschiedene photosensitive Systeme anhand statischer und zeitaufgelöster optischer Spektroskopiemethoden charakterisiert. Das Hauptaugenmerk dieser Arbeit lag in der Entwicklung und Untersuchung neuer Quantenpunkt-basierter Hybridsysteme. Es war möglich die optischen Eigenschaften der Quantenpunkte über Optimierung der Syntheseschritte zu variieren und so auf geplante Projekte anzupassen.
Im Projekt „Quantenpunkte als Zwei-Photonen Antenne“ sollten die hohen Zwei-Photonen Einfangquerschnitte von Quantenpunkten ausgenutzt werden um in Kombination mit einer photolabilen Schutzgruppe, ein Uncaging im NIR-Bereich zu realisieren. Es wurden ZnSe/ZnS Partikel synthetisiert, die eine starke Emission im Bereich der Absorption der Schutzgruppe zeigen. Anhand von zeitaufgelösten transienten Absorptionsexperimenten mit einer Anregungswellenlänge bei 775 nm wurde eine Zwei-Photonen Absorption der Partikel nachgewiesen. Jedoch wurden starke Emissionsbeiträge aus Fallenzuständen und eine geringe Stabilität beobachtet. Die Synthese von CdS/ZnS Quantenpunkten lieferte stabile Partikel mit geringer trap state Emission. Diese Partikel wurden in einem Modellhybridsystem als Energiedonoren eingesetzt. Als Akzeptor wurde der Farbstoff Cumarin343 gewählt. In statischen Absorptions- und Emissionsmessungen, zeitkorrelierten Einzelphotonenmessungen sowie in fs-zeitaufgelösten transiente Absorptionsmessungen konnte ein ultraschneller Energietransfer nach Ein-Photonen Anregung des Hybridsystems beobachtet werden. Über TPiF Messungen wurde die Zwei-Photonen Absorption der Quantenpunkte detektiert. Ein Energietransfer nach Zwei-Photonen Anregung der Quantenpunkte wurde beobachtet. Schließlich wurde ein Hybridsystem aus CdS/ZnS und der photolabilen Schutzgruppe Az-NDBF (Synthese im AK Heckel, Goethe Universität, Frankfurt a. M.) untersucht. Auch in diesem System wurde ein Energietransfer von Quantenpunkt auf die Schutzgruppe nach Ein- und Zwei-Photonen Anregung beobachtet. Anhand von TA Experimenten wurde eine Zeitkonstante von <100 ps für den Energietransfer nach Ein-Photonen Anregung ermittelt. Es konnte anhand der vorgestellten Resultate gezeigt werden, dass sich Quantenpunkte, aufgrund der guten Anpassung ihrer optischen Eigenschaften generell sehr gut als Antennen für organische Verbindungen eigenen.
Des Weiteren wurde ein Hybridsystem aus CdSe/ZnS Quantenpunkten und einer Dyade (Verbindung eines DTE Photoschalters und BODIPY Derivats), entworfen und charakterisiert. Ein ultraschneller EET von BODIPY auf den geschlossenen DTE Schalter wurde in vorangegangenen Studien beobachtet. Dieser EET führte zur Löschung der BODIPY-Emission. Sobald der Photoschalter im offenen Zustand vorliegt, findet aufgrund des fehlenden spektralen Überlapps kein EET statt und es wird die BODIPY-Emission detektiert. Die Erweiterung der Dyade um einen Quantenpunkt zeigte nach Anregung des Quantenpunkts dessen Fluoreszenzlöschung. Da die Emissionsbande der Quantenpunkte im Absorptionsbereich des BODIPY Farbstoffes liegt, konnte über statische und zeitaufgelöste Experimente ein ultraschneller EET von CdS/ZnS auf den Farbstoff ermittelt werden. Dies führte zu der Erweiterung des Anregungsspektrums des BODIPY Farbstoffs. Die Kopplung der Dyade an die Quantenpunktoberfläche lieferte eine Verbindung mit dem breiten Anregungsspekrum des Quantenpunkts und der schaltbaren Fluoreszenz der Dyade.
Das Hybridsystem aus CdSe Quantenpunkten und PDI zeigte vom Verhältnis der Quantenpunkte zu gekoppelten PDI Molekülen abhängige Fluoreszenzsignale. In TA Experimenten wurde ein ultraschneller EET ermittelt. Für hohe PDI Konzentrationen wurde ein weiterer EET von höher angeregten Elektronen auf das PDI identifiziert. Neben der EET Charakterisierung konnte ein zusätzlicher Prozess innerhalb des Hybridsystems mit hoher PDI Konzentration beobachtet werden. Auf den EET von Quantenpunkt auf PDI folgt ein ET aus dem Valenzband des Quantenpunkts in das HOMO des PDI*. In vorangegangene Arbeiten zu Hybridsystemen aus CdSe/ZnS und PDI wurde kein ET beobachtet. In dem beschriebenen Projekt konnte der Einfluss einer passivierenden Schale auf die elektronischen Eigenschaften von CdSe Quantenpunkten gezeigt werden.
Im letzten Teil dieser Thesis wurde die spektroskopische Charakterisierung einer NVOC und zweier NDBF Schutzgruppen beschrieben. Es konnten anhand statischer Absorptionsmessungen eine Freisetzungsquantenausbeute für NVOC-Adenin von 1,1 % ermittelt werden. Die Charakterisierung der Schutzgruppen mit einer NDBF Grundstruktur (DMA-NDBF und Az-NDBF) ergab eine Abhängigkeit der Freisetzungs- und Fluoreszenzausbeute von der Polarität des Lösungsmittels. In polarer Umgebung reduzierten sich die Quantenausbeuten deutlich...
In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT-containing diribonucleotides with native species in RNA hydrolysates by high-resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT-specific iodine-desulfurization, we disprove the existence of PTs in RNA from E. coli, S. cerevisiae, human cell lines, and mouse brain. Furthermore, we discuss how an MS artifact led to the initial misidentification of 2′-O-methylated diribonucleotides as RNA phosphorothioates. To aid structure validation of new nucleic acid modifications, we present a detailed guideline for MS analysis of RNA hydrolysates, emphasizing how the chosen RNA hydrolysis protocol can be a decisive factor in discovering and quantifying RNA modifications in biological samples.
Candida boidinii NAD+-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential applications in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of the wild-type CbFDH at cryogenic and ambient temperatures as well as Val120Thr mutant at cryogenic temperature determined at the Turkish Light Source "Turkish DeLight". The structures reveal new hydrogen bonds between Thr120 and water molecules in the mutant CbFDH's active site, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, our findings provide invaluable insights into future protein engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.
Candida boidinii NAD+-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential applications in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of the wild-type CbFDH at cryogenic and ambient temperatures as well as Val120Thr mutant at cryogenic temperature determined at the Turkish Light Source "Turkish DeLight". The structures reveal new hydrogen bonds between Thr120 and water molecules in the mutant CbFDH's active site, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, our findings provide invaluable insights into future protein engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.
The covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to proteins generates a signal that regulates transcription, response to cell stress, and differentiation. Ufmylation is initiated by ubiquitin-like modifier activating enzyme 5 (UBA5), which activates and transfers UFM1 to ubiquitin-fold modifier-conjugating enzyme 1 (UFC1). The details of the interaction between UFM1 and UBA5 required for UFM1 activation and its downstream transfer are however unclear. In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/γ-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.
The health status of every nucleated cell in the human body is monitored through peptides presented by major histocompatibility complex class I (MHC I) to T-cell receptors of CD8+ T-cells. Thereby, the adaptive immune system ensures the recognition and elimination of infected or cancerous cells. MHC I molecules comprise the polymorphic heavy chain (hc) and the light chain β2-microglobulin (β2m). More than 13,000 allomorphs of the MHC I hc have been identified. All MHC I hcs associate with β2m but differ in their binding preferences for peptides, ensuring the presentation of a large peptide pool. After maturation of MHC I hc/β2m heterodimers in the endoplasmic reticulum (ER), most of the peptide-deficient MHC I molecules are recruited to the peptide-loading complex (PLC). There, they go through peptide loading and editing before they are released as stable peptide-MHC I (pMHC I) complexes and traffic to the cell surface for antigen presentation.
During the stringent quality control of MHC I peptide loading and editing within the PLC, the chaperone tapasin in conjunction with the oxidoreductase ERp57 stabilizes peptide-receptive MHC I molecules and alters the peptide cargo for high immunogenicity by catalyzing peptide-exchange. The tapasin-homologue TAP-binding protein related (TAPBPR) is involved in downstream quality control, editing the peptide repertoire of MHC I molecules that slipped through peptide proofreading by tapasin. Both chaperones were shown to adopt similar binding-modes for MHC I, suggesting related mechanisms of peptide editing. Nevertheless, the MHC I specific chaperones operate in different subcellular locations with differing assistance. While TAPBPR mediates peptide-exchange solely in the peptide-poor environment of the cis-Golgi and ER-Golgi intermediate compartment (ERGIC), tapasin functions mainly within the PLC together with ERp57 and the lectin-like chaperone calreticulin. Calreticulin with its lectin-, arm- and C-terminal domain contacts the MHC I heterodimer, ERp57 and the C-terminal domain of tapasin, respectively. Notably, the interaction site between calreticulin and tapasin has not yet been elucidated experimentally at molecular detail. The depletion of tapasin leads to a compromised immune response and a change in the pool of peptide cargo. The numerous MHC I allomorphs vary in their plasticity and their dependence on tapasin for the loading of optimal peptides. Moreover, the conformational plasticity of MHC I correlates with their dependence on tapasin. However, the molecular basis on how tapasin edits the various MHC I allomorphs and the structural features that are essential for peptide exchange catalysis at atomic resolution remained elusive.
In the first part of this thesis, the trimeric complex of tapasin–ERp57/calreticulin was analyzed. To this end, laser induced liquid bead ionization mass spectrometry (LILBID-MS) was performed as part of a collaboration and revealed the trimeric assembly for tapasin–ERp57 and calreticulin. Furthermore, additional to a wildtype construct of calreticulin, a second construct, lacking the acidic helix of calreticulin that was found to come to close contact with tapasin, was utilized for isothermal titration calorimetry (ITC). A micromolar affinity of wildtype calreticulin to tapasin–ERp57 was determined. Previous biochemical and NMR studies utilizing the P-domain of calreticulin and solely ERp57 provided a micromolar affinity for the complex of calreticulin and ERp57. In this study, no interaction of calreticulin lacking the acidic helix with tapasin–ERp57 could be measured by ITC. However, these results undergo with findings that calreticulin lacking the acidic helix impairs the function of the PLC. Most likely, the negatively charged acidic helix is located in a groove of tapasin, carrying a more positive charge. Taken together, the functional data demonstrates the importance of the acidic helix of calreticulin for assembly of the trimeric subunit of calreticulin/tapasin–ERp57.
In the main part of this study an MHC I–tapasin–ERp57 complex was structurally analyzed. Therefore, a photo-triggered approach was chosen to assemble the transient complex of MHC I–tapasin–ERp57. Various allomorphs were screened for complex formation with the tapasin–ERp57 heterodimer after photocleavage by size exclusion chromatography (SEC), resulting in mouse MHC I H2-Db as the suited allomorph. Microseed matrix screening was performed. Crystals diffracting X-rays to a resolution of 2.7 Å were obtained showing one tetrameric tapasin–ERp57–MHC I complex per asymmetric unit.
The MHC I-chaperone structure shows molecular rearrangements upon MHC I engagement and unveils structural features of tapasin, involved in peptide-exchange catalysis...
Upon antibiotic stress Gram-negative pathogens deploy resistance-nodulation-cell division-type tripartite efflux pumps. These include a H+/drug antiporter module that recognizes structurally diverse substances, including antibiotics. Here, we show the 3.5 Å structure of subunit AdeB from the Acinetobacter baumannii AdeABC efflux pump solved by single-particle cryo-electron microscopy. The AdeB trimer adopts mainly a resting state with all protomers in a conformation devoid of transport channels or antibiotic binding sites. However, 10% of the protomers adopt a state where three transport channels lead to the closed substrate (deep) binding pocket. A comparison between drug binding of AdeB and Escherichia coli AcrB is made via activity analysis of 20 AdeB variants, selected on basis of side chain interactions with antibiotics observed in the AcrB periplasmic domain X-ray co-structures with fusidic acid (2.3 Å), doxycycline (2.1 Å) and levofloxacin (2.7 Å). AdeABC, compared to AcrAB-TolC, confers higher resistance to E. coli towards polyaromatic compounds and lower resistance towards antibiotic compounds.
The members of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) transporter superfamily mediate export of a wealth of molecules of physiological and pharmacological importance. According to the Transporter Classification Database (TCDB), the MOP superfamily is mainly categorized into six distantly related families functionally characterized families: the multidrug and toxic compound extrusion (MATE), the polysaccharide transporter (PST), the oligosaccharidyl-lipid flippase (OLF), the mouse virulence factor (MVF) the agrocin 84 antibiotic exporter (AgnG), and the progressive ankylosis (Ank) family. Among these, the multidrug resistance MATE family transporters are most ubiquitous, being present in all domains of life: Archaea, Bacteria and Eukarya. As secondary active transporters, they utilize transmembrane electrochemical ion gradients of Na+ and/or H+ in order to drive the efflux of xenobiotics or cytotoxic metabolic waste products with specificity mainly for polyaromatic and cationic substrates. Active efflux of drugs and toxic compounds carried out by multidrug transporters is one of the strategies developed by bacterial pathogens to confer multidrug resistance. MATE proteins provide resistance to, e.g., fluoroquinolone, aminoglycoside antibiotics, and anticancer chemotherapeutical agents, thus serving as promising pharmacological targets for tackling a severe global health issue. Based on their amino acid sequence similarity, the MATE family members are classified into the NorM, the DNA-damage-inducible protein F (DinF), and the eukaryotic subfamilies. Structural information on the alternate conformational states and knowledge of the detailed mechanism of the MATE transport are of great importance for the structure-aided drug design. Over the past decade, the crystal structures of representative members of the NorM, DinF and eukaryotic subfamilies have been presented. They all share similar overall architecture comprising 12 transmembrane helices (TMs) divided into two domains, the N-terminal domain (TMs 1-6) and the C-terminal domain (TMs 7-12), connected by a cytoplasmic loop between TM6 and TM7 (Fig. II.1). Since all available MATE family structures are known only in V-shaped outward-facing states with the central binding cavity open towards the extracellular side, a detailed understanding of the complete transport cycle has remained elusive. In order to elucidate the underlying steps of the MATE transport mechanism, structures of distinct intermediates, particularly inward-facing conformation, are required.In my PhD project, structural and functional studies have been performed on a MATE family (DinF subfamily) transporter, PfMATE, from the hyperthermophilic and anaerobic archaeon Pyrococcus furiosus. This protein was produced homologously in Pyrococcus furiosus as well as heterologously in Escherichia coli, and used for the subsequent purification and crystallization trials by the vapor diffusion (VD) and lipidic cubic phase (LCP) method. To the best of my knowledge, PfMATE is the first example of a successful homologous production of a membrane protein in P. furiosus. Due to the very low final amount of the purified protein from the native source, the heterologously produced PfMATE samples were typically used for the extensive structural studies. Crystal structures of PfMATE have been previously determined in an outward-facing conformation in two distinct states (bent and straight) defined on the arrangement of TM1. A pH dependent conformational transition of this helix regulated by the protonation state of the conserved aspartate residue Asp41 was proposed. However, it has been discussed controversially, leading to the hypothesis about TM1 bending to be rather affected by interactions with exogenous lipids (monoolein) present under the crystallization conditions. Based on these open questions, an experimental approach to investigate the role of lipids as structural and functional modulators of PfMATE has been taken in the course of my PhD project. The interplay between membrane proteins and lipids can affect membrane protein topology, structure and function. Considering differences between archaeal and bacterial lipid composition, cultivation of P. furiosus cells and extraction of its lipids was followed by the mass spectrometry (MS) based lipidomics for identification of individual lipid species in the archaeal extract. In order to assess the effects of lipids on PfMATE, different lipid molecules were used for co-purification and co-crystallization trials. This dissertation presents a workflow leading to the structure determination of a MATE transporter in the long sought-after inward-facing state, which has been achieved upon purification and crystallization of the heterologously produced PfMATE in the presence of lipids from its native source P. furiosus. Also, the PfMATE outward-facing state obtained from the crystals grown at the acidic pH conditions sheds light on the previously proposed pH-dependent structural alterations within TM1. It is interesting to note that the inward and outward-facing states of PfMATE were obtained from the crystals grown under similar conditions, but in the presence and absence of native lipids, respectively. This observation supports the hypothesis about physiologically relevant lipids to act as conformational modulators or/and a new class of substrates, expanding the substrate spectrum of the MATE family transporters. Comparative analysis of two PfMATE states reveals that transition from the outward to the inward-facing state involves rigid body movements of TMs 2-6 and 8-12 to form an inverted V, facilitated by a loose binding of TMs 1 and 7 to their respective bundles and their conformational flexibility. Local fluctuations within TM1 in the inward-facing structure, including bending and unwinding in the intracellular half of the helix, invoke its highly flexible nature, which is suitable for ion and substrate gating.
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The electron transport chain (ETC) is used by cells to create an electrochemical proton gradient which can be used by the ATP synthase to produce ATP. ETC, also called respiratory chain, is formed in mitochondria by four complexes (complex I-IV) and mediated by two electron carriers: cytochrome c and ubiquinone. Electrons are passed from one complex to another in a series of redox reactions coupling proton pumping from the negative (N) side of the membrane to the positive (P) side. Complex I can introduce electrons into the ETC by oxidizing NADH to NAD+ and reducing quinone (Q) to quinol (QH2). The process accomplishes pumping of four protons across the membrane. Complex II is another electrons entry point. It catalyzes the oxidation of succinate to fumarate while reducing Q to QH2. Complex III, also called cytochrome bc1 complex, can transfer the electrons from QH2 to cytochrome c and couple to proton pumping. In complex III the Q-cycle contributes four proton translocations: two protons are required for the reduction of one quinone to a quinol and two protons are released to the P side. Complex IV (cytochrome c oxidase), the terminal complex of the ETC, catalyzes the electron transfer to oxygen and pumps four protons to the P side. Structures of ETC complexes are available. However, the structure of a hyperthermophilic cytochrome bc1 complex has not been elucidated till now. Additionally, the dimeric crystal structure of cytochrome c oxidase from bovine has been discussed controversially.
To build up a functional complex, cofactors are required. The active site of A- and B-type cytochrome c oxidases contain the high spin heme a which is synthesized by the integral membrane protein heme A synthase (HAS). HAS can form homooligomeric complexes and its oligomerization is essential for the biological function of HAS. HAS is evolutionarily conserved among prokaryotes and eukaryotes. Despite its importance, little is known about the detailed structural properties of HAS oligomers.
During my PhD studies, I focused on the cytochrome c oxidase (AaCcO), the cytochrome bc1 complex (Aabc1) and the heme A synthase (AaHAS) from Aquifex aeolicus. This organism is one of the most hyperthermophilic ones and can live at extremely high temperatures, even up to 95 °C. Respiratory chain complexes provide energy for the metabolism of organisms, and their structures have been studied extensively in the past few years. However, there has been a lack of atomic structures of complexes from hyperthermophilic and ancient bacteria, so little is known about the mechanism of these macromolecular machines under hyperthermophilic conditions. Therefore, my PhD studies had four main objectives: 1) to structurally and functionally characterize AaCcO, 2) to reveal the mechanism of Aabc1 thermal stability based on its structure, 3) to determine the oligomerization of AaHAS, 4) to provide valuable insights into the relationship between function and oligomerization of AaHAS.
1) Structure of AaCcO
Heme-copper oxidases (HCOs) catalyze the oxygen reduction reaction being the terminal enzymes in the plasma membranes in many prokaryotes or of the aerobic respiratory chain in the inner mitochondrial membrane. By coupling this exothermic reaction to proton pumping across the membrane to the P side, they contribute to the establishment of an electrochemical proton gradient. The energy in the proton electrochemical proton gradient is used by the ATP synthase to generate ATP. HCOs are classified into three major families: A, B and C, based on phylogenetic comparisons. The well-studied aa3-type cytochrome c oxidase from Paracoccus denitrificans (P. denitrificans) represents A-family HCOs. So far, the only available structure of the ba3-type cytochrome c oxidase from Thermus thermophilus represents the B-family of HCOs. This family contains a number of bacterial and archaeal oxidases. The C-family contains only cbb3-type cytochrome c oxidases.
The AaCcO is one of the ba3-type cytochrome c oxidases. Based on the genomic DNA sequence analysis, it has been revealed that A. aeolicus possesses two operons coding for cytochrome c oxidases (two different subunit I genes, two different subunit II genes and one subunit III gene). So far, only subunits CoxB2 and CoxA2 were identified. The presence of the additional subunit IIa was reported in 2012. Moreover, a previous paper reported that AaCcO can use horse heart cytochrome c and decylubiquinol as electron donors and the typical cytochrome c oxidase inhibitor cyanide does not block the reaction completely.
In the course of my PhD studies, I performed heterologous expression of AaCcO in Pseudomonas stutzeri (P. stutzeri) and co-expression with AsHAS in Escherichia coli, respectively. The subcomplex CoxA2 and CoxB2 can be purified from P. stutzeri, however, it lacks heme A. Additionally, a protocol for the heterologous production of cytochrome c555 from A. aeolicus was established. In parallel, I also purified the AaCcO from native membranes according to previously reported methods with some modifications. The activity of AaCcO with its native substrate, cytochrome c555, was 14 times higher than with horse heart cytochrome c.
To enable a detailed investigation and comparison of AaCcO and other cytochrome c oxidases, the cryo-EM structure of AaCcO was determined to 3.4 Å resolution. It shows that the three subunits CoxA2, CoxB2, and IIa are tightly bound together to form a dimer in the membrane. Surprisingly, CoxA2 contains two additional TMHs (TMH13 and TMH14) to enhance the protein stability. The cofactors heme a3, heme b, CuA and CuB are also identified. Interestingly, two molecules of 1,4-naphthoquinone and cardiolipin were observed in the dimer interface. Based on the structure analysis, the AaCcO possesses only the K-pathway for proton delivery to the active site and proton pumping.
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The ATP-binding cassette half-transporter Mdl1 from Saccharomyces cerevisiae has been proposed to be involved in the quality control of misassembled respiratory chain complexes by exporting degradation products generated by the m-AAA proteases from the matrix. Direct functional or structural data of the transport complex are, however, not known so far. After screening expression in various hosts, Mdl1 was overexpressed 100-fold to 1% of total mitochondrial membrane protein in S. cerevisiae. Based on detergent screens, Mdl1 was solubilized and purified to homogeneity. Mdl1 showed a high binding affinity for MgATP (Kd = 0.26 μm) and an ATPase activity with a Km of 0.86 mm (Hill coefficient of 0.98) and a turnover rate of 2.6 ATP/s. Mutagenesis of the conserved glutamate downstream of the Walker B motif (E599Q) or the conserved histidine of the H-loop (H631A) abolished ATP hydrolysis, whereas ATP binding was not affected. Mdl1 reconstituted into liposomes showed an ATPase activity similar to the solubilized complex. By single particle electron microscopy, a first three-dimensional structure of the mitochondrial ATP-binding cassette transporter was derived at 2.3-nm resolution, revealing a homodimeric complex in an open conformation.
Membrane proteins are a diverse group of proteins that serve a multitude of purposes with one of the most important ones being transport. All kinds of substrates are shuffled over biological membranes with the help of dedicated proteins enabling the transport along and against a concentration gradient. Within the group of actively transporting proteins a diverse set of proteins that rely on an electrochemical gradient to facilitate transport of a substrate against its concentration gradient can be found. Those so-called secondary active
transporters are a group on integral membrane proteins ubiquitous to all cells. They allow the transport of all kinds of substrates like nutrients, ions, other metabolites and drugs over the hydrophobic barrier created by the cellular and organellar membrane. The gradients that provide the main driving force for most of the transporters are either sodium ions or protons, although transporters utilizing other ions or organic compounds are found as well. In case of exchangers two very similar substrates are transported in opposing direction over the membrane, one against its electrochemical gradient driven by the other.
Along with a structural diversity of the transporters concerning overall shape, oligomerization and number of transmembrane elements comes a mechanistic variety though still following the principle of alternating access. In humans the malfunction of secondary active transporters can lead to a physiological disorders such as epilepsy, depression or obesity.
The focus of this thesis was the structural and functional characterization of the secondary active transporter SeCitS from Salmonella enterica, a symporter of the 2-hydroxycarboxylate family. The transport of citrate as a bivalent ion is facilitated by the flux of sodium ions that have an inward-facing gradient over the inner membrane of Salmonella enterica. Transport experiments showed that the transport ratio is two sodium ions per citrate molecule, netting in an electroneutral transport. Compared to other members of the family the specificity of the transporter towards its main substrate is very high.
Structural information on the protein was initially obtained through 2D electron crystallography, which allowed the identification of the oval shaped dimer and a first hint towards a significant conformational change that the protein undergoes during its transport cycle. Using 3D crystallography, the X-ray structure of the transporter was solved. The protein crystalizes as a stable, but conformationally asymmetric dimer. As bound citrate can be readily identified in both protomers they can be assigned into an outward- and an inward-facing conformation, with the main citrate binding site in the outward-facing conformation.
One interesting feature of the crystal structure was the large surface available for multimerization, providing a platform for tight dimerization of the two protomers. On the other hand, SeCitS did not show a true cooperativity of transport. With those two aspects taken into account the question arose if any potential crosstalk between the monomers within the dimer takes place and influences transport (negative cooperativity) or the conformational distribution within the dimer (stabilization of the protein within the membrane).
The functional approach in answering this question was the use of mutated variants of the protein for cross-linking within one monomer. Two residues were chosen respectively to lock one of either conformation to be able to test for transport activity in the remaining protomer. The suitability of the residues was derived from the crystal structure (D112 – R205 to lock the inward-facing conformation and L337 – S412 for the outward-facing conformation). After initial promising results the final variants were not stable enough to be analyzed in transport assays.
To analyze the distribution of relative conformations within the dimer the protein was reconstituted into native-like lipid environment such as nanodiscs or saposin nanoparticles to be analyzed by cryo-electron microscopy. The first images were recorded and did yield promising 2D classes where the general features of the transporter were identified. Yet, an improved preparation is required to obtain a high resolution structure.
The key functional aspects of a transporter are its ability to bind and transport its substrates. In a set of experiments those features were investigated by a radioligand transport assay and by isothermal titration calorimetry (ITC). The transport properties of the protein were assessed in a filter assay using a radioactively labeled citrate as a read-out. The protein was reconstituted into proteoliposomes and subjected to different substrate conditions. Different ions were tested in its ability to drive or inhibit transport, but only sodium ions were able to drive transport and also not hindered by the presence of other ions...
In der vorgelegten kumulativen Arbeit wurden strukturelle und funktionale Untersuchungen an Nukleinsäuren durchgeführt, hauptsächlich, aber nicht ausschließlich unter Verwendung von NMR-Spektroskopie (Kernspin Resonanzspektroskopie) als Analysemethode. Die untersuchten Biomoleküle umfassten kleinere und größere biologisch relevante RNAs sowie einen artifiziellen DNA G-Quadruplex. Hierbei konnten Ergebnisse im Bereich der Bestimmung der molekularen Struktur, der Aufklärung der biologischen Funktion und der Wirkstoffentwicklung gewonnen werden, die in sechs verschiedenen Publikationen dargelegt sind, an deren Erstellung der Autor maßgeblich oder hauptverantwortlich beteiligt war. Des Weiteren wird in einem mehrgliedrigen Einleitungssegment auf den Stand der aktuellen Forschung in den jeweiligen Teilgebieten eingegangen.
Proteostasis stressors that destabilize the cellular proteome, like heat shock, trigger transcription and translational reactions leading to the accumulation of heat shock proteins, also called molecular chaperones. During stress, induction of stress response genes is prioritized so that molecular chaperones and other stress response proteins are synthesized to cope with proteome misfolding and aggregation. In order to promote the selective translation of stress-specific genes, translation of others genes that are nonessential for cell survival has to stop. Nonessential protein-coding mRNAs accumulate in the cytosol with the associated proteins to form granular structures called stress granules (SG). These membrane-less organelles are thought to be involved in cell survival, mRNA stabilization and mRNA triage. They were proposed to form via the liquid-liquid phase separation which can be triggered by the high local concentration of RNA-binding proteins. mRNAs were long thought to simply play a scaffolding role by bringing RNA-binding proteins together and allowing their concentration and local aggregation. Recently, the active role of mRNAs in the SG assembly became apparent, too. For example, the spontaneous assembly of total yeast RNA into granules was observed, and these RNA granules showed a large overlap with SG transcriptome. Furthermore, cytosolic mRNAs can be released from polyribosomes under stress and be exposed to the cytosolic contents as free mRNAs. It has been suggested that this massive increase of free mRNA in the cytosol might overload the capacities of RNA-stabilizing proteins. The remaining free mRNA molecules would then become exposed to misfolded and aggregation-prone proteins and trigger granulation.
We investigated the role of free mRNAs in different stress conditions during the early and chronic phases of stress response and explored their involvement in SGs assembly and amlyoidogenesis. We identified and studied the interactome of a free mRNA probe incubated with heat shocked cell lysate by means of quantitative mass spectrometry. Proteomics analysis allowed us to identify 79 interactors of free mRNA. Among these interactors, we focused on the translation initiation factor eIF2α and on the RNA methyltransferase TRMT6/61A. Both interactions were verified biochemically, which confirmed that the association is enhanced in heat shocked lysate. In vitro reconstitution showed that free mRNA and TRMT6 interact directly. Ex vivo pulldowns revealed that eIF2α and TRMT6/61A interact under stress conditions and that this interaction is RNA-dependent.
TRMT6/61A is a tRNA methytransferase responsible for the methylation of the adenosine 58 at the position 1 producing m1A. However, also mRNAs have been recently found to be methylated by TRMT6/61A. Our bioinformatics analyses revealed that significantly more mRNAs enriched in SG contain the motif for methylation than SG-depleted mRNAs. We hypothesized that m1A methylation of mRNAs could constitute a tag for the mRNAs targeting to SGs. TRMT61A knock-down (KD) cell lines were generated using the CRISPR-Cas9 technique. In TRMT61A KD cells, m1A was significantly reduced on mRNAs, which correlated with an increased sensitivity of the cells to proteostasis stress. KD cells also showed defects in SG assembly. In heat shocked cells, an m1A motif-containing mRNA recovered better after returning to normal temperature than a control mRNA with mutated motif. In addition, we could isolate SGs and analyze their m1A and m6A content by mass spectrometry. While m6A content in SG mRNAs was very similar to cytosolic mRNAs, m1A was almost 8 times enriched in SGs. Thus, we could confirm experimentally the results of the bioinformatics analysis and directly support the hypothesis that m1A is a tag to direct mRNAs for sequestration. Finally, we compared amyloidogenesis in wild-type and TRMT61A KD cell lines. Cells with reduced levels of TRMT61A demonstrated an increased accumulation of transfected Aβ and an impaired aggregate clearance. Various assays led us to conclude that the lack of m1A deposition on mRNAs enhanced RNA co-aggregation with amyloids.
Based on our results, we propose a model explaining the fate of free mRNA during proteostasis stress. Upon polysome disassembly, free mRNA is released and becomes free to interact with other proteins, including the methyltransferase TRMT6/61A. TRMT6/61A methylates the freed mRNAs containing the cognate motif. The m1A tag then targets mRNAs to SGs promoting sequestration. Upon stress release, SGs disassemble, thus releasing rescued mRNAs which could now reenter translation and support cell recovery. On the other hand, non-sequestered mRNAs increasingly co-aggregate with aggregating proteins. Thus, deficiency of the N1-adenine methylation of mRNAs due to the lack of TRMT6/61A increases the amount of unpacked mRNAs. The deposition of m1A on mRNAs could then be a way to protect them during exposure to stress, to limit their co-aggregation with misfolded proteins and to allow a faster recovery upon stress release.
The focus of this thesis is the integral membrane protein Escherichia coli diacylglycerol kinase (DGK). It is located within the inner membrane, where it catalyzes the ATP-dependent phosphorylation of diacylglycerol (DAG) to phosphatic acid (PA). DGK is a unique enzyme, which does not share any sequence homology with typical kinases. In spite of its small size, it exhibits a notable complexity in structure and function. The aim of this thesis is the investigation of DGK’s structure and function at an atomic level directly within the native-like lipid bilayer using MAS NMR. This way, a deeper understanding of DGK’s catalytic mechanism should be obtained.
First, the preparation of DGK was optimized, leading to a sample, which provides well-resolved MAS NMR spectra. The high quality MAS NMR spectra formed the foundation for the second step, the resonance assignment of DGK’s backbone and side chains. The assignment was performed at high magnetic field (1H frequency 850 MHz). The sequential assignment of immobile domains was carried out using dipolar coupling based 3D experiments, NCACX, NCOCX and CONCA. The measurement time could be reduced by paramagnetic doping with Gd3+-DOTA in combination with an E-free probehead. The sequential assignment was mainly performed using a uniformly labelled sample (U-13C,15N-DGK). Residual ambiguities could be resolved by reverse labelling (U-13C,15N-DGK-I,L,V). Resonances could be assigned for 82% of the residues, from which 74% were completely assigned. For validation, ssFLYA was applied, which is a generally applicable algorithm for the automatic assignment of protein solid state NMR spectra. Its principal applicability for demanding systems as membrane proteins could be proven for the first time. Overall, ~90% of the manually obtained assignments could be confirmed by ssFLYA. For the completion of DGK’s assignment, J-coupling based 2D experiments, 1H-13C/15N HETCOR and 13C-13C TOBSY, were carried out to detect highly mobile residues. This way, residues of the two termini and the cytosolic loop, which were not detectable by dipolar coupling based experiments, could be assigned tentatively. Whereupon, peaks for arginine and lysine were assigned unambiguously to Arg9 and Lys12. Overall, ~84% of the residues could be assigned by the applied NMR strategy. Furthermore, a secondary structure analysis was carried out. It showed substantial similarities between wild-type DGK, its thermostable mutant determined both by MAS NMR and the crystal structure of wtDGK. However, there are few differences around the flexible regions most likely caused by the high mobility of these regions. During the assignment procedure, no systematic peak doublets or triplets were detected, indicating that the DGK trimer adopts a symmetric conformation. This is in contrast to the X-ray structure, which shows asymmetries between the three subunits. Especially, crystal packing may be a potential source for these structural asymmetries.
On the basis of the nearly complete assignment of DGK, the apo state was compared with the substrate bound states. Perturbations in peak position and intensity of the substrate bound states were analysed for all assigned residues in 3D and 2D spectra. The nucleotide-bound state was emulated by adenylylmethylenediphosphonate (AMP-PCP), a non-hydrolysable ATP analogue, whereas the DAG-bound state was mimicked by 1,2-dioctanoyl-sn-glycerol (DOG, chain length n = 8). Upon nucleotide binding, extensive chemical shift perturbations could be observed. These data provide evidence for a symmetric DGK trimer with all of its three active sites concurrently occupied. Additionally, it could be demonstrated that the nucleotide substrate induces a substantial conformational change. This most likely supports the enzyme in binding of the lipid substrate, indicating positive heteroallostery. In contrast, the overall alterations caused by DOG are very minor. They involve mainly changes in peak intensities. For DGK bound with either AMP-PCP+DOG or only AMP-PCP, a similar spectral fingerprint was observed. This implies that binding of the nucleotide seems to set the enzyme into a catalytic active state, triggering the actual phosphoryl transfer reaction.
The investigation of DGK’s remarkable stability and the cross-talk between its subunits forms the last part of this thesis. This demands for the identification of key intra- and interprotomer contacts, which are of structural or functional importance. For this purpose, 13C-13C DARR and 2D NCOCX spectra with long mixing times were recorded using high field MAS NMR. Additionally, DNP-enhanced 13C−15N TEDOR experiments were conducted on mixed labelled DGK trimers to enable the visualization of interprotomer contacts. With the applied NMR strategy, intra- (Arg32 - Trp25/ Glu28/ Ala29 and Trp112 - Ser61) and interprotomer (ArgNn,e - AspCg/ GluCd/ AsnCg) long-range interactions could be identified.
Malfunction of the actin cytoskeleton is linked to numerous human diseases including neurological disorders and cancer. LIMK1 (LIM domain kinase 1) and its paralogue LIMK2 are two closely related kinases that control actin cytoskeleton dynamics. Consequently, they are potential therapeutic targets for the treatment of such diseases. In the present review, we describe the LIMK conformational space and its dependence on ligand binding. Furthermore, we explain the unique catalytic mechanism of the kinase, shedding light on substrate recognition and how LIMK activity is regulated. The structural features are evaluated for implications on the drug discovery process. Finally, potential future directions for targeting LIMKs pharmacologically, also beyond just inhibiting the kinase domain, are discussed.
The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR/DEER distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain is no prerequisite for dimerization. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers.
The heterotetrameric human transfer RNA (tRNA) splicing endonuclease (TSEN) catalyzes the excision of intronic sequences from precursor tRNAs (pre-tRNAs)1. Mutations in TSEN and its associated RNA kinase CLP1 are linked to the neurodegenerative disease pontocerebellar hypoplasia (PCH)2–8. The three-dimensional (3D) assembly of TSEN/CLP1, the mechanism of substrate recognition, and the molecular details of PCH-associated mutations are not fully understood. Here, we present cryo-electron microscopy structures of human TSEN with intron-containing pre-tRNATyrgta and pre-tRNAArgtct. TSEN exhibits broad structural homology to archaeal endonucleases9 but has evolved additional regulatory elements that are involved in handling and positioning substrate RNA. Essential catalytic residues of subunit TSEN34 are organized for the 3’ splice site which emerges from a bulge-helix configuration. The triple-nucleotide bulge at the intron/3’-exon boundary is stabilized by an arginine tweezer motif of TSEN2 and an interaction with the proximal minor groove of the helix. TSEN34 and TSEN54 define the 3’ splice site by holding the tRNA body in place. TSEN54 adapts a bipartite fold with a flexible central region required for CLP1 binding. PCH-associated mutations are located far from pre-tRNA binding interfaces explaining their negative impact on structural integrity of TSEN without abrogating its catalytic activity in vitro10. Our work defines the molecular framework of pre-tRNA recognition and cleavage by TSEN and provides a structural basis to better understand PCH in the future.
Electron microscopy (EM) demarcates itself from other structural biology techniques by its applicability to a large range of biological objects that spans from whole cells to individual macromolecules. In single-particle cryo-EM, frozen-hydrated samples, prepared by vitrification with liquid ethane, retain macromolecules in a medium that approximates their natural aqueous environment and that, in this way, preserves high-resolution structural information. Nonetheless, the sensitivity of biological specimens to the high-energy electron beam introduces restrictions on the total dose that can be used during imaging while avoiding significant radiation damage. Consequently, the signal-to-noise ratio attained in each individual image is very low, and structures with high-resolution detail must be recovered by averaging thousands of projections in random orientations. This is achieved through the use of image processing algorithms capable of aligning and classifying particle images through the evaluation of cross-correlation functions between each particle and a reference.
In recent years, several innovations took place in the field of single-particle cryo-EM, among which the development of direct electron detectors must be highlighted. Direct electron detectors have a better detective quantum efficiency (DQE) than both photographic film and CCD cameras, and offer a fast readout, compatible with the acquisition of movie stacks. Additionally, new image processing software has become available, with more sophisticated algorithms and designed to take advantage of the specific characteristics of the movies produced with direct electron detectors. These technological advances in both hardware and software catalyzed a revolution in single-particle cryo-EM, which is now routinely used for the determination of near-atomic structures. As a result, the range of macromolecules accessible to cryo-EM has increased drastically, as targets that were unsuitable before for imaging due to their small dimensions can now be adequately visualized and refined to high-resolution.
During my doctoral work, I have used single-particle cryo-EM to structurally characterize challenging membrane proteins, with a strong emphasis on protein complexes from aerobic respiratory chains. In chapter I of this thesis, I present my results on the bovine respirasome, a mitochondrial supercomplex composed of complexes I, III and IV. Chapter II is dedicated to the analysis of the structure of alternative complex III (ACIII) from Rhodothermus marinus, a bacterial quinol:cytochrome c/HiPIP oxidoreductase unrelated to the canonical cytochrome bc1 complex (complex III). In addition, in chapter III I describe the structure of KimA, a high-affinity potassium transporter that drives the transport of its substrate by using the energy stored in the form of a proton gradient. These three membrane proteins, with molecular weights ranging from 140 kDa to 1.7 MDa, illustrate the possibilities and limitations faced in single-particle cryo-EM.
The aerobic respiratory chain is responsible for the generation of a transmembrane difference of electrochemical potential that is then used by ATP synthase for the production of ATP or for driving solute transport over the membrane. They catalyze the transfer of electrons from a substrate, such as NADH or succinate, to molecular oxygen and use the chemical energy released in these redox reactions to drive the translocation of protons, or in some cases sodium ions, to the intermembrane space in mitochondria or the periplasm in bacteria.
In mitochondria, the respiratory chain is composed of four complexes: complex I (NADH:ubiquinone oxidoreductase), complex II (succinate dehydrogenase), complex III (cytochrome bc1 complex) and complex IV (cytochrome c oxidase). While it was for a long time believed that these complexes existed as single entities in the membrane, the use of milder procedures for protein purification and analysis revealed that respiratory complexes associate into well-ordered structures, known as supercomplexes. These have been proposed to offer different structural and functional advantages that are still controversial, including substrate channeling, stabilization of individual complexes and reduction of reactive oxygen species (ROS) production. The most thoroughly studied respiratory supercomplex has been the respirasome, conserved in higher eukaryotes and composed of one copy of complex I, a complex III dimer and one complex IV. By single-particle cryo-EM analysis, I retrieved a 9 Å map of the respirasome from Bos taurus, which allowed the accurate docking of atomic models of the three component complexes. The structure shows that complex III associates to the concave side of the membrane arm of complex I, while complex IV is located between the end of the complex I hydrophobic arm and complex III. Several defined protein-protein contacts are observed between the component complexes, which are mediated predominantly by supernumerary subunits and close to the membrane surfaces. The interactions established between complex I and complex III are extensive and may support the argument that the association of complex I into supercomplexes is required for the stabilization or even the biogenesis of this complex.
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The p53 protein family is the most studied protein family of all. Sequence analysis and structure determination have revealed a high
similarity of crucial domains between p53, p63 and p73. Functional studies, however, have shown a wide variety of different tasks in
tumor suppression, quality control and development. Here we review the structure and organization of the individual domains of
p63 and p73, the interaction of these domains in the context of full-length proteins and discuss the evolutionary origin of this
protein family.
FACTS:
● Distinct physiological roles/functions are performed by specific isoforms.
● The non-divided transactivation domain of p63 has a constitutively high activity while the transactivation domains of p53/p73
are divided into two subdomains that are regulated by phosphorylation.
● Mdm2 binds to all three family members but ubiquitinates only p53.
● TAp63α forms an autoinhibited dimeric state while all other vertebrate p53 family isoforms are constitutively tetrameric.
● The oligomerization domain of p63 and p73 contain an additional helix that is necessary for stabilizing the tetrameric states.
During evolution this helix got lost independently in different phylogenetic branches, while the DNA binding domain became
destabilized and the transactivation domain split into two subdomains.
OPEN QUESTIONS:
● Is the autoinhibitory mechanism of mammalian TAp63α conserved in p53 proteins of invertebrates that have the same function
of genomic quality control in germ cells?
● What is the physiological function of the p63/p73 SAM domains?
● Do the short isoforms of p63 and p73 have physiological functions?
● What are the roles of the N-terminal elongated TAp63 isoforms, TA* and GTA?
Die vorliegende Dissertation mit dem Titel “Structural dynamics of eukaryotic H/ACA RNPs from Saccharomyces cerevisiae & Structural dynamics of the Guanidine-II riboswitch from Escherichia coli” besteht aus zwei Projekten. Das erste Projekt befasst sich mit den eukaryotischen H/ACA Ribonukleoproteinen (RNP) aus der Hefe. Diese können sequenzspezifisch in der RNA ein Uridin Nukleotid in das Rotationsisomer Pseudouridin (Ψ) umwandeln. Die H/ACA RNPs bestehen aus einer Leit-RNA und vier Proteinen, der katalytisch aktiven Pseudouridylase Cbf5, Nhp2, Gar1 und Nop10. Die Leit-RNA besteht in Eukaryoten konserviert aus zwei Haarnadelstrukturen, die von einem H-Box oder ACA-Box Sequenzmotiv gefolgt sind. In jeder dieser Haarnadeln befindet sich ein ungepaarter Bereich, die sogenannte Pseudouridylierungstasche, wo durch komplementäre Basenpaarung die Ziel-RNA gebunden wird. Fehlerhafte H/ACA RNPs können beim Menschen zu schweren Krankheiten wie verschiedenen Krebsarten oder dem Knochenmarksversagen Dyskeratosis congenita führen, aber sie bieten auch Möglichkeiten zum Einsatz als Therapiemethode. In dieser Arbeit wurde hauptsächlich der zweiteilige Aufbau der H/ACA RNPs untersucht.
Dafür wurden zunächst die einzelnen Komponenten hergestellt werden. Cbf5, Nop10 und Gar1 wurden zusammen heterolog in E. coli exprimiert und gereinigt. Außerdem wurden mehrere Deletionsvarianten von Gar1 hergestellt. Zusätzlich wurde die Leit-RNA unmarkiert über T7 Transkription synthetisiert, sowie sechs verschiedene FRET-Konstrukte mit verschiedenen Markierungschemas der Fluorophore Cy3 und Cy5 über DNA-geschiente Ligation. Anschließend wurde über Größenausschlusschromatographie und radioaktiven Aktivitätsassays geprüft, dass sich die aktiven H/ACA RNPs in vitro aus den einzelnen Komponenten rekonstituieren lassen.
In smFRET Experimenten wurden einzelne Haarnadelstrukturen mit dem zweiteiligen Komplexen verglichen. Dabei konnte gezeigt werden, dass die H3 Haarnadel durch die Anwesenheit von H5 dynamischer und heterogener wurde, während H5 überwiegend unbeeinflusst war. Außerdem konnte die dreidimensionale Orientierung der Haarnadelstrukturen in verschiedenen Assemblierungsschritten mittels smFRET untersucht werden. Hier deutete sich an, dass in Abwesenheit von Proteinen beide Haarnadeln eher entgegengesetzt stehen als in einer parallelen Konformation. Cbf5 scheint den Linker zwischen den Beiden auszustrecken bzw. zu orientieren und die Haarnadelstrukturen etwas gegeneinander zu neigen. Ein Zusammenspiel von Nhp2 und Gar1 war nötig um die oberen Bereiche der Haarnadeln zusammenzuziehen. Es konnte auch ein Modell für den vollen H/ACA RNP vorgeschlagen werden. Im kompletten Komplex könnte das Zusammenziehen der Haarnadelstrukturen durch Nhp2 und Gar1 mit dem Effekt von Cbf5 konkurrieren und könnte hauptsächlich den oberen Bereich von H3 betreffen. Zum Schluss wurde das Zusammenspiel von Gar1 und Nhp2 auf eine Abhängigkeit von den RGG Domänen von Gar1 hin untersucht. Hier besteht möglicherweise eine Hierarchie, die eine Kooperativität von den N- und C-terminalen Domänen benötigt.
Das zweite Projekt befasst sich mit dem Guanidin-II Riboschalter aus E. coli. Der Riboschalter kann das toxische Molekül Guanidinium (Gdm+) spezifisch in seiner Aptamerdomäne binden und dadurch die Genexpression von Proteinen zur Detoxifizierung von Gdm+ aktivieren. Der Riboschalter besteht aus zwei Haarnadelstrukturen, mit einer Schleife, die aus der Sequenz ACGR besteht, wobei R ein Purin ist. In einem vorgeschlagenen Modell soll die Ribosomenbindestelle (Shine-Dalgarno Sequenz) in Abwesenheit von Ligand mit dem Linker komplementär Basenpaaren und so die Translation verhindern. Mit Ligand würde sich dann eine Schleifen-Schleifen Interaktion mit den beiden CG Basen ausbilden, wodurch die Anti-Shine-Dalgarno Sequenz nicht mehr zugänglich wäre. Bisherige Studien arbeiteten zumeist nur mit der Aptamerdomäne, den einzelnen Haarnadeln oder noch kleineren Elementen. In dieser Arbeit wurden die Strukturdynamiken von verschiedenen Längen, auch mit der Expressionsplatform, untersucht. Außerdem wurden verschiedene Mutationen analysiert und die Effekte auf den Riboschalter in seiner natürlichen Umgebung in E. coli.
Zunächst mussten insgesamt 24 FRET-Konstrukte hergestellt werden, die sich in Länge, Markierungsschema und Mutationen unterschieden. Hierfür wurde DNA-geschiente Ligation verwendet. Dank der verschiedenen Fluorophorpositionen konnte ein konformationelles Modell für die Aptamerdomäne vorgeschlagen werden. In diesem Modell könnte in Abwesenheit von Ionen das Aptamer offen vorliegen. Durch Mg2+ würde sich bereits eine lockere Schleifen-Schleifen Interaktion ausbilden. Zusätzlich deuten die Ergebnisse auf eine neue Konformation hin, der stabilisierten Schleifen-Schleifen Interaktion, bei der der Linker zusätzlich mit den Haarnadelstrukturen interagiert, beispielswese mit den Purinen an der vierten Schleifenposition...
N6-methyladenosine (m6A) is the most abundant and well understood modification in eukaryotic mRNA and was first identified in polyadenylated parts of the mRNA.The distinct distribution of m6A in the transcriptome with special enrichment in long internal exons, 39UTRs and around stop codons was uncovered by early biochemical work and later on antibody based sequencing techniques. The so called m6A writer, reader and eraser machinery is responsible for the dynamic and with that regulatory nature of the m6A modification. As m6A writer, the human N6-methyltransferase complex (MTC) cotranscriptionally methylates the central adenine within a RRACH (preferably GGACU) sequence context to form m6A in the nascent RNA chain.9–15 The catalytic core of the complex is formed by the two proteins METTL3 and METTL14, with the active site located in the methyltransferase domain (MTD) of METTL3.16–18 The DPPW motif near the methyl donor S-adenosylmethionine (SAM) binding site in this MTD was postulated to bind the target adenine during catalysis. Moreover, a positively charged groove in the METTL3-METTL14 interface, the C-terminal RGG domain in METTL14 and the zinc finger motifs in METTL3 were identified as important domains for RNA binding. However, to date there are no full-length or substrate-RNA-bound structures of the catalytic METTL3-METTL14 complex.
In addition, a set of accessory proteins assembles to the METTL3-METTL14 heterodimer to form the full MTC, mediated by WTAP that firmly binds to the N-terminal leader helix in METTL3.20 WTAP was shown to locate the whole complex to the nuclear speckles and can modulate m6A deposition to specific sites in the RNA. Moreover, WTAP acts as binding platform for other accessory proteins including VIRMA, RBM15, ZC3H13 and HAKAI that are mostly identified to mediate position specific methylation. For example, RBM15 was shown to mediates region-selective methylation in a WTAP dependent manner, directing specificity towards U-rich sequences.
The observed specificity of the methyltransferase complex to methylate only site specific DRACH sequenced is still poorly understood. Some possible modulators like the role of the accessory proteins are under investigation, however, the structural context of the RNA methylation sites or a structural preference of the complex have been mainly neglected so far. Moreover, the structural dynamics of this methylation process still remain elusive. This thesis contributes to the afore-mentioned aspects by analysis of the methylation process regarding RNA structure sensitivity with enzymatic activity assays and its dynamic nature by implementing a smFRET approach.
We hypothesized the target RNA secondary structure to be an additional important modulator of methylation efficiency, based on the RNA binding elements of the complex (positively charged binding groove, zinc finger domain, RGG domain) and the supposed target adenine binding in the active site. Here, we postulated the possibility for a flipped-out adenine to be of special relevance, which is closely related to the local stability of the target adenine containing structure. Moreover, efficient binding of the protein complex to the RNA should require the ability to anchor the RNA on both sides of the target sequence.
YEATS-domain-containing MLLT1 is an acetyl/acyl-lysine reader domain, which is structurally distinct from well-studied bromodomains and has been strongly associated in development of cancer. Here, we characterized piperazine-urea derivatives as an acetyl/acyl-lysine mimetic moiety for MLLT1. Crystal structures revealed distinct interaction mechanisms of this chemotype compared to the recently described benzimidazole-amide based inhibitors, exploiting different binding pockets within the protein. Thus, the piperazine-urea scaffold offers an alternative strategy for targeting the YEATS domain family.
The nsP3 macrodomain is a conserved protein interaction module that plays essential regulatory roles in host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus, targeting this protein domain may offer a therapeutic strategy to combat the current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of macrodomain crystal structures complexed with diverse naturally-occurring nucleotides, small molecules as well as nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir. The presented data strengthen our understanding of the SARS-CoV-2 macrodomain structural plasticity and it provides chemical starting points for future inhibitor development.
Cytochrome c oxidases are among the most important and fundamental enzymes of life. Integrated into membranes they use four electrons from cytochrome c molecules to reduce molecular oxygen (dioxygen) to water. Their catalytic cycle has been considered to start with the oxidized form. Subsequent electron transfers lead to the E-state, the R-state (which binds oxygen), the P-state (with an already split dioxygen bond), the F-state and the O-state again. Here, we determined structures of up to 1.9 Å resolution of these intermediates by single particle cryo-EM. Our results suggest that in the O-state the active site contains a peroxide dianion and in the P-state possibly an intact dioxygen molecule, the F-state may contain a superoxide anion.
Signal transduction via phosphorylated CheY towards the flagellum and the archaellum involves a conserved mechanism of CheY phosphorylation and subsequent conformational changes within CheY. This mechanism is conserved among bacteria and archaea, despite substantial differences in the composition and architecture of archaellum and flagellum, respectively. Phosphorylated CheY has higher affinity towards the bacterial C-ring and its binding leads to conformational changes in the flagellar motor and subsequent rotational switching of the flagellum. In archaea, the adaptor protein CheF resides at the cytoplasmic face of the archaeal C-ring formed by the proteins ArlCDE and interacts with phosphorylated CheY. While the mechanism of CheY binding to the C-ring is well-studied in bacteria, the role of CheF in archaea remains enigmatic and mechanistic insights are absent. Here, we have determined the atomic structures of CheF alone and in complex with activated CheY by X-ray crystallography. CheF forms an elongated dimer with a twisted architecture. We show that CheY binds to the C-terminal tail domain of CheF leading to slight conformational changes within CheF. Our structural, biochemical and genetic analyses reveal the mechanistic basis for CheY binding to CheF and allow us to propose a model for rotational switching of the archaellum.
The endoplasmic-reticulum-associated protein degradation pathway ensures quality control of newly synthesized soluble and membrane proteins of the secretory pathway. Proteins failing to fold into their native structure are processed in a multistep process and finally ubiquitinated and degraded by the proteasome in order to protect the cell from proteotoxic stress. My thesis covers structural as well as functional studies of various protein components that constitute the protein complexes that are responsible for this process.
One sub-project addressed the mechanism of glycan recognition by Yos9 as part of the ERAD substrate selection. NMR solution structures of the mannose-6-phosphate homology (MRH) domain of Yos9 both in a free and glycan bound conformation reveal a gripping movement of loop regions upon binding of correctly processed glycan structures.
The main projects focused on revealing the mechanism of efficient ubiquitin chain assembly by the ERAD ubiquitination machinery. This included the investigation of the role of the ERAD components Cue1 and Ubc7 in processive ubiquitin chain formation, how ubiquitin chain conformations change during elongation, how the conformation of a chain is impacted by interacting proteins and finally understand the activity regulation of the ERAD E2 enzyme Ubc7 by its cognate RING E3 ligases. Nuclear magnetic resonance (NMR) analysis and fluorescence-based ubiquitination assays show that the CUE domain of Cue1 contributes with its proximal binding preference as well as with its position dependent accelerating effect to efficient ubiquitin chain formation. This is required to efficiently drive degradation of substrates. Specific ubiquitin binding events dictate and coordinate the spatial arrangement of the E2 enzyme relative to the distal tip of a chain. This process can be further accelerated by RING E3 ligases that promote Ubc7 activity by more than ~20 fold via inducing allosteric changes around the catalytic cysteine. My results additionally suggest a model where Ubc7 dimerization results in proximity induced activation of the E2. This data ensures rapid diubiquitin formation that is followed by a CUE domain assisted chain elongation mechanism where Cue1 acts in an E4 like fashion.
How ubiquitin binding events can modulate the conformations of a ubiquitin chain were investigated by pulsed electron-electron double resonance (PELDOR) spectroscopy combined with molecular modeling. This shows that K48-linked diubiquitin samples a broad conformational space which can be modulated in distinct ways. The CUE domain of Cue1 uses conformational selection of pre-populated open conformations to support ubiquitin chain elongation. In contrast, deubiquitinating enzymes shift the conformational distribution to weakly or even non-populated conformations to allow cleavage of the isopeptide bond that connects adjacent ubiquitins. Ubiquitin chain elongation increases the sampled conformational space and suggests that this high conformational flexibility might contribute to efficient proteasomal recognition.
Human protein kinases play essential roles in cellular signaling pathways and - if deregulated - are linked to a large diversity of diseases such as cancer and inflammation or to metabolic diseases. Because of their key role in disease development or progression, kinases have developed into major drug targets resulting in the approval of 52 kinase inhibitors by the Food and Drug Administration (FDA) so far.
Within the drug discovery process, the affinity of the inhibitors is the parameter that is used most often to predict the later efficacy in humans. However, the kinetics of binding have recently emerged as an important but largely neglected factor of kinase inhibitor efficacy. To efficiently suppress a signaling pathway, the targeted kinase needs to be continuously inhibited. Thus, it has been hypothesized that fast binding on-rates and slow off-rates would be the preferred property of an efficacious inhibitor. Despite optimizing the potency of kinase inhibitors, in the past decade optimization of kinetic selectivity has therefore gained interest as a molecule cannot be active unless it is bound, as Paul Ehrlich once stated. There is increasing evidence of correlations between prolonged drug-target residence time and increased drug efficacy, and that inhibitor selectivity in cellular contexts can be modulated by altered residence times. In order to contribute to the understanding of the effect of long residence times on cellular targets we initiated two projects.
The first of these projects is related to the STE20 kinase Serine/threonine kinase 10 (STK10) and its close relative STE20 like kinase (SLK) which have been reported to be frequent off-targets for kinase inhibitors used in the clinics. Also, an inhibition of STK10 and SLK has been linked to a common side-effect of severe skin rash developed upon treatment with the EGFR inhibitor erlotinib, but not gefitinib and the severity of this rash correlated with the treatment outcome, which fits the known biology of STK10 and SLK to be regulators of lymphocyte migration and PLK kinases. However, there are yet no explanations why these two proteins show such high hit-rates across the kinome among the kinase inhibitors. Using structural analysis, we identified the flexibility of STK10 to be the main reason for this hit-rate. The observed strong in vitro potencies did however not translate to the cellular system which is why we investigated the inhibitors residence time on STK10. We found the same flexibility to be the main reason for slow residence times among several inhibitors. We observed large rearrangements in the hydrophobic backpocket of STK10 including the αC, the P-loop enclosing the inhibitor like a lid and strong π-π-stackings to be the main reasons for prolonged residence times on STK10. Interestingly, we observed an increased residence time for erlotinib, which showed skin-related side-effects, giving rise whether the binding kinetics should be investigated for weak cellular off-target effects in future drug discovery efforts.
In the second project we initiated, we illuminate a structural mechanism that allows kinetic selection between two closely related kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2). Using an inhibitor series designed to probe the mechanism, residence times measured in vitro and in cells showed a strong correlation. Crystal structures and mutagenesis identified hydrophobic interactions with L567, adjacent to the DFG-motif, as being crucial to kinetic selectivity of FAK over PYK2. This specific interaction was observed only when the DFG-motif was stabilized into a helical conformation upon ligand binding to FAK. The interplay between the protein structural mobility and ligand-induced effect was found to be the key regulator of kinetic inhibitor selectivity for FAK over PYK2.
These two projects showed that the parameter residence time should be considered for different problems among the drug discovery process. First, in an open in vivo system not only the potency of a drug alone, but as well its residence time might be of importance. Here we showed that the weak cellular potency translated to prolonged residence times for several inhibitors in cells and established a link between the phenotypic outcome of skin rash after erlotinib treatment and the residence time of this inhibitor on STK10 in cells. On the other hand, medicinal chemistry efforts should consider structure kinetic relationships (SKR) in the optimization process and aim to understand the molecular basis for prolonged target residence times. Here, we showed that a hydrophobic interaction that is enforced upon inhibitor binding is crucial for an unusual helical DFG conformation which arrests the inhibitor and prolongs its residence time providing the molecular basis for understanding the kinetic selectivity of two closely related protein kinases. Establishing the SKRs will help medicinal chemists to kinetically optimize their drug candidates to select a suitable molecule to proceed into further optimization programs. Hence, the projects showed that the target residence time parameter needs to be considered both as a molecular optimization parameter to improve compound potency and binding behavior as well as a parameter to be understood for proceeding to the open system of in vivo models to later modulate the in vivo efficacy of protein kinase targeting drugs.
The post-transcriptional modification of the canonical nucleoside uridine into its rotational isomer pseudouridine occurs in non-coding as well as coding RNA and is the most abundant post-transcriptional modification in all kingdoms of life. While the occurrence of pseudouridine has been linked to the enhancement of stability and the codon-anticodon interaction in tRNAs, enhancement of the translation efficiency in rRNAs, regulatory functions in spliceosomal snRNA and nonsense codon suppression in mRNA, its exact role in many RNAs is still ambiguous. The uridine to pseudouridine isomerization can either be catalyzed by one of various standalone pseudouridylases or it can be performed in an RNA-guided manner by H/ACA ribonucleoproteins. In eukaryotes, the guide RNA always adapts a conserved bipartite, double-hairpin conformation. Each hairpin contains an internal RNA-loop motif, which can recruit a specific substrate RNA via base pairing. The catalytically active RNP is formed by the interactions of the guide RNA with four proteins. While Cbf5 forms the catalytically active center, Nop10 and Nhp2 perform auxiliary functions and Gar1 is involved in substrate turnover. Up until now, most structural knowledge about H/ACA RNPs has been derived from archaeal complexes, while the exact structure-function-relationships between RNA and proteins in eukaryotic RNPs is still ambiguous. While archaeal H/ACA RNPs share many similarities with eukaryotic RNPs and act as good model system, there are also many differences between them like eukaryotic specific protein domains as well as the overall bipartite complex structure, dictated by the snoRNA. Investigating pseudouridylation by eukaryotic H/ACA RNPs opens up a broad area of research and helps to gain a better understanding of this enzyme class – especially since malfunction of H/ACA RNPs has been linked to the genetic disease Dyskeratosis congenita as well as several types of cancer.
The main goal of this thesis was to gain new insights into the RNA/protein interactions in the eukaryotic snR81 H/ACA snoRNP from Saccharomyces cerevisiae on a structural as well as dynamical level. In the first part of this thesis, the main goal was to in vitro prepare a functionally active snR81 H/ACA RNP. The guiding snoRNA was prepared by in vitro transcription and purification, while the Saccharomyces cerevisiae proteins were recombinantly expressed from Escherichia coli. Apart from the full length, bipartite snR81 snoRNP, several sub-complexes of the RNP were reconstituted. Therefore, snoRNA constructs were designed and prepared, which only contained a single hairpin motif of the complex. Furthermore, snoRNA constructs in which the apical hairpin stem was replaced by a stable tetraloop were prepared, to investigate the influence of the apical stem on protein binding and activity. Also, for the eukaryotic proteins, a shortened version of Gar1 (Gar1Δ) was utilized, which lacks the eukaryotic specific RGG domains, that have been characterized as accessory RNA binding motifs. Reconstituted snoRNPs were utilized in catalytic activity assays, monitoring the turnover rate of uridine to pseudouridine. For this purpose, radioactively labeled substrate RNAs were prepared by phosphorylation and splinted ligation of oligonucleotides and were objected to reconstituted H/ACA RNPs under single as well as multiple turnover conditions. In the second part of this thesis, the RNA/protein interactions were dissected via single molecule FRET spectroscopy. Therefore, the snoRNA was labeled with an acceptor fluorophore via NHS ester/amine-reaction. Furthermore, the snoRNA contained a biotin-handle, allowing immobilization of the complex during the experimental time-window of the spectroscopic analysis. Eukaryotic specific protein Nhp2 was labeled with a donor fluorophore via “click” chemistry, which included the chemical synthesis and incorporation by genetic code expansion of non-canonical amino acids. The interactions of Nhp2 with the different snoRNA constructs (standalone-hairpins “H5” and “H3”, as well as hairpins lacking the apical binding motif “H5Δ” and “H3Δ”) were monitored on a single molecule level.
In summary, it was possible to gain new insights into the complex structure and the dynamical behavior of the still sparsely characterized eukaryotic H/ACA RNPs. Especially, new knowledge could be obtained about the hairpin specific behavior on the bipartite RNA complex structure, including the rather ambiguous role of the protein Nhp2 and the contribution of the eukaryotic specific features of Gar1 in their interaction with the guide/substrate RNA.
To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.
Während meiner Promotion habe ich zwei Projekte unter der Aufsicht von Dr. Misha Kudryashev durchgeführt. Im ersten Projekt habe ich die Strukturen des Ryanodinrezeptors 1 (RyR1) in Apo- und Ryanodin-Bindungszuständen in der nativen Membran durch Tomographie und Subtomogramm-Mittelung bei 12,6 bzw. 17,5 Å bestimmt. Im Vergleich zur Struktur von gereinigtem RyR1 unter Verwendung der Einzelpartikel-Kryo-Elektronenmikroskopie (Cryo-EM) können zusätzliche Dichten in der cytoplasmatischen Domäne und der sarkoplasmatischen Retikulum (SR)-Membran bzw. im SR-Lumen beobachtet werden. Die Auflösung der Struktur von RyR1 im Apo-Zustand wurde von den Kollegen in meinem Labor mithilfe der Hybridmethode auf 9,5 Å verbessert. Diese Arbeit hat unser Verständnis für die Mechanismen von RyR1 in nativen Membranen erweitert. Im zweiten Projekt habe ich die Struktur des Proteins SdeC der SidE-Familie durch Einzelpartikel-Kryo-EM bei 4,6 Å bestimmt. Die Kristallstruktur des C-Terminus von SdeA wurde von meinem Forschungspartner Dr. Mohit Misra gelöst. Durch Überlagerung einer gemeinsamen Helix dieser beiden Strukturen konnten wir ein kombiniertes Modell erstellen und ein allgemeines Verständnis der Proteine der SidE-Familie erhalten.
In this thesis, we characterized megasynthases such as fatty acid synthases (FASs) and polyketide synthases. The obtained insights into structure and function were used to engineer such systems to produce new-to-nature compounds.
The in vitro characterization of megasynthases requires reproducible access to these enzymes in high quality. Therefore, we established purification strategies for the yeast FAS and the methylsalicylic acid synthase (MSAS) from Saccharopolyspora erythraea (SerMSAS) and applied the latter one on MSAS from Penicillium patulum (PenPaMSAS) and on 6-deoxyerythronolide B synthase (DEBS) module 6. With the purified samples, we were able to obtain initial structural data for SerMSAS and solve the complete structure of the yeast FAS (PDB: 6TA1). On the example of the yeast FAS, we could show that the sample can suffer from adsorption to the water-air interface during the grid preparation for electron microscopy and presented how the use of graphene-based grids can overcome this problem. The combined structural and functional analysis of the yeast FAS showed that the structural domains trimerization module and dimerization module 2 are not essential for the assembly of the whole system. Therefore, they can potentially be used for domain exchange approaches. The in-depth functional analysis of SerMSAS revealed that not SerMSAS itself releases the product, but a 3-oxoacyl-(acyl-carrier protein) synthase like enzyme within the gene cluster transfers 6-methyl salicylic acid from SerMSAS to another carrier protein for subsequent modifications. In contrast, we showed that PenPaMSAS can release its product by hydrolysis and that non-native substrates can be incorporated although at significantly slower turnover rates compared to the native starter substrate. Our further investigation demonstrated that the substrate specificity of the acyltransferase (AT) is a critical factor for the incorporation of non-native substrates.
With the insight from the functional and structural characterization, we engineered megasynthases for the biosynthesis of natural product derivatives. We targeted the AT of PenPaMSAS for active site mutagenesis and discovered a mutant which can transfer non-native substrates significantly faster (~200-300%). Additionally, the malonyl/acetyl transferase (MAT) of the mammalian FAS was used as a promising target for protein engineering because of its previously reported properties including polyspecificity, fast transfer kinetics, robustness, and plasticity. We showed that the MAT can transfer fluorinated substrates and accept the acyl carrier protein of DEBS module 6. By exchanging the substrate specific AT of DEBS with the polyspecific MAT of the mammalian FAS, we demonstrated an efficient DEBS/FAS hybrid and an optimal truncation site for the applied ATs. In contrast to the wild type system, the DEBS/FAS enzyme was able to synthesize demethylated and fluorinated derivatives. The production and purification of a fluoro-methyl-disubstituted polyketide was of particular interest, as it has a high potential for the generation of new drugs and shows the potential of protein engineering. Furthermore, the incorporation of the disubstituted substrate had important implication in the mechanistic details of the ketosynthase-mediated C-C bond formation.
Dysfunction of YEATS-domain-containing MLLT1, an acetyl/acyl-lysine dependent epigenetic reader domain, has been implicated in the development of aggressive cancers. Mutations in the YEATS domain have been recently reported as a cause of MLLT1 aberrant reader function. However, structural basis for the reported alterations in affinity for acetyled/acylated histone has remained elusive. Here, we report the crystal structures of both insertion and substitution present in cancer, revealing significant conformational changes of the YEATS-domain loop 8. Structural comparison demonstrates that such alteration not only altered the binding interface for acetylated/acylated histones, but the sequence alterations in the T1 loop may enable dimeric assembly consistent inducing self-association behavior. Nevertheless, we show that also the MLLT1 mutants can be targeted by developed acetyllysine mimetic inhibitors with affinities similarly to wild type. Our report provides a structural basis for the altered behaviors and potential strategy for targeting oncogenic MLLT1 mutants.
ATP-binding cassette (ABC) transporters constitute an omnipresent superfamily of integral membrane proteins, which catalyze the translocation of a multitude of chemically diverse substrates across biological membranes. In humans, ABC transporters typically act as highly promiscuous exporters, responsible for many physiological processes, multi-drug resistance, and severe diseases, such as hypercholesterolemia, lipid trafficking disorders, and immune deficiency. In all ABC transporters, ATP-driven movements within two highly conserved nucleotide-binding domains (NBDs) are coupled to conformational changes of two transmembrane domains (TMDs), which provide a framework for substrate binding and release on the opposite side of the membrane and enable the transporter to cycle between inward-facing and outward-facing orientations. Several structures of ABC transporters determined either by X-ray crystallography or single-particle electron cryo-microscopy (cryo-EM) have been reported, mostly exhibiting a variation of the inward-facing state, which highlights their dynamic behavior. However, for a complete understanding of the conformational dynamics, further structural information on intermediates is needed – especially for heterodimeric ABC transporters, which are predominant in humans and for which only limited structural information is available.
One prime example of such human heterodimeric ABC transport complexes is the transporter associated with antigen processing (TAP). TAP is a key player of the adaptive immune response, because it translocates proteasomal degradation products into the ER lumen for loading of MHC I molecules. Many functional aspects of TAP have been disclosed in recent years. However, structural information is lacking far behind and a major challenge in the field of medical relevant transporters. Recently, the heterodimeric ABC export system TmrAB (Thermus thermophilus multidrug resistance proteins A and B) was identified as an ortholog of TAP, by sharing structural homology with TAP and, intriguingly, being able to restore antigen presentation in human TAP-deficient cells. Thus, TmrAB is a biochemically well-characterized ABC exporter that can be regarded as a functional ortholog of TAP and serves as a model system for (heterodimeric) ABC export systems in general.
Thus, to illuminate the molecular basis of substrate translocation by single-particle cryo-EM, one of the main objectives of this work was the generation of stabilizing chaperones (synthetic antibodies, nanobodies, cyclic peptides) to reduce the conformational heterogeneity of TAP and TmrAB. Selected antibodies were analyzed with respect to stable complex formation, conformational trapping, and the ability to serve as alignment tools for structural studies by single-particle cryo-EM. Both antibody types were shown to form sufficiently stable complexes to serve as a rigid body for EM analyses. However, all selected antibodies bound to the inward-facing state exclusively.
Hence, for EM studies, various ligands were added to elucidate the full spectrum of conformational states during the catalytic cycle. For TAP, first attempts by negative-stain EM revealed a homogenous distribution of particles on the grid. Surprisingly, no transporter-like features were observed although various attempts were applied to increase the overall sample quality.
For TmrAB, in contrast, the complete conformational space in a native-like lipid environment under turnover conditions was mapped. Cryo-EM analysis of TmrAB incubated with ATP-Mg2+ and substrate revealed two distinct inward-facing conformations (IFwide and IFnarrow) as well as two asymmetric conformations with dimerized NBDs, which were markedly different from all previously reported structures. Here, the catalytically active site was slightly wider and contained ADP, while ATP was still bound at the catalytically-inactive site within the NBDs, demonstrating an asymmetric post-hydrolysis state. Intriguingly for the inward-facing conformations, a weak additional density close to residues M139TmrB and W297TmrB was observed in the inward-facing conformation, which displayed a higher degree of cytosolic gate opening (IFwide) indicating the presence of substrate. To verify that this density corresponds to substrate, single alanine mutations of M139TmrB and W297TmrB were introduced, leading to a strong reduction in substrate binding and transport. Since substrate release requires the opening of the extracellular gate, the absence of an outward-facing open conformation indicated that the opening must be highly transient. In order to explore the outward-facing open conformation, a cryo-EM analysis of the catalytically-inactive TmrAE523QB mutant upon incubation with ATP-Mg2+ was performed. Remarkably, within the same dataset, two different outward-facing conformations (occluded and open) were resolved, both in an ATP-bound state, which indicated that binding of ATP is sufficient to drive the large-scale conformational transition from inward-facing to outward-facing open. To explore the effect of nucleotide hydrolysis, TmrAB was trapped by vanadate. Again, two populations were observed, representing the outward-facing open and outward-facing occluded conformation.
Based on several structures of key intermediates, determined under turnover conditions or trapped in the pre-hydrolysis and hydrolysis transition state, for the first time the complete description of the ATP hydrolysis and translocation cycle of a heterodimeric ABC transport complex was elucidated in one single study. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates, fundamental and so-far hidden steps of the substrate translocation cycle of asymmetric ABC transporters were resolved and a general template for (heterodimeric) ABC exporter-catalyzed substrate translocation was provided.
Biological membranes serve as physical barriers in cells and organelles, enabling the maintenance of chemical or ionic gradients that are essential for triggering various integral, peripheral, or lipid-anchored membrane proteins, necessary for their life-essential functions. The study of membrane proteins has unique challenges due to their hydrophobic nature, limited expression levels, and inherent flexibility. Single-particle analysis (SPA) enables the determination of high-resolution three-dimensional structures using minimal amounts of specimen without the need for crystallization. Additionally, cryogenic electron tomography (cryo-ET) and subtomogram averaging (StA) offer the ability to study membrane protein complexes, cellular architecture, and molecular interactions while preserving close-to-life conditions. With ongoing improvements in cryo-EM technologies, obtaining high-resolution structures of membrane proteins in vitro can allow people to understand their mechanisms and functions, and to facilitate the design and optimization of new therapeutic agents. Furthermore, there has been significant growth in the structural characterization of membrane proteins in situ, as studying biomolecules within their physiological context is an ultimate goal in structural biology for a comprehensive understanding of molecular networks in cells.
Due to the amphipathic nature of membrane proteins, their production, purification, and isolation pose significant challenges compared to soluble proteins. To maintain the membrane protein fold in an aqueous buffer after disrupting lipid membranes, the use of detergents, amphipols, lipid nanodiscs, saposin-lipoprotein (salipro), styrene-maleic acid co-polymer lipid particles (SMALPS) is common and often essential. A limitation of the membrane-mimetic systems is the absence of an actual lipid bilayer environment. To address this issue, membrane proteins can be reconstituted into liposomes, and this closed membrane environment closely mimics the physiological conditions of the proteins. The use of liposomes for structure determination is expected to significantly expand in the in vitro study of membrane proteins and membrane-associated proteins, particularly for capturing transient complexes in specific functional states.
Resolving the structures of membrane proteins in their native cellular context is considered the ideal approach for understanding their functions and associated molecular networks. While single-particle cryo-EM can achieve higher resolution than subtomogram averaging, it often requires at least partial purification of the target molecules from their native environment inside cells and tissues. By combining averaging tools on subvolumes obtained through cryo-ET, structures can currently be determined at resolutions of 10-30 Å. With ongoing advancements and refinements in cryo-ET methodologies, routine high-resolution structure determination in situ is poised to become a valuable tool for both structural and cell biologists in the long run, and the field holds great promise for further expanding our understanding of cellular structures and processes at the molecular level.
The main aim of this thesis is to further our knowledge of the structure and function of a small prokaryotic voltage-gated sodium ion channel, NaChBac in liposomes, and a large knob complex found on the surface of Plasmodium falciparum-infected human erythrocyte by cryo-ET and StA.
Chapter 2 presents the first StA map of the 120-kDa NaChBac embedded in liposomes under a resting membrane potential at a modest resolution of 16 Å. The approach presented in this study, which can be widely applied to cryo-EM analysis of membrane proteins, with a specific focus on membrane proteins with small soluble domains, lays the foundation for cryo-ET and StA of integral or peripheral membrane proteins whose functions are affected by transmembrane electrochemical gradients and/or membrane curvatures. Chapter 3 shows the first cryo-EM structure of the supramolecular knob complex in P. falciparum-infected human erythrocyte. While a previous study provided an overall architectural view of knobs using negative stain tomography, the in situ structure bridges this gap, guiding future investigations into the molecular composition and the role of these native knobs in Plasmodium infection and immunity.
This thesis opens up several promising lines for future studies of membrane proteins in vitro and in situ, where other membrane proteins can be studied in physiologically relevant environments. Already with the present generation of cryo-EM hardware and software, this thesis represents pioneering research in the field of membrane protein structural biology.
A method for the ab initio crystal structure determination of organic compounds by a fit to the pair distribution function (PDF), without prior knowledge of lattice parameters and space group, has been developed. The method is called ‘PDF-Global-Fit’ and is implemented by extension of the program FIDEL (fit with deviating lattice parameters). The structure solution is based on a global optimization approach starting from random structural models in selected space groups. No prior indexing of the powder data is needed. The new method requires only the molecular geometry and a carefully determined PDF. The generated random structures are compared with the experimental PDF and ranked by a similarity measure based on cross-correlation functions. The most promising structure candidates are fitted to the experimental PDF data using a restricted simulated annealing structure solution approach within the program TOPAS, followed by a structure refinement against the PDF to identify the correct crystal structure. With the PDF-Global-Fit it is possible to determine the local structure of crystalline and disordered organic materials, as well as to determine the local structure of unindexable powder patterns, such as nanocrystalline samples, by a fit to the PDF. The success of the method is demonstrated using barbituric acid as an example. The crystal structure of barbituric acid form IV solved and refined by the PDF-Global-Fit is in excellent agreement with the published crystal structure data.
H/ACA-RNPs are involved in RNA guided pseudouridylation of rRNAs and snRNAs. In this thesis I reconstituted active and labeled archaeal as well as eukaryotic H/ACA-RNPs and studied the structural dynamics of complex assembly and pseudouridine formation. Single molecule FRET spectroscopy was used as method of analysis to study structure, assembly and dynamics of these important complexes.
Salt-inducible kinases (SIKs) are key metabolic regulators. Imbalance of SIK function is associated with the development of diverse cancers, including breast, gastric and ovarian cancer. Chemical tools to clarify the roles of SIK in different diseases are, however, sparse and are generally characterized by poor kinome-wide selectivity. Here, we have adapted the pyrido[2,3-d]pyrimidin-7-one-based PAK inhibitor G-5555 for the targeting of SIK, by exploiting differences in the back-pocket region of these kinases. Optimization was supported by high-resolution crystal structures of G-5555 bound to the known off-targets MST3 and MST4, leading to a chemical probe, MRIA9, with dual SIK/PAK activity and excellent selectivity over other kinases. Furthermore, we show that MRIA9 sensitizes ovarian cancer cells to treatment with the mitotic agent paclitaxel, confirming earlier data from genetic knockdown studies and suggesting a combination therapy with SIK inhibitors and paclitaxel for the treatment of paclitaxel-resistant ovarian cancer.
The transcriptional regulator RcsB controls the expression of a minimum of 20 different genes having diverse functionalities and biosynthetic operons in the family of Enterobacteriaceae. While in the heterodimeric complex with the co activator RcsA, the RcsAB box consensus is recognized, DNA binding sites for RcsB without RcsA have also been identified. The conformation of RcsB might therefore be modulated upon interaction with various co activators, resulting in recognition of different DNA targets. In this study the interaction of RcsB with some of these DNA targets have been analysed by a diverse array of techniques including gel shift assay and SPR. The solution structure of the C-terminal DNA-binding domain of RcsB from Erwinia amylovora spanning amino acid residues 129-215 has been solved in this study by heteronuclear NMR spectroscopy. The C-terminal domain is composed of four α-helices where the two central helices of the H-T-H motif are similar to the structures of the regulatory proteins GerE, NarL and TraR. The DNA-binding activity of the C-terminal domain alone is established for the first time in this study and was specified by fluorescence spectroscopy, SPR and NMR titration experiments. The molecular interaction between the individual RcsB domains was analysed by cross-linking experiments and heteronuclear NMR spectroscopy and the amino acid residues of the C-terminal domain involved in this interaction were identified precisely. Another important part of this project was the cell-free production of different Trp analogue labelled RcsB protein. RcsB protein was produced in quite a good yield with different Trp analogue having spectrally enhanced properties. The isolated RcsB alloproteins proved to be ideal for protein interaction studies by fluorescence spectroscopy and the very first evidence of an oligomerization of RcsB due to molecular association has been put forth from these studies. The phosphorylated state of the RcsB protein was mimicked by a beryllofluoride complex in order to study its role in transcriptional regulation. It was found that RcsB alone could bind to DNA targets upon this modification by the beryllofluoride complex. Thus the phosphorylation of the protein that involves the Asp 56 residue induces a structural change of the protein followed probably by a domain movement also, so that the C-terminal domain having the H-T-H DNA binding motif that was previously eclipsed by the N-terminal domain is relieved of this constraint.
Structure-function relationships in substrate binding protein dependent secondary transporters
(2023)
This work provides new insights into the relevance of SBP dependent secondary transport systems, especially in the thus far under-researched subgroup of TAXI transporters. Importantly, we identified and characterized the TAXI transport system TAXIPm-PQM from Proteus mirabilis. We demonstrated that, in contrast to previously characterized SBP dependent secondary transport systems, TAXIPm-PQM is a proton coupled system and transports the C5-dicarboxylate α- ketoglutarate. Since initially the transport of α-ketoglutarate could only be demonstrated in vivo but not in vitro using established protocols (Mulligan et al. 2009), we investigated in detail the differences between the in vivo and in vitro assay. This resulted in a bioinformatic analysis of TRAP and TAXI signal peptides, which strongly implied that TAXIPm-P requires a transmembrane anchor to allow for transport. We then provided TAXIPm-P surface tethered to the membrane in in vitro transport assays and confirmed the prediction of our bioinformatic analysis that TAXIPm-PQM deploys a membrane-anchored instead of a soluble SBP. Furthermore, the TAXI transport system TAXIMh-PQM from Marinobacter hydrocarbonoclasticus transports fumarate only if both membrane domains Q and M are present. For further characterization, Michaelis-Menten kinetics and affinities were determined for both TAXI transport systems TAXIPm-PQM from Proteus mirabilis and TAXIMh-PQM from Marinobacter hydrocarbonoclasticus. In addition, nanobodies were selected for the membrane domain TAXIPm-QM from Proteus mirabilis to stabilize different conformations which can serve in subsequent structural elucidation studies. Furthermore, the TRAP SBP TRAPHi-SiaP from Haemophilus influenzae was shown to interact not only with its corresponding membrane domain TRAPHi-SiaQM but with at least one additional transporter. It was thereby excluded that TRAPHi- SiaP transfers N-acetylneuraminic acid to the only native E. coli TRAP transporter TRAPEc-YiaMNO and suggested to rather interact with a SBP dependent ABC transport system as this protein family represents the largest SBP dependent protein group in E. coli (Moussatova et al. 2008).
The enzyme 5-lipoxygenase (5-LO) occupies a central role in the biosynthesis of inflammatory leukotrienes and thus takes part in the pathogenesis of related diseases. Its occurrence is mainly restricted to cells of the immune system including granulocytes, monocytes/macrophages or B-lymphocytes and can be induced by cell differentiation of myeloid cells after treatment with differentiating agents, such as DMSO, retinoic acid or the combination of TGFβ/1,25(OH)2D3. The latter contribute to the highest level of induction of mRNA and protein expression. Its cell specific occurrence is at least partly due to DNA methylation in cells that do not exhibit 5-LO activity and genetic regulation is further dependent on histone acetylation. 5-LO expression is controlled by transcription factors binding to the promoter sequence of the ALOX5 gene that induce basal promoter activity, as well as promoter independent effects including transcript initiation and elongation, which are mostly attributed to TGFβ/1,25(OH)2D3 signaling. The ALOX5 gene resembles a typical housekeeping gene, hence lacks TATA- or CAAT-boxes for transcriptional regulation, but displays a high GC-content with eight GC-boxes, five of which are arranged in tandem, that provide binding sites for transcription factors Sp1, Sp3 and Egr-1.
The proximal ALOX5 promoter is furthermore a target for additional factors, such as TGFβ effector proteins SMADs or the vitamin D receptor and possesses additional consensus sequences for transcriptional regulators, including NF-κB or PU.1. However, as yet no actual binding of these proteins to the promoter sequence was demonstrated and an unbiased screening for identifying further ALOX5 promoter interacting proteins, which might have impact on 5-LO expression, is still lacking. For this purpose, the present study focused on the identification of significantly interacting proteins, employing DNA-affinity enrichment coupled to label-free quantitative proteomics, spanning a sequence of about 270 base pairs of the proximal ALOX5 promoter. For the elucidation of potential cell specific differences in protein patterns and compositions, DNA pulldowns were performed by using oligonucleotide stretches comprising the core promoter sequence including the 5-fold GC-box, which were incubated with different cell lines and differentiation states of myeloid, as well as B-lymphocytic lineages. In order to compare different mass spectrometric quantification strategies that would allow for identification of interactors, dimethyl labeling and label-free techniques were used. Since the label-free approach outperformed the label-based one in initial experiments, it was established as standard quantification strategy in all DNA pulldowns performed. The pulldowns of myeloid cell lines in both undifferentiated and differentiated state and B-lymphocytes resulted in a cell-unspecific protein pattern whose composition was similar, regardless of cell lineage. Additionally, further DNA sequences comprising either a vitamin D response element or a SMAD binding element were investigated in the promyelocytic model cell line HL-60 in both undifferentiated and differentiated state. The identified proteins confirmed known interaction partners and furthermore revealed novel potential regulators of the 5-LO promoter. Out of these, the most prominently identified and promising proteins included transcription factors of the KLF- and CCAAT/enhancer binding protein-family. In this context, KLF5 and KLF13 are both involved in the regulation of inflammatory processes, the former additionally being an effector protein of TGFβ-signaling, whose functional characterization is of utmost interest in terms of regulation of 5-LO expression. Further protein characterization will be inevitable for the CCAAT/enhancer binding proteins C/EBPα, C/EBPβ and C/EBPε. These transcription factors are involved in the regulation of inflammatory processes and heterodimers thereof (C/EBPα/β) are known to control TGFβ/1,25(OH)2D3-mediated effects of the CD14 gene.
Several of the identified proteins of the pulldowns containing the tandem GC-box represented interactors of G-quadruplex DNA, including the helicases BLM and DHX36, the ribonucleoproteins hnRNP D and hnRNP K and transcription factor MAZ. Since G-quadruplexes form in G-rich DNA sequences as secondary DNA structures and exhibit substantial regulatory effects on the transcription of their target genes, the potential formation thereof in the ALOX5 core promoter sequence was investigated in a second project. Out of the proteins mentioned above, MAZ is shown to exert resolving effects on G4-DNA and synergistically induce Sp1-dependent gene activation of oncogene h-RAS, which displays analogous promoter characteristics to the ALOX5 gene. A DNA stretch comprising the tandem GC-box was used for elucidating the potential of secondary DNA structure formation. Intriguingly, both immune-based and spectroscopic methods provided clear evidence for the in vitro G-quadruplex formation of the proximal promoter sequence for the first time. In order to provide additional information on a possible regulatory effect of existing G-quadruplex structures on 5-LO transcription, differentiated HL-60 cells were subsequently treated with two distinct G4-DNA stabilizing agents. A porphyrin analogon (TMPyP4) did not exhibit any effects on 5-LO mRNA and protein expression after cell treatment. A second G4-DNA stabilizing agent (pyridostatin) on the other hand revealed significant reduction on 5-LO protein expression after cellular treatment. These mixed results render further experiments inevitable, in order to provide a clear assertion as to whether 5-LO expression is regulated by G-quadruplex structures or not.
Altogether, this study enlarges the knowledge of ALOX5 proximal promoter interacting proteins by corroborating the binding of already known transcription factors and identifying novel interactors. It yields essential groundwork for subsequent functional studies of proteins involved in 5-LO transcription and introduces G-quadruplexes as a new potential mechanism in ALOX5 gene regulation.