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G-protein-coupled receptors (GPCRs) from the largest family of receptors in the human body. They contain seven transmembrane helices. There are roughly 800-900 GPCR genes expressed in humans encoded by 4-5% of the human genome. These receptors are the most important signal transducers and play a crucial role in cell physiology and pathology, by using various extracellular stimuli to start complex intracellular signaling. GPCRs interact with a wide variety of stimuli from small molecules (photons, ions, amines) to large molecules (peptides, small proteins), and trigger downstream cascade effects by interacting with G-proteins, GPCR kinases, and ß-arrestin. Because of their crucial roles in many cellular functions, GPCRs are the most important drug targets for the pharmaceutical industry. Approximately 30% of the clinically approved drugs available in the market are against GPCRs. In this work achieved successful expression and purification of GPCRs from class-C and class-A families. Combined with biochemical experiments, DNP-ssNMR, and molecular simulation helped to decipher the mechanism of crosstalk between the allosteric modulator, and the orthosteric binding sites of the peptide receptor. The main findings and major highlights of this dissertation are outlined in the following paragraphs.
The calcium-sensing receptor (CaSR) belongs to the GPCR class-C family and contains a large extracellular domain. This receptor regulates Ca2+ homeostasis in blood and its absorption in the kidney and bone. To understand the molecular and structural mechanisms of these receptors their cDNAs were cloned into the pPICZ and pOET1 vectors to express them in Pichia pastoris and in Sf9 insect cells respectively. The CaSR was successfully expressed heterologously in Pichia pastoris and in the insect cell with high yield. The purified receptor purified in LMNG shows no aggregation in a monomeric state. Further optimization was performed to use it for cryo-EM sample preparation and structure determination. In 2nd part of the thesis, different mini G (mini Gs, mini Gi, mini Gqs, and mini Gsi) DNA constructs were made and expressed in E. coli. It's challenging to obtain active GPCR structures due to the instability of G-protein or G-protein-bound receptors. In this work, all mini-G proteins and chimera mini-G-protein-maltose binding protein (MBP) were cloned and expressed in E. coli and purified with a His-trap column with high purity.
In the last part of the thesis, to decipher the mechanism of allosteric modulation of orthosteric binding sites in the bradykinin receptor was produced and characterized in insect cells. Angiotensin I converting enzyme inhibitors (ACEIs), are very important drugs and are widely used for the treatment of hypertension, congestive heart failure, and diabetic neuropathy. These drugs target primarily the catalytic zinc center of the ACE. It has been shown that enalaprilat, a well-known ACEI, binds to a proposed zinc-binding site on hB1R and even directly activates the receptor. To obtain information on the influence of ACEIs on the receptor-peptide complex, and to have a better understanding of the molecular mechanism and structural plasticity of the bradykinin receptor and PAM, we used the three commercially available ACEIs captopril, enalaprilat, and lisinopril for our studies. An important result of this thesis is that though enalaprilat, captopril, and lisinopril all have similar functional properties in humans, each one regulates the orthosteric binding site of hB1R in a unique way. These findings provide atomic insights into the allosteric modulation of the bradykinin receptor. This study along with the effects of ACEI on the binding sites of receptors also deciphers the effects of the Zn2+ as well as the crosstalk between zinc binding sites and ACEI compounds. The binding of allosteric modulators induces distinct endogenous binding, which might aid in creating new possibilities in the pharmaceutical field.
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.
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.
...
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.
...
Funktionelle und strukturelle Charakterisierung von SLC-Transportern in eukaryotischen Systemen
(2018)
Die evolutionäre Voraussetzung für die Entwicklung komplexer, differenzierter Organismen bildet die Separierung der Zelle in Reaktionsräume, die so genannte Kompartimentierung. Das Prinzip der Kompartimentierung ermöglicht zahlreiche lebensnotwendige, biochemische Prozesse, wie die Konservierung von Energie durch Protonengradienten in der Atmungskette oder parallele, gegenläufige Stoffwechselwege. Zelluläre Kompartimente werden häufig durch Biomembranen gebildet, welche aus einer zweilagigen Lipidschicht bestehen. Lipidmoleküle in einer Zelle sind meistens amphipathisch, das bedeutet, sie bestehen aus einer polaren, hydrophilen Kopfgruppe und einem unpolaren, hydrophopen Ende (Abbildung 1). Die Lipidzusammensetzung in einer Biomembran ist sehr divers und unterscheidet sich in verschiedenen Organismen und Organellen. Phosphoglyceride bilden den Hauptbestandteil der Lipidschicht. Phosphoglyceride besteht aus einem Glycerin Rückgrat, welches an dem C1- und C2-Atom mit zwei Fettsäuren verestert und an dem C3-Atom mit einem Phosphorsäurediester verbunden ist. ...
NADH:ubiquinone oxidoreductase (Complex Ⅰ) is the first and largest enzyme in the respiratory chain. It catalyzes the transfer of two electrons from NADH to ubiquinone via a series of enzyme-bound redox centers - Flavin mononucleotide (FMN) and iron-sulfur (Fe-S) clusters – and couples the exergonic reaction with the endergonic translocation of four protons across the membranes. Bacteria contain the minimal form of complex I, which is composed of 14 conserved core subunits with a molecular mass of around 550 kDa. Complex Ⅰ has an L-shaped structure which can be subdivided into two major parts (arms). The hydrophilic arm protruding into the bacterial cytosol (or mitochondrial matrix) harbors the binding site for the substrate NADH, the two- to one-electron switch FMN and all one-electron transferring Fe-S clusters and therefore considered as the catalytic unit. The membrane arm consists of the membranespanning subunits and conducts the proton pumping process. The Quinone binding site is located at the interface of both arms. ...
Membrane proteins are biological macromolecules that are located in a cell’s membrane and are responsible for essential functions within an organism, which makes them to prominent drug targets. The extraction of membrane proteins from the hydrophobic membrane bilayer to determine high-resolution crystal structures is a difficult task and only 2% of all solved proteins structures are membrane proteins. Computational methods may help to gain deeper insights into membrane protein structures and their functions. This study will give an overview of such computational methods on a representative set of membrane proteins and will provide ideas for future computational and experimental research on membrane proteins.
In a first step (chapter 2), I updated an earlier, manually-curated data set of homologous membrane proteins (HOMEP) to more recent versions in 2010 (HOMEP2) and 2013 (HOMEP3) using an automated clustering approach. High-resolution structures of membrane proteins listed in the PDB_TM database were structurally aligned and subsequently clustered using structural similarity scores. Both data sets were used as a standard gold reference set for subsequent work.
Subsequently, I have updated and applied the sequence alignment program AlignMe to determine protein descriptors that are suitable for detecting evolutionary relationship between homologous a-helical membrane proteins. Single input descriptors were tested alone and in combination with each other in different modes of AlignMe by optimizing gap penalties on the HOMEP2 data set. Most accurate alignments and homology models on the HOMEP2 data set were observed when using position-specific substitution information (P), secondary structure propensities (S) and transmembrane propensities (T) in the AlignMe PST mode. An evaluation on an independent reference set of membrane protein sequence alignments from the BAliBASE collection showed that different modes of AlignMe are suitable for different sequence similarity levels. The AlignMe PST mode improved the alignment accuracy significantly for distantly related proteins, whereas for closely-related proteins from the BAliBASE set the AlignMe PS mode was more suitable. This work was published in March 2013 in PLOS ONE. In order to allow also an easier usage of the AlignMe program, I have implemented a web server of AlignMe (chapter 4) that provides the optimized settings and gap penalties for the AlignMe P, PS and PST modes. A comparison to other recent alignment web server shows that the alignments of AlignMe are similar or even more accurate than those of other methods, especially for very distantly related proteins for which the inclusion of membrane protein information has been shown to be suitable. This work was published in the NAR web server issue in July 2014.
Although membrane-specific information has been shown to be suitable for aligning distantly related membrane proteins on a sequence level, such information was not incorporated into structural alignment programs making it unclear which method is the most suitable for aligning membrane proteins. Thus, I compared 13 widely-used pairwise structural alignment methods on an updated reference set of homologous membrane protein structures (HOMEP3) and evaluated their accuracy by building models based on the underlying sequence alignments and used scoring functions (e.g., AL4 or CAD-score) to rate the model accuracy (chapter 5). The analysis showed that fragment-based approaches such as FR-TM-align are the most useful for aligning structures of membrane proteins that have undergone large conformational changes whereas rigid approaches were more suitable for proteins that were solved in the same or a similar state. However, no method showed a significant higher accuracy than any other. Additionally, all methods lack a measure to rate the reliability of the accuracy for a specific position within a structure alignment. In order to solve these problems, I propose a consensus-type approach that combines alignments from four different methods, namely FR-TM-align, DaliLite, MATT and FATCAT and assigns a confidence value to each position of the alignment that describes the agreement between the methods. This work has been published 2015 in the journal “PROTEINS: structure, function and bioinformatics”.
Consensus alignments were then generated for each pair of proteins of the HOMEP3 data set and subsequently analyzed for single evolutionary events within membrane spanning segments and for irregular structures (e.g., 310- and p-helices) (chapter 6). Interestingly, single insertions and deletions could be observed with the help of consensus alignments in the conserved membrane-spanning segments of membrane proteins in four protein families. The detection of such single InDels might help to identify crucial residues for a proteins function.
G protein coupled receptors (GPCRs) constitute the largest family of cell-surface receptors in mammals and are key players in signal transduction. By responding to a plethora of extracellular stimuli ranging from photons to amines to fatty acids to peptides and proteins, these receptors trigger intracellular signalling cascades and regulate a variety of cellular responses. Approximately 800 genes in humans encode GPCRs which are classified according to sequence conservation into rhodopsin-like, glutamate, adhesion, frizzled/taste2 and secretin receptors. GPCRs share a seven transmembrane domain fold undergoing a conformational change upon ligand binding which is translated to the intracellular surface of the receptor thereby allowing a heterotrimeric G protein to couple. Heterotrimeric G proteins consist of a Ga, Gb and Gg subunit and dissociate into their Ga and Gbg entities upon activation by a GPCR. Subsequently, distinct signalling cascades are triggered by each G protein protomer.
Membrane proteins and GPCRs in particular, are highly important targets in drug design and development as currently approximately 60% of all marketed drugs target membrane proteins. Although these classes of proteins are of high therapeutic interest, our understanding of their mechanism of action and structure remains limited. The first structure of a human GPCR was determined in 2007 and required the development of protein engineering and innovative crystallisation techniques. Since then, approximately 130 GPCR structures of less than 40 individual receptors have been determined providing insights into the structural arrangement of the transmembrane helices, ligand binding pockets and G protein interactions. Combined with spectroscopic methods, these studies allowed a more detailed understanding of the molecular aspects of GPCR activation and signalling. Despite the tremendous advances in GPCR structural biology, certain aspects of GPCR function still remain poorly understood. Due to their size and inherent flexibility, the interaction of protein and peptide ligands with their receptors remains a challenging aspect in the structural characterisation of GPCRs. Moreover, structural information on subtype selectivity of peptide ligands continues to be scarce. To contribute functional and structural information on the molecular mechanisms of peptide interactions with GPCRs, this thesis focused on characterising receptors from the chemoattractant cluster using radioligand binding assays as well as NMR spectroscopy.
The chemoattractant cluster mainly groups the kinin, angiotensin, anaphylatoxin chemotactic complement and apelin receptors according to conserved residues in their ligand binding cavities. All receptors in this cluster bind to peptide ligands deriving from high molecular weight protein precursors upon proteolytic processing. Comparable to the conserved binding pocket of the chemoattractant receptors, the peptide ligands display a certain sequence conservation although they differ strongly in size. The largest ligands used in this thesis are the anaphylatoxins complement 3a and 5a, comprising 77 or 74 residues, respectively. Due to their size and complex fold involving three intramolecular disulphide bonds, solid phase synthesis is impossible, which prompted us to develop a modified cell-free expression system to produce these ligands in tritiated form for subsequent functional characterisation of the complement receptors. To demonstrate the versatility of the developed system, it was applied to another disulphidebond containing peptide ligand, the 21 amino acid endothelin-1. We describe a reliable and multifaceted tool to generate custom labelled peptide ligands for the structural and functional characterisation of GPCRs. The system allows the production of custom radioligands, peptides labelled for NMR studies or with fluorescent amino acids.
Apart from the modulation of GPCR activity by orthosteric ligands, GPCR signalling has long been described to be regulated by allosteric ligands including peptides, small molecules and ions. In this thesis, the influence of sodium ions on the activity state of the chemoattractant cluster receptors and in particular on the apelin, bradykinin 2 and angiotensin II type 1 receptors was examined. In recent high resolution crystal structures an allosteric sodium ion pocket beneath the orthosteric ligand binding cavity was identified and residues contributing to the coordination of sodium ions are conserved throughout the chemoattractant cluster receptors. This allosteric sodium ion coordinated within the transmembrane domain bundle has been described to negatively influence the affinity of agonists but not of antagonists. It was found that sodium ions have distinct influences on the affinity state as well as the available number of binding sites of the chemoattractant receptors. In case of the apelin and bradykinin 2 receptors, sodium ions drastically reduced the number of available binding sites whereas the affinity of peptide ligands to the bradykinin 2 receptors remained constant and the ligand binding affinities to the apelin receptor were completely abolished. In contrast, the angiotensin II type 1 receptor affinity state towards the endogenous peptide ligand angiotensin II is highly dependent on the presence of sodium ions, whereas binding of the synthetic peptide antagonist Sar1-Ile8-angiotensin II remained unaffected by the sodium ion concentration. As differential effects irrespective of the efficacy class but dependent on the amino acid composition of the applied ligands are observed, it can be concluded that electrostatic interactions between charged residues of the peptide ligands and amino acids on the extracellular surface of the receptors are influenced by sodium ions thereby adding another layer of complexity on GPCR signalling.
To elucidate the structure-function relationship of ligand selectivity between the kinin receptors, the structure of desArg10-kallidin (DAK) bound to the bradykinin 1 receptor was determined using solid state NMR (SSNMR) in the course of this thesis. The kinin peptides DAK and bradykinin bind with high affinity and high selectivity to either the bradykinin 1 or bradykinin 2 receptor, respectively. The binding pockets of the receptors are highly conserved and the two peptide ligands only differ in one amino acid at their N- and C-termini whereas the remaining eight amino acids are fully conserved. DAK adopts a U-shaped structure when bound to the bradykinin 1 receptor which resembles a horse shoe-like conformation. Using 2D TEDOR spectroscopy it could furthermore be demonstrated that positively charged residues at the N-terminal part of the peptide engage in ionic interactions with negatively charged amino acids on the extracellular surface of the bradykinin 1 receptor. In contrast, bradykinin displays a distinct b-turn at the C-terminus and an S-shaped conformation of the N-terminal segment when bound to the bradykinin 2 receptor. By using SSNMR to study the binding mode of DAK on the bradykinin 1 receptor we could determine that subtype selectivity between the kinin receptors is conferred by distinct conformational restraints within the peptide ligands and by the formation of specific ionic interaction between charged residues on the peptide and receptor, respectively.
In brief, this thesis contributes structural and functional data on the binding mechanisms and binding mode of different peptide-ligand GPCRs helping to understand subtype selectivity and allosteric modulation of the chemoattractant cluster receptors. In addition, a versatile cell-free expression system was developed that allows the custom synthesis of isotopically labelled peptides containing disulphide bonds for the functional characterisation of GPCRs.
Heme-copper oxidases (HCOs) are the terminal enzymes of the aerobic respiratory chain in the inner mitochondrial membrane or the plasma membrane in many prokaryotes. These multi-subunit membrane protein complexes catalyze the reduction of oxygen to water, coupling this exothermic reaction to the establishment of an electrochemical proton gradient across the membrane in which they are embedded. The energy stored in the electrochemical proton gradient is used e.g. by the FOF1-ATP synthase to generate ATP from ADP and inorganic phosphate. The superfamily of HCOs is phylogenetically classified into three major families: A, B and C. The A-family HCOs, represented by the well-studied aa3-type cytochrome c oxidases (aa3-CcOs), are found in mitochondria and many bacteria. The B-family of HCOs contains a number of bacterial and archaeal oxidases. The C-family comprises only the cbb3-type cytochrome c oxidase (cbb3-CcO) and is most distantly related to the mitochondrial respiratory oxidases.
This work presents a biochemical, functional and structural characterization of Aquifex aeolicus F1FO ATP synthase obtained using both a native form (AAF1FO) and a heterologous form (EAF1FO) of this enzyme.
F1FO ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane and therefore play a key cellular function. Because of their central role in supporting life, F1FO ATP synthases are ubiquitous and have been remarkably conserved throughout evolution. For their biological importance, F1FO ATP synthases have been extensively studied for many decades and many of them were characterized from both a functional and a structural standpoint. However, important properties of ATP synthases – specifically properties pertaining to their membrane embedded subunits – have yet to be determined and no structures are available to date for the intact enzyme complex. Therefore, F1FO ATP synthases are still a major focus of research worldwide. Our research group had previously reported an initial characterization of AAF1FO and had indicated that this enzyme presents unique features, i.e. a bent central stalk and a putatively heterodimeric peripheral stalk. Based on such a characterization, this enzyme revealed promising for structural and functional studies on ATP synthases and became the focus of this doctoral thesis. Two different lines of research were followed in this work.
First, the characterization of AAF1FO was extended by bioinformatic, biochemical and enzymatic analyses. The work on AAF1FO led to the identification of a new detergent that maintains a higher homogeneity and integrity of the complex, namely the detergent trans-4-(trans-4’-propylcyclohexyl)cyclohexyl-α-D-maltoside (α-PCC). The characterization of AAF1FO in this new detergent showed that AAF1FO is a proton-dependent, not a sodium ion-dependent ATP synthase and that its ATP hydrolysis mechanism needs to be triggered and activated by high temperatures, possibly inducing a conformational switch in subunit γ. Moreover, this approach suggested that AAF1FO may present unusual features in its membrane subunits, i.e. short N-terminal segments in subunits a and c with implications for the membrane insertion mechanism of these subunits.
Investigating on these unique features of A. aeolicus F1FO ATP synthase could not be done using A. aeolicus cells, because these require a harsh and dangerous environment for growth and they are inaccessible to genetic manipulations. Therefore, a second approach was pursued, in which an expression system was created to produce the enzyme in the heterologous host E. coli. This second approach was experimentally challenging, because A. aeolicus F1FO ATP synthase is a 500-kDa multimeric membrane enzyme with a complicated and still not entirely determined stoichiometry and because its encoding genes are scattered throughout A. aeolicus genome, rather than being organized in one single operon. However, an artificial operon suitable for expression was created in this work and led to the successful production of an active and fully assembled form of Aquifex aeolicus F1FO ATP synthase. Such artificial operon was created using a stepwise approach, in which we expressed and studied first individual subunits, then subcomplexes, and finally the entire F1FO ATP synthase complex. We confirmed experimentally that subunits b1 and b2 form a heterodimeric subcomplex in the E. coli membranes, which is a unique case among ATP synthases of non-photosynthetic organisms. Moreover, we determined that the b1b2 subcomplex is sufficient to recruit the soluble F1 subcomplex to the membranes, without requiring the presence of the other membrane subunits a and c. The latter subunits can be produced in our expression system only when the whole ATP synthase is expressed, but not in isolation nor in the context of smaller FO subcomplexes. These observations led us to propose a novel mechanism for the assembly of ATP synthases, in which first the F1 subcomplex attaches to the membrane via subunit b1b2, and then cring and subunits a assemble to complete the FO subcomplex. Furthermore, we could purify the heterologous ATP synthase (EAF1FO) to homogeneity by chromatography and electro-elution. Enzymatic assays showed that the purified form of EAF1FO is as active as AAF1FO. Peptide mass fingerprinting showed that EAF1FO is composed of the same subunits as AAF1FO and all soluble and membrane subunits could be identified. Finally, single-particle electron microscopy analysis revealed that the structure of EAF1FO is identical to that of AAF1FO. Therefore, the EAF1FO expression system serves as a reliable platform for investigating on properties of AAF1FO.
Specifically, in this work, EAF1FO was used to study the membrane insertion mechanism of rotary subunit c. Subunits c possess different lengths and levels of hydrophobicity across species and by analyzing their N-terminal variability, four phylogenetic groups of subunits c were distinguished (groups 1 to 4). As a member of group 2, the subunit c from A. aeolicus F1FO ATP synthase is characterized by an N-terminal segment that functions as a signal peptide with SRP recognition features, a unique case for bacterial F1FO ATP synthases. By accurately designing mutants of EAF1FO, we determined that such a signal peptide is strictly necessary for membrane insertion of subunit c and we concluded that A. aeolicus subunit c inserts into E. coli membranes using a different pathway than E. coli subunit c. Such a property may be common to other ATP synthases from extremophilic organisms, which all cluster in the same phylogenetic group.
In conclusion, the successful production of the fully assembled and active F1FO ATP synthase from A. aeolicus in E. coli reported in this work provides a novel genetic system to study A. aeolicus F1FO ATP synthase. To a broader extent, it will also serve in the future as a solid reference for designing strategies aimed at producing large multi-subunit complexes with complicated stoichiometry.