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Structural and functional consequences of the H180A mutation of the light-driven sodium pump KR2
(2022)
Krokinobacter eikastus rhodopsin 2 (KR2) is a light-driven pentameric sodium pump. Its ability to translocate cations other than protons and to create an electrochemical potential makes it an attractive optogenetic tool. Tailoring its ion pumping characteristics by mutations is therefore of great interest. In addition, understanding the functional and structural consequences of certain mutations helps to derive a functional mechanism of ion selectivity and transfer of KR2. Based on solid-state NMR spectroscopy, we report an extensive chemical shift resonance assignment of KR2 within lipid bilayers. This data set was then used to probe site-resolved allosteric effects of sodium binding, which revealed multiple responsive sites including the Schiff base nitrogen and the NDQ motif. Based on this data set, the consequences of the H180A mutation are probed. The mutant is silenced in the presence of sodium while in its absence, proton pumping is observed. Our data reveal specific long-range effects along the sodium transfer pathway. These experiments are complemented by time-resolved optical spectroscopy. Our data suggest a model in which sodium uptake by the mutant can still take place, while sodium release and backflow control are disturbed.
According to the World Health Organization (WHO) bacterial resistance to antibiotic drug therapy is emerging as a major public health problem around the world. Infectious diseases seriously threaten the health and economy of all countries. Hence, the preservation of the effectiveness of antibiotics is a world wide priority. The key to preserving the power of antibiotics lies in maintaining their diversity. Many microorganisms are capable of producing these bioactive products, the so called antibiotics. Specifically in microorganisms, polyketide synthases (PKS) and non-ribosomal peptide synthases (NRPS) produce these natural bioactive compounds. Besides being used as antibiotics these non-ribosomal peptides and polyketides display an even broader spectrum of biological activities, e.g. as antivirals, immunosuppressants or in antitumor therapy. The wide functional spectrum of the peptides and ketides is due to their structural diversity. Mostly they are cyclic or branched cyclic compounds, containing non-proteinogenic amino acids, small heterocyclic rings and other unusual modifications such as epimerization, methylation, N‐formylation or heterocyclization. It is has been shown that these modifications are important for biological activity, but little is known about their biosynthetic origin.
PKS and NRPS are multidomain protein assembly lines which function by sequentially elongating a growing polyketide or peptide chain by incorporating acyl units or amino acids, respectively. The growing product is attached via a thioester linkage to the 4’-phosphopantetheine (4’-Ppant) arm of a holo acyl carrier protein (ACP) in PKSs or holo peptidyl carrier protein (PCP) in NRPSs and is passed from one module to another along the chain of reaction centers. The modular arrangement makes PKS and NRPS systems an interesting target for protein engineering. More than 200 novel polyketide compounds have already been created by module swapping, gene deletion or other specific manipulations. Unfortunately, however, engineered PKS often fail to produce significant amounts of the desired products. Structural studies may faciliate yield improvement from engineered systems by providing a more complete understanding of the interface between the different domains. While some information about domain-domain interactions, involving the most common enzymatic modules, ketosynthase and acyltransferase, is starting to emerge, little is known about the interaction of ACP domains with other modifying enzymes such as methyltransferases, epimerases or halogenases.
To further improve the understanding of domain-domain interactions this work focuses on the curacin A assembly line. Curacin A, which exhibits anti-mitotic activity, is from the marine cyanobacterium Lyngbya majuscula. This outstanding natural product contains a cyclopropane ring, a thiazoline ring, an internal cis double bond and a terminal alkene. The biosynthesis of curacin A is performed by a 2.2 Mega Dalton (MDa) hybrid PKS-NRPS cluster. A 10-enzyme assembly catalyzes the formation of the cyclopropane moiety as the first building block of the final product. Interestingly, for these enzymes the substrate is presented by an unusual cluster of three consecutive ACPs (ACPI,II,III). Little is known about the function of multiple ACPs which are supposed to increase the overall flux for enhanced production of secondary metabolites.
The first task in this work was to elucidate the structural effect of the triplet ACP repetition by nuclear magnetic resonance (NMR). The initial data show that the excised ACPI, ACPII or ACPIII proteins resulted in [15N, 1H]-TROSY spectra with strong chemical shift perturbations (CSPs), suggesting an effect on the structure. The triplet ACP domains display a high sequence identity (93- 100%) making structural investigation using usual NMR techniques due to high peak overlap impossible. To enable the investigation of the triplet ACP in its native composition we developed a powerful method, the three fragment ligation. Segmental labeling allows incorporating isotopes into one single domain in its multidomain context. As a result we could prepare the triplet ACP with only one domain isotopically labeled and therefore assign the full length protein. In this way our method paved the way to study the structural effects of the triplet ACP repetition. We could show unexpectedly, that, despite the fact that the triplet repeat of CurA ACPI,II,III has a synergistic effect in the biosynthesis of CurA, the domains are structurally independent.
In the second part of this work, we studied the structure of the isolated ACPI domain. Our results show that the CurA ACPI undergoes no major conformational changes upon activation via phosphopantetheinylation and therefore contradicts the conformational switching model which has been proposed for PCPs. Further we report the NMR solution structures of holo-ACPI and 3-hydroxyl-3-methylglutaryl (HMG)-ACPI. Data obtained from filtered nuclear overhauser effect (NOE) experiments indicate that the substrate HMG is not sequestered but presented on the ACP surface.
In the third part of this work we focussed on the protein-protein interactions of the isolated ACPI with its cognate interaction partners. We were especially interested in the interaction with the halogenase (Cur Hal), the first enzyme within the curacin A sub-cluster, acting on the initial hydroxyl-methyl-glutaryl (HMG) attached to ACPI. Primarily we studied the interaction using NMR titration and fluorescence anisotropy measurements. Surprisingly no complex between ACPI and Cur Hal could be detected. The combination of an activity assay using matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy and mutational analysis revealed several amino acids of ACPI that strongly decrease the activity of CurA Hal. Mapping these mutations according to their effect on the Cur Hal activity onto the structure of HMG-ACPI displays that these amino acids surround the substrate and form a consecutive surface. These results suggest that this surface is important for Cur Hal recognition and selectivity. Our research presented herein is an excellent example for protein-protein interactions in PKS systems underlying a specific recognition process.
Rotary adenosine triphosphate (ATP)ases are ubiquitous, membrane-bound enzyme complexes involved in biological energy conversion. The first subtype, the so-called F1Fo ATP synthase, predominantly functions as an ATP synthesizing machinery in most bacteria, mitochondria and chloroplasts. The vacuolar subtype of enzyme, the V1Vo ATPase, operates as an ATP driven ion pump in eukaryotic membranes. The subtype found in archaea and some bacteria is called A1Ao ATP (synth)ase and is capable of working in both directions either to synthesize ATP or to generate an ion motive force by consuming the same.
All the three above-mentioned subtypes of rotary ATPases work as nanomolecular machines sharing a conserved mechanism to perform the energy conservation process. The simplest form of these enzymes is the bacterial F1Fo ATP synthase. Here, ions are channelled via the membrane stator subunit a to the rotor ring of the enzyme. After almost a complete rotation of the ring the ions are released again on the other side of the membrane. This rotation is further transmitted via the central stalk to the soluble part of the enzyme, the F1-complex, where conformational changes within the nucleotide binding sites result in the synthesis of ATP from ADP and Pi.
The rotor or c-ring of the enzyme is the key protein complex in mediating transmembrane ion translocation. Several structural and biochemical methods have been applied in the past years to study the rotor rings from many different organisms. The results revealed that the stoichiometry of a c-ring of a given species is constant while it can vary between different species within a range of 8 to 15 c subunits. The c-ring stoichiometry determines directly the number of ions transported through Fo per rotation whereby three molecules of ATP are concurrently synthesized in the water-soluble F1 headgroup. Hence the number of c subunits has an important influence on the bioenergetics of the corresponding enzyme and thus the entire organism.
The c-ring of a rotary ATPase is able to specifically bind either protons (H+) or sodium ions (Na+) as the coupling ion for the enzyme. Several structures are already available revealing the coordination network of both types of rotor rings. In each case ion binding includes a highly-conserved carboxylic acid residue (glutamate or aspartate), in addition to a more varying combination of amino acid residues, whereby Na+ coordination is structurally more demanding than H+ binding.
In the first part of my PhD thesis, I aimed to characterize the F1Fo ATP synthase rotor ring of the opportunistic pathogenic bacterium Fusobacterium nucleatum on a functional and structural level. F. nucleatum is an anaerobic bacterium which uses peptides and amino acids as a primary energy source. It is one of the most frequently occuring bacteria in human body infections and involved in human periodontal diseases.
The protein complex was heterologously expressed within a hybrid ATP synthase in Escherichia coli and purified without an affinity tag for further analysis. Two high resolution X-ray structures of the c-ring were solved at low (5.3) and high (8.7) pH to 2.2 and 2.64 Å, respectively. In both structures, the conserved glutamate is in an ion-locked conformation, revealing that the conformational state of the ion binding carboxylate is not depending on the pH of the crystallization condition, which is in good agreement with previous structural and biochemical studies of other c-rings.
A Na+ ion is present within the c-ring binding site and directly coordinated by four amino acid residues and a structural water molecule. Remarkably, the Na+ is bound by two glutamate residues instead of one as is the case in the I. tartaricus Na+ binding c-ring, of which the first high resolution X-ray structure of a c-ring has been solved in 2005. Thus, a new type of Na+ coordination in an ATP synthase rotor ring with a two-carboxylate ion binding motif is described here, which also occurs in other bacteria, including several pathogens. Na+ specificity of the investigated c-ring was further confirmed by a competitive biochemical labeling reaction performed with a fluorescent ATP synthase inhibitor molecule (N-cyclohexyl-N`-[4(dimethylamino)-α-naphtyl] carbodiimide, NCD-4).
We furthermore complemented our functional and structural data of the F. nucleatum c-ring by computational studies to explore the ion translocation mechanism of this enzyme in more details. We therefore analyzed the protonation state of the second, additional glutamate in the ion binding site. Molecular dynamics (MD) simulations and free-energy calculations indicated that this glutamate is constitutively protonated, in the ion-locked as well as in a simulated, more hydrated open-conformation of the ion binding glutamate as when it is travelling through the a/c-ring interface upon c-ring rotation.
Cells perform a wide range of functions such as signalling, transportation, immunoprotection and metabolism. Unravelling the molecular mechanism behind those processes will provide a platform for more targeted and rational drug design. This is achieved by discerning the structural and functional aspects of the biological macromolecules involved. This thesis discusses about the biophysical characterization of protein structures and the biological importance of protein dynamics. Membrane receptors and enzymes which are ubiquitously present in our biological systems and regulate wide variety of functions are excellent choice for such study. From a pharmaceutical point of view, receptor and enzymes are exceptionally important drug targets as they represent the major share (receptor, 30% and enzymes, 47%) of all marketed drugs. Therefore, apart from biological insights, the detailed study of receptors and enzymes will provide the basis for new pharmaceutical applications. Most information about receptor activation and enzyme activity come from the structural and functional analysis of target members of the above mentioned systems.
In “Chapter 1 – General Introduction” the readers are introduced to the world of proteins with special focus on G-protein coupled receptors (GPCRs) and methyltransferases. The first part of this chapter discusses about GPCRs with emphasis on their classification, structural features and functions. GPCRs are the most abundant membrane receptors present in mammalian cells, accounting for almost 15% of all membrane proteins. The GPCR superfamily consists of ~800 members and can be subdivided into six classes (A-F). Class A containing rhodopsin, peptide hormones, olfactory GPCRs, is the most abundant with a large share of 85% of GPCR protein family. GPCRs share a common architecture of 7 transmembrane a-helices, with different ligand binding sites. Although a variety of ligands ranging from subatomic particles (a photon) to large proteins can activate a GPCR, their mechanism of signal transduction is almost similar. There are two major signal transduction pathways identified for GPCRs: the cAMP pathway and the phosphatidylinositol pathway. The therapeutic relevance of GPCRs has also been pointed out here since a large share (30%) of modern marketed drugs target GPCRs.
In the second part of this chapter, the structural and functional characterizations of methyltransferases (MTs) are discussed in detail. Several important biological processes in cells e.g. drug metabolism, gene transcription, epigenetic regulations are modulated by methylation of targets ranging from small biomolecules to large proteins. MTs are the proteins which catalyze this methylation reaction and transfer the methyl group to an acceptor molecule through SN2 like nucleophilic substitution reaction. The MTs can be classified on the basis of the substrate atoms they methylate: O (54% of all MTs), N (23%), C (18%), S (3%) and other acceptors (such as halides; 2%). They can also be categorized into five different classes (Class I-V) depending upon distinctive structural features facilitating substrate binding or catalytic activity. Rossmann fold and SET (acronym acquired from the Drosophila Su(var)3-9 and 'Enhancer of zeste' proteins) domain are the two characteristic structural motifs commonly found in MTs. Similar to GPCRs, MTs dysfunction has been shown to be involved in various diseases including neuropsychiatric diseases and cancer. Therefore they are also interesting targets for drug development. The final part of this chapter discusses the importance of structural biology in gathering information related to structure and conformational dynamics of proteins. The two prominent biophysical techniques used in structural biology, X-ray crystallography and NMR, are discussed with focus on their advantages and limitation. The importance of NMR spectroscopic techniques to investigate different dynamic processes of protein at atomic resolution under physiological conditions is also discussed. Real time NMR spectroscopy required for the analysis of slow protein dynamic processes (protein folding, enzyme catalysis, domain rearrangement) has been explained in detail.
The second part of the thesis (Chapters 3-4), which is the cumulative part, comprises the original publications grouped into 2 chapters according to their topic:
• NMR-spectroscopic characterization of the transiently populated photointermediates of bovine rhodopsin and it’s interaction with arrestin (Chapter 3)
• Structural and biophysical characterization of PaMTH1, a putative SAM dependent O-methyltransferase from filamentous fungi Podospora anserina (Chapter 4)
Each chapter is initiated by a detailed introduction to the topic, providing the framework for the following papers. The personal contribution of this thesis’ author to each publication is stated in the introduction to the respective article.
Respiration is one of the key processes of energy transduction used by the cell. It consists of two components: electron transfer and ATP production. The electron transfer chain converts the energy released from several biochemical redox reactions into an electrochemical proton gradient across membranes. This stored energy is used as the driving force for the production of ATP by the ATP synthase. The mitochondrial electron transfer chain contains four major protein complexes called complexes I-IV, with counting starting at the lower side of the redox potentials. It has been discussed for a long time how these protein complexes are organized in the membranes. Do they diffuse freely in the membrane? Alternatively, do they form a supercomplex built up of several neighboring complexes? The evidence supporting the free diffusion mode is that both electron transfer intermediates (cytochrome c and quinone) behave as “pool”. However, respiratory supercomplexes have been detected in membranes from bacteria, fungi, yeast, plant and animal during the last decade, and sometimes the respiratory complexes are only stable inside a supercomplex. Therefore, the idea of supercomplex formation has become more popular. The argument that the supercomplex arises from solubilization and is a detergent artifact could be rejected because: 1) supercomplexes can be isolated from many organisms in an active form; 2) supercomplexes have been proven to stabilize the individual complexes in some cases; 3) supercomplexes can be very stable after chromatographic isolation in some cases....
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.
PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. The crystal structure of a Drosophila PERIOD (dPER) fragment comprising two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B) and two additional C-terminal alpha-helices (alphaE and alphaF) has revealed a homodimer mediated by intermolecular interactions of PAS-A with tryptophane 482 in PAS-B and helix alphaF. Here we present the crystal structure of a monomeric PAS domain fragment of dPER lacking the alphaF helix. Moreover, we have solved the crystal structure of a PAS domain fragment of the mouse PERIOD homologue mPER2. The mPER2 structure shows a different dimer interface than dPER, which is stabilized by interactions of the PAS-B beta-sheet surface including tryptophane 419 (equivalent to Trp482dPER). We have validated and quantitatively analysed the homodimer interactions of dPER and mPER2 by site-directed mutagenesis using analytical gel filtration, analytical ultracentrifugation, and co-immunoprecipitation experiments. Furthermore we show, by yeast-two-hybrid experiments, that the PAS-B beta-sheet surface of dPER mediates interactions with TIMELESS (dTIM). Our study reveals quantitative and qualitative differences between the homodimeric PAS domain interactions of dPER and its mammalian homologue mPER2. In addition, we identify the PAS-B beta-sheet surface as a versatile interaction site mediating mPER2 homodimerization in the mammalian system and dPER-dTIM heterodimer formation in the Drosophila system.
The transporter associated with antigen processing (TAP) is a heterodimeric ATP-binding cassette (ABC) transport complex, which selects peptides for export into the endoplasmic reticulum (ER) and subsequent loading onto major histocompatibility complex class I (MHC I) molecules to trigger adaptive immune responses against virally or malignantly transformed cells. Due to its pivotal role in adaptive immunity, TAP is a target for infectious diseases and malignant disorders, such as bare lymphocyte syndrome type I and cancer. A detailed knowledge about the TAP structure and transport mechanism is fundamental for the development of therapies or drugs against such diseases, but numerous aspects are insufficiently determined to date. The aim of this PhD thesis was to elucidate several structural details of TAP using powerful biochemical and biophysical methods and thereby to contribute to the understanding of the translocation machinery functionality.
High protein yields, an efficient isolation from the lipid environment and subsequent purification of a stoichiometric, stable, and functional TAP complex are prerequisites to get detailed insights into TAP functionality. The natural product digitonin is typically used as detergent to isolate TAP, but suffered from fluctuating purity and high costs. The novel detergent GDN was selected from a number of potential detergents upon their ability to isolate and purify TAP overcoming the limitations of digitonin without compromising on functional integrity. State-of-the-art biophysical techniques, such as solid-state nuclear magnetic resonance (NMR), require highly concentrated protein samples. A new and mild procedure to concentrate TAP was established within this thesis. Freeze drying is superior to conventional concentration techniques, such as ultrafiltration, resulting in TAP inactivation and aggregation already at concentrations of 10 mg/mL. This new procedure enables stabilizing TAP in a condensed glycerol matrix and to concentrate the transport complex up to 30 mg/mL active transporter. The functional integrity of the freeze-dried TAP complex was verified by determining equilibrium dissociation constants, peptide dissociation and ATP-hydrolysis rates as well as long-term stabilities identical to untreated TAP. The combined application of the detergent GDN and the freeze drying procedure facilitates the cost-efficient isolation of functional and highly concentrated TAP and enables to study the structure and mechanism of the peptide transporter TAP using modern analyses methods.
Information on peptide-TAP interactions at atomic level have not been obtained so far. This lack of knowledge hampered the mechanistic understanding of the initial steps of substrate translocation catalyzed by TAP. Dynamic nuclear polarization (DNP) enhanced magic angle spinning (MAS) solid-state NMR on highly concentrated TAP samples prepared with the freeze-drying procedure was used within this thesis to study this challenging membrane protein-substrate complex. The affinity and specificity of peptide binding by TAP are mediated by multiple recognition sites in the N- and C-terminal regions. Side-chains of positions 1, 3, and 9 are most substantially affected upon binding to TAP, revealing recognition principles of the translocation machinery. The nonamer peptide binds to TAP in an extended conformation with an N-to-C terminus distance of ~2.5 nm. Molecular docking revealed that the peptide substrate is locked with its N and C termini between TAP1 and TAP2 and adopts a tilted pose with respect to the membrane plane. The identified contact sites of TAP are consistent with results from earlier crosslinking and mutational analyses on the TAP complex.
The inadequate structure determination and insufficient knowledge about the dynamics of substrate translocation impedes a detailed comprehension of the TAP transport mechanism. Advanced biophysical methods, such as pulsed electron paramagnetic resonance (EPR) or single-molecule Förster resonance energy transfer (FRET), enable to locate the peptide-binding pocket and to elucidate dwell-times, conformational states and dynamics within the translocation cycle of TAP. The specific introduction of spin or fluorescent labels via single cysteines for such studies requires a cysteine-less TAP complex. The endogenous cysteine 213 in TAP2 remained to create a pseudo Cys-less TAP complex within this thesis due to its altered substrate repertoire when mutated to serine as shown in previous studies. Latter complex was used to introduce single-Cys mutations in the cytosolic extensions of transmembrane helices of TAP1. Their functional integrity with respect to peptide binding and translocation was comparable to pseudo Cys-less TAP. All pseudo single cysteines were efficiently labeled, but unintentionally C213TAP2 was labeled as well and TAP concomitantly inactivated. These unsatisfactory initial experiments required the generation of a functional, entirely Cys-less TAP transporter within this thesis. Therefore, C213TAP2 was replaced by all 19 proteinogenic amino acids. All analyzed mutants were capable to bind a high-affinity peptide of TAP, but with varying affinities and binding capacities. The replacement of C213 by isoleucine enabled the generation of a cysteine-less TAP complex with functional characteristics similar to the wild-type transporter and will promote the elucidation of the translocation mechanism of the peptide transporter TAP in future studies using pulsed EPR and single-molecule FRET.
Megasynthases are large multienzyme proteins that produce a plethora of important natural compounds by catalyzing the successive condensation and modification of precursor units. Within the class of megasynthases, polyketide synthases (PKS) are responsible for the production of a large spectrum of bioactive polyketides (PK), which have frequently found their way into therapeutic applications. Rational engineering approaches have been performed during the last 25 years that seek to employ the "assembly-line synthetic concept" of megasynthases in order to deliver new bioactive compounds. Here, we highlight PKS engineering strategies in the light of the newly emerging structural information on megasynthases, and argue that fatty acid synthases (FAS) are and will be valuable objects for further developing this field.
Membrane proteins (MPs) constitute about 30% of the genome and are essential in many cellular processes. In particular structural characterisation of MPs is challenged by their hydrophobic nature resulting in expression difficulties and structural instability upon extraction from the membrane. Despite these challenges, progress in sample preparation and the techniques to solve MP structures has led to 281 unique MP structures as of January 2011. Through the combination of a cell-free expression system and selective labelling strategies, this thesis aimed to advance the structure determination of α-helical MPs by NMR spectroscopy and resulted in the structure determination of a seven-ransmembrane-helix protein. Results were obtained for the 5-lipoxygenase-activating protein (FLAP) and proteorhodopsin (PR). The detergent-based cell-free expression mode proved most efficient for production of both targets, but optimisation of FLAP and PR followed different routes. The presence of a retinal cofactor in PR greatly facilitated the search for an appropriate hydrophobic environment. For structural studies, NMR spectra of FLAP indicated favourable properties of the lysolipid LPPG. In contrast, PR was stable and homogenous in the short-chain lipid diC7PC. As NMR spectra of α-helical MPs are generally characterised by broad lines and signal overlap, selective labelling strategies were essential in the assignment process of both targets. For the backbone assignment of FLAP the transmembrane segment-enhanced (TMS) labelling was developed, employing the six amino acids AFGILV. These residues cluster predominantly in transmembrane helices and form long stretches allowing a large extent of backbone assignment. Besides that, the combinatorial labelling enables identification of unique pairs in the sequence based on a mixture of 15N and 1-13C-labelled amino acids. To find the optimal labelling pattern for a given primary structure, the UPLABEL algorithm has been made available and successfully applied in the backbone assignment of PR. Both selective labelling approaches greatly benefitted from the use of a cell-free expression system to reduce isotope scrambling. Additionally, the de novo structure of PR was determined with an average backbone rmsd of 1.2 Å based on TALOS-derived backbone torsion angles, intrahelical hydrogen bond restraints and distance restraints from the NOE and paramagnetic relaxation enhancement (PRE). A major bottleneck in the NMR structure determination of MPs concerns the number of long-range distances which are often limited. In PR, side chain assignment was enabled by stereo-array isotope labelling as well as selective labelling which provided 33 long-range NOEs. These NOEs stabilised the symmetry of the seven helix bundle. With a total number of 1031, the majority of long-range distances were derived from PREs. The structure of PR reveals differences to its homologues such as the absence of an anti-parallel β-sheet between helices B and C and allows conclusions towards the mechanism of colour tuning.
The recently developed stereospecific sodium salt glycosylation procedure has been successfully applied to the synthesis of the β-ᴅ-2′-deoxyribofuranosides of benzimidazole, 5,6-dihalogeno benzimidazoles, and some 2-substituted analogues in high yield. The 5,6-dibromo analogue was obtained by bromination of the parent nucleoside. These have all been characterized by spectroscopic methods, including 1H NMR, which permitted analyses of their solution conformations and comparison with those of the corresponding ribofuranosides. Some biological aspects, including preliminary results on cytotoxicity and antiviral activity, are briefly considered.
The centrosome linker proteins C-Nap1, rootletin, and CEP68 connect the two centrosomes of a cell during interphase into one microtubule-organizing center. This coupling is important for cell migration, cilia formation, and timing of mitotic spindle formation. Very little is known about the structure of the centrosome linker. Here, we used stimulated emission depletion (STED) microscopy to show that each C-Nap1 ring at the proximal end of the two centrioles organizes a rootletin ring and, in addition, multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers originating from the two centrosomes form a web-like, interdigitating network, explaining the flexible nature of the centrosome linker. The rootletin/CEP68 filaments are repetitive and highly ordered. Staggered rootletin molecules (N-to-N and C-to-C) within the filaments are 75 nm apart. Rootletin binds CEP68 via its C-terminal spectrin repeat-containing region in 75-nm intervals. The N-to-C distance of two rootletin molecules is ∼35 to 40 nm, leading to an estimated minimal rootletin length of ∼110 nm. CEP68 is important in forming rootletin filaments that branch off centrioles and to modulate the thickness of rootletin fibers. Thus, the centrosome linker consists of a vast network of repeating rootletin units with C-Nap1 as ring organizer and CEP68 as filament modulator.
On the basis of the results obtained in a previous paper it is shown that in the thermodynamic limit the analogues of the Massieu-Plandc functions are linked with each other by means of the Legendre transformation. The existence of the limiting function φk(∞) implies the existence of the limiting function φl(∞) (l<k) under the same assumptions. Passage to the limit and derivation with respect to all independent variables commute. A statistical derivation of the thermodynamic stability condition in its most general form is given which leads naturally to a statistical interpretation of the concept of thermodynamic stability.
It is shown that, for all conceivable ensembles of statistical thermodynamics, at the thermodynamic limit, the frequency function of the fluctuations of macroscopic extensive parameters equals a Gaussian. The proof is based on a generalisation of Khinchin's method using the concept of "smoothed frequency functions."
The group of neurodegenerative diseases, Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) all exhibit inclusions containing amyloid-type α-synuclein (α-syn) aggregates within degenerating brain cells. α-syn also exists as soluble oligomeric species that are hypothesized to represent intermediates between its native and aggregated states. These oligomers are present in brain extracts from patients suffering from synucleinopathies and hold great potential as biomarkers. Although easily prepared in vitro, oligomers are metastable and dissociate over time, thereby complicating α-syn oligomer research. Using the small amine-reactive cross-linker, formaldehyde (FA), we successfully stabilized α-syn oligomers without affecting their size, overall structure or antigenicity towards aggregate-conformation specific α-syn antibodies FILA and MJFR-14-6-4-2. Further, cross-linked α-syn oligomers show resistance towards denaturant like urea and SDS treatment and remain fully functional as internal standard in an aggregation-specific enzyme-linked immunosorbent assay (ELISA) despite prior incubation with urea. We propose that FA cross-linked α-syn oligomers could serve as important calibrators to facilitate comparative and standardized α-syn biomarker studies going forward.
Split intein enabled protein trans-splicing (PTS) is a powerful method for the ligation of two protein fragments, thereby paving the way for various protein modification or protein function control applications. PTS activity is strongly influenced by the amino acids directly flanking the splice junctions. However, to date no reliable prediction can be made whether or not a split intein is active in a particular foreign extein context. Here we describe SPLICEFINDER, a PCR-based method, allowing fast and easy screening for active split intein insertions in any target protein. Furthermore we demonstrate the applicability of SPLICEFINDER for segmental isotopic labeling as well as for the generation of multi-domain and enzymatically active proteins.
The thermal decomposition of 1,2-diadamantyldioxetane was studied by kinetic and spectroscopic methods. Spectra of the chemiluminescence emitted during the thermally induced decomposition of 1,2-diadamantyldioxetane, tetramethyldioxetane and trimethyldioxetane were obtained and the influence of quenchers and radical-scavengers, and the presence of "heavy atoms" in the surrounding of the emitting species was investigated. The kinetics of the decay mechanism was followed by measuring the time dependence of the chemiluminescence. The influence of radical-scavengers, quenchers and "external heavy atoms" on the kinetics was assessed. Experimental results were discussed in terms of a biradical decay mechanism.
Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene BCR-ABL1T315I differ from BCR-ABL1 native (BCR-ABL1) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin β3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1T315I+ tumor burden and significantly prolonged survival of mice with BCR-ABL1T315I+ CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin β3/ILK-mediated signaling pathway in BCR-ABL1T315I+ cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting.
The transporter associated with antigen processing (TAP) plays a key role in adaptive immunity by translocating proteasomal degradation products from the cytosol into the endoplasmic reticulum lumen for subsequent loading onto major histocompatibility (MHC) class I molecules. For functional and structural analysis of this ATP-binding cassette complex, we established the overexpression of TAP in the methylotrophic yeast Pichia pastoris. Screening of optimal solubilization and purification conditions allowed the isolation of the heterodimeric transport complex, yielding 30 mg of TAP/liter of culture. Detailed analysis of TAP function in the membrane, solubilized, purified, and reconstituted states revealed a direct influence of the native lipid environment on activity. TAP-associated phospholipids, essential for function, were profiled by liquid chromatography Fourier transform mass spectrometry. The antigen translocation activity is stimulated by phosphatidylinositol and -ethanolamine, whereas cholesterol has a negative effect on TAP activity.
Optogenetic approaches using light-activated proteins like Channelrhodopsin-2 (ChR2) enable investigating the function of populations of neurons in live Caenorhabditis elegans (and other) animals, as ChR2 expression can be targeted to these cells using specific promoters. Sub-populations of these neurons, or even single cells, can be further addressed by restricting the illumination to the cell of interest. However, this is technically demanding, particularly in free moving animals. Thus, it would be helpful if expression of ChR2 could be restricted to single neurons or neuron pairs, as even wide-field illumination would photostimulate only this particular cell. To this end we adopted the use of Cre or FLP recombinases and conditional ChR2 expression at the intersection of two promoter expression domains, i.e. in the cell of interest only. Success of this method depends on precise knowledge of the individual promoters' expression patterns and on relative expression levels of recombinase and ChR2. A bicistronic expression cassette with GFP helps to identify the correct expression pattern. Here we show specific expression in the AVA reverse command neurons and the aversive polymodal sensory ASH neurons. This approach shall enable to generate strains for optogenetic manipulation of each of the 302 C. elegans neurons. This may eventually allow to model the C. elegans nervous system in its entirety, based on functional data for each neuron.