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Orthopoxviruses are large DNA viruses that replicate within the cytoplasm of infected cells encoding over a hundred different proteins. The orthopoxviral 68k ankyrin‐like protein (68k‐ank) is highly conserved among orthopoxviruses, and this study aimed at elucidating the function of 68k‐ank. The 68k‐ank protein is composed of four ankyrin repeats (ANK) and an F‐box‐like domain; both motifs are known proteinprotein interaction domains. The F‐box is found in cellular F‐box proteins (FBP), crucial components of cellular E3 ubiquitin (Ub) ligases. With yeast‐two‐hybrid screens and subsequent co‐immunoprecipitation analyses, it was possible to identify S‐phase kinase‐associated protein 1a (Skp1a) as a cellular counterpart of 68k‐ank via binding to the F‐box‐like domain. Additionally, Cullin‐1 was co‐precipitated, suggesting the formation of a viral‐cellular SCF E3 Ub ligase complex. Modified Vaccinia virus Ankara (MVA) ‐ being attenuated and unable to replicate in most mammalian cell lines due to a block in morphogenesis – nevertheless, expresses its complete genetic information attributing to its properties as promising vector vaccine. Conservation of 68k‐ank as the only ANK protein encoded by MVA implied a substantial role of this viral factor. Hence, its function in the viral life cycle was assessed by studying a 68k‐ank knock‐out MVA. A mutant phenotype manifested in nonpermissive mammalian cells characterized by a block succeeding viral early gene expression and by a reduced ability of the virus to shutoff host protein synthesis. Studies with MVA encoding a 68k‐ank F‐box‐like domain truncated protein revealed that viral‐cellular SCF complex formation and maintenance of viral gene expression are two distinct, unrelated functions fulfilled by 68k‐ank. Moreover, K1, a well‐described VACV host range factor of the ANK protein family, is able to complement 68k‐ank function. This suggests that gene expression of MVA putatively depends on the ANKs encoded in 68k‐ank. In addition to the important findings in vitro, first virulence studies with the mouse pox agent, ectromelia virus (ECTV) deleted of the 68k‐ank ortholog (C11) suggested that this factor contributes to ECTV virulence in vivo.
During the last decade of the 20th century, the field of mass spectrometry has seen a revolutionary change in its application and scope. The introduction of soft ionization methods for the analysis of biological molecules has expanded the area of mass spectrometry from its early roots in the analysis of inorganic and organic species into the fields of biology and medicine.
Today, the use of the mass spectrometry is extended to a wide range of applications in biotechnology and pharmaceutical industry, in geological, environmental and clinical research. In biochemistry, the principles of mass spectrometry are, however, broadly applicable in accurate molecular weight determination, reaction monitoring, amino acid sequencing, oligonucleotide sequencing and protein structure.
In order to carry out their biological activities, proteins interact most often to each other and form transient or stable complexes. In addition, some proteins specifically interact also with other proteins or with non-protein molecules, such as DNA, RNA or metabolites, these interactions being critical for their function. Hence, defining the composition of protein complexes, as well as understanding how protein complexes are assembled and regulated yield invaluable insights into protein function. Coupled with an isolation technique to purify a specific protein complex of interest, mass spectrometry can rapidly and reliably identify the components of complexes. In addition, quantitative MS techniques offer the possibility of studying dynamically regulated interactions....
Characterization of mouse NOA1 : subcellular localizaion, G-Quadruplex binding and proteolysis
(2013)
Mitochondria contain their own protein synthesis machinery with mitoribosomes that are similar to prokaryotic ribosomes. The thirteen proteins encoded in the mitochondrial genome are members of the respiratory chain complexes that generate a proton gradient, which is the electromotoric force for ATP synthesis.
NOA1 (Nitric Oxide Associated Protein-1) is a nuclear encoded GTPase that positively influences mitochondrial respiration and ATP production. Although a role in mitoribosome assembly was assigned to NOA1 the underlying molecular mechanism is poorly understood. This work shows that the multi-domain protein NOA1 serves multiple purposes for the function of mitochondria. NOA1 is a dual localized protein that makes a detour through the nucleus before mitochondrial import. The nuclear shuttling is mediated by a nuclear localization signal and the now identified nuclear export signal. SELEX (Systemic Evolution of Ligands by Exponential Enrichment) analysis revealed a G-quadruplex binding motif that characterizes NOA1 as ribonucleoprotein (RNP). G-quadruplex binding was coupled to the GTPase activity and increased the GTP hydrolysis rate. The sequence of localization events and the identification of NOA1 being a RNP lead to the discussion of an alternative import pathway for RNPs into mitochondria. The short-lived NOA1 contains ClpX recognition motifs and is specifically degraded by the mitochondrial matrix protease ClpXP. NOA1 is the first reported substrate of ClpXP in higher eukaryotes and augments the contribution of the ClpXP protease for mitochondrial metabolism. To assess the direct action of NOA1 on the mitoribosome co-sedimentation assays were performed. They showed that the interaction of NOA1 and the mitoribosome is dependent on the GTPase function and the nascent peptide chain. In vitro, NOA1 facilitated the membrane insertion of newly translated and isotope labeled mitochondrial translation products into inverted mitochondrial inner membrane vesicles. In conclusion, NOA1 is a G-quadruplex-RNP that acts as mitochondrial membrane insertion factor for mtDNA-encoded proteins.
This thesis provides a comprehensive model of the molecular function of NOA1 and is the basis for future research. The identification of NOA1 as ClpXP substrate is a major contribution to the field of mitochondrial research.
HDAC inhibitors (HDACI), a new class of anticancer agents, induce apoptosis in many cancer entities. JNJ-26481585 is a second generation class І HDACI that displays improved efficacy in preclinical studies compared to the established HDACI SAHA (Vorinostat). Therefore, this study aims at evaluating the effects of JNJ-26481585 on human rhabdomyosarcoma (RMS) and at identifying novel synergistic interactions of JNJ-26481585 or the more common HDACI SAHA with different anticancer drugs in RMS cells. Indeed, we show that JNJ-26481585 and SAHA significantly increase chemotherapeutic drug-induced apoptosis in embryonal and alveolar RMS cell lines, when used in combination with chemotherapeutic agents (i.e. doxorubicin, etoposide, vincristine, and cyclophosphamide) which are currently used in the clinic for the treatment of RMS.
We demonstrate that JNJ-26481585 as single agent and in combination with doxorubicin induces apoptosis, which is characterized by activation of the caspase cascade, PARP cleavage, and DNA fragmentation. Induction of caspase-dependent apoptotic cell death is confirmed by the use of the broad-range caspase inhibitor zVAD.fmk, which significantly decreases both JNJ-26481585-triggered and combination treatment-mediated DNA fragmentation, and in addition completely abrogates loss of cell viability. Importantly, JNJ-26481585 significantly inhibits tumor growth in vivo in two preclinical RMS models, i.e. the chicken chorioallantoic membrane (CAM) model and a xenograft mouse model, supporting the notion that JNJ-26481585 hampers tumor maintenance. Also, in combination with doxorubicin JNJ-26481585 significantly reduces tumor growth in in vivo experiments using the CAM model.
Mechanistically, we identify that JNJ-26481585-induced apoptosis is mediated via the intrinsic apoptotic pathway, since we observe increased loss of mitochondrial membrane potential and activation of the proapoptotic Bcl-2 family members Bax and Bak. Interestingly, we find that JNJ-26481585 triggers induction of Bim, Bmf, Puma, and Noxa on mRNA level as well as on protein level, pointing to an altered transcription of BH3-only proteins as important event for the Bax/Bak-mediated loss of mitochondrial membrane potential as well as mitochondrial apoptosis induction upon JNJ-26481585 treatment. JNJ-26481585-initiated activation of Bax and Bak is not prevented with the addition of zVAD.fmk, suggesting that JNJ-26481585 first disrupts the mitochondria and subsequently activates the caspase cascade. When JNJ-26481585 is used in combination with doxorubicin, we observe not only an increase of proapoptotic Bcl-2 proteins, but also a decrease in the level of the antiapoptotic mitochondrial proteins Bcl-2, Mcl-1, and Bcl-xL. This indicates that Bax, Bak, Bim, and Noxa are crucial for JNJ-26481585-induced as well as JNJ/Dox treatment-induced apoptosis, since RNAi mediated silencing of Bax, Bak, Bim, and Noxa significantly impedes DNA fragmentation upon those treatments.
Furthermore, ectopic overexpression of Bcl-2 profoundly impairs both JNJ-26481585 and combination treatment-mediated apoptosis, abrogates caspase cleavage, and reduces activation of Bax and Bak, underlining the hypothesis that JNJ-26481585 initially targets the mitochondria and then activates caspases.
With the more commonly used HDACI SAHA we confirm the results obtained with the HDACI JNJ-26481585, since combination treatment with SAHA and doxorubicin also induces intrinsic apoptosis, which can be significantly diminished by zVAD.fmk or ectopic overexpression of Bcl-2. Treatment with SAHA and doxorubicin also affects expression levels of pro- and antiapoptotic mitochondrial proteins, thus shifting the balance towards the proapoptotic mitochondrial machinery, resulting in Bax/Bak activation, caspase activation, and subsequently apoptosis.
Taken together, we provide evidence that the HDACIs JNJ-26481585 and SAHA are promising therapeutic agents for the treatment of RMS and that combination regimens with HDACIs represent an efficient strategy to prime RMS cells for chemotherapy-induced apoptosis. These findings have important implications for mitochondrial apoptosis-targeted therapies of RMS.
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.
Characterization of a dual BET/HDAC inhibitor for treatment of pancreatic ductal adenocarcinoma
(2020)
Pancreatic ductal adenocarcinoma (PDAC) is resistant to virtually all chemo‐ and targeted therapeutic approaches. Epigenetic regulators represent a novel class of drug targets. Among them, BET and HDAC proteins are central regulators of chromatin structure and transcription, and preclinical evidence suggests effectiveness of combined BET and HDAC inhibition in PDAC. Here, we describe that TW9, a newly generated adduct of the BET inhibitor (+)‐JQ1 and class I HDAC inhibitor CI994, is a potent dual inhibitor simultaneously targeting BET and HDAC proteins. TW9 has a similar affinity to BRD4 bromodomains as (+)‐JQ1 and shares a conserved binding mode, but is significantly more active in inhibiting HDAC1 compared to the parental HDAC inhibitor CI994. TW9 was more potent in inhibiting tumor cell proliferation compared to (+)‐JQ1, CI994 alone or combined treatment of both inhibitors. Sequential administration of gemcitabine and TW9 showed additional synergistic antitumor effects. Microarray analysis revealed that dysregulation of a FOSL1‐directed transcriptional program contributed to the antitumor effects of TW9. Our results demonstrate the potential of a dual chromatin‐targeting strategy in the treatment of PDAC and provide a rationale for further development of multitarget inhibitors.
Structural characterization of a polymethylsilsesquioxane (PMSQ) and a DT-type methyl silicone resin (MeDT) has been carried out by various instrumental analyses including GPC, NMR, gas chromatography, and gas chromatography-mass spectrometry. Although the PMSQ had a Mw around 5000, the resin contained a significant amount of low molecular weight species consisting of T2 [MeSi(OH)O2/2] and T3 [MeSiO3/2] units, ranging from T34T23 to T38T22 including many isomers. One isomer of T36T22 was isolated of which structure was determined as a cage structure. The species are supposed to consist mainly of cyclotetra- and cyclopentasiloxanes, but presence of strained rings such as cyclotrisiloxane rings also was suggested. In MeDT, species in which the T2 units in the molecules from PMSQ is replaced with D2 [Me2SiO2/2] were found, for example, T36D22, suggesting that general silicone resins consist of similar structures as silsesquioxanes. The Mark-Houwink exponent for these methyl resins was ~0.3, indicating the molecular shape to be compact. Investigation on the formation chemistry of the cubic octamers indicates that siloxane bond rearrangement is an important mechanism in the molecule build-up process.
The human endothelin receptors, ETA and ETB, are two members of the G-protein coupled receptors family (GPCRs) and they are key players in cardiovascular regulation. The characterization of their functionality in vitro has been limited by the possibility to obtain high quality samples using conventional expression systems. The Cell-Free expression system is an alternative technique for the production of membrane protein as well as GPCRs and can overcome some of the limitations that are commonly encountered using an in vivo approach. Cell-Free expression protocols for the two receptors ETA and ETB have been optimized by implementing post- and co-translational association to lipid bilayers. The efficiency of the reconstitution or association to liposomes and nanodiscs has systematically been studied and the ligand binding properties of the two receptors have been analyzed using a set of different complementary techniques. In several different conditions a high affinity binding of the peptide ligand ET-1 to both endothelin receptors could be obtained and the highest activity values were detected in sample prepared using a co-translational approach in presence of nanodiscs. Furthermore, the characteristic differential binding pattern of selected agonists and antagonists to the two receptors was confirmed. In samples obtained from several Cell-Free expression conditions, two intrinsic properties of the functionally folded ETB receptor, such as the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were detected. ETA and ETB are able to induce in vivo the activation of hetrotrimeric G proteins upon stimulation with an agonist, leading to the dissociation of the heterotrimeric complex and the exchange of GDP to GTP in the Galpha subunit. The Cell-Free expression system was chosen for the production of two G alpha subunit, Galpha s and Galpha q. Soluble expression of the two proteins was achieved and the production of active Galpha s was confirmed using fluorescent as well as radioactive assays. In conclusion, the obtained results document a new process for the production of ligand binding competent endothelin receptors, as well as Galpha proteins, using a Cell-Free expression system. The combination of this expression system and the nanodiscs technology appears to be a promising tool for the further characterization of membrane proteins as well as GPCRs.
Alzheimer’s disease (AD), which was first reported more than a century ago by Alhzeimer, is one of the commonest forms of dementia which affects >30 million people globally (>8 million in Europe). The origin and pathogenesis of AD is poorly understood and there is no cure available for the disease. AD is characterized by the accumulation of senile plaques composed of amyloid beta peptides (Ab 37-43) which is formed by the gamma secretase (GS) complex by cleaving amyloid precursor protein. Therefore GS can be an attractive drug target. Since GS processes several other substrates like Notch, CD44 and Cadherins, nonspecific inhibition of GS has many side effects. Due to the lack of crystal structure of GS, which is attributed to the extreme difficulties in purifying it, molecular modeling can be useful to understand its architecture. So far only low resolution cryoEM structures of the complex has been solved which only provides a rough structure of the complex at low 12-15 A resolution Furthermore the activity of GS in vitro can be achieved by means of cell-free (CF) expression.
GS comprises catalytic subunits namely presenilins and supporting elements containing Pen-2, Aph-1 and Nicastrin. The origin of AD is hidden in the regulated intramembrnae proteolysis (RIP) which is involved in various physiological processes and also in leukemia. So far growth factors, cytokines, receptors, viral proteins, cell adhesion proteins, signal peptides and GS has been shown to undergo RIP. During RIP, the target proteins undergo extracellular shredding and intramembrane proteolysis.
This thesis is based on molecular modeling, molecular dynamics (MD) simulations, cell-free (CF) expression, mass spectrometry, NMR, crystallization, activity assay etc of the components of GS complex and G-protein coupled receptors (GPCRs).
First I validated the NMR structure of PS1 CTF in detergent micelles and lipid bilayers using coarse-grained MD simulations using MARTINI forcefield implemented in Gromacs. CTF was simulated in DPC micelles, DPPC and DLPC lipid bilayer. Starting from random configuration of detergent and lipids, micelle and lipid bilyer were formed respectively in presence of CTF and it was oriented properly to the micelle and bilyer during the simulation. Around DPC molecules formed micelle around CTF in agreement of the experimental results in which 80-85 DPC molecules are required to form micelles. The structure obtained in DPC was similar to that of NMR structure but differed in bilayer simulations showed the possibility of substrate docking in the conserved PAL motif. Simulations of CTF in implicit membrane (IMM1) in CHAMM yielded similar structure to that from coarse grained MD.
I performed cell-free expression optimization, crystallization and NMR spectroscopy of Pen-2 in various detergent micelles. Additionally Pen-2 was modeled by a combination of rosetta membrane ab-initio method, HHPred distant homology modeling and incorporating NMR constraints. The models were validated by all atom and coarse grained MD simulations both in detergent micelles and POPC/DPPC lipid bilayers using MARTINI forcefield.
GS operon consisting of all four subunits was co-expressed in CF and purified. The presence of of GS subunits after pull-down with Aph-1 was determined by western blotting (Pen-2) and mass spectrometry (Presenilin-1 and Aph-1). I also studied interactions of especially PS1 CTF, APP and NTF by docking and MD.
I also made models and interfaces of Pen-2 with PS1 NTF and checked their stability by MD simulations and compared with experimental results. The goal is to model the interfaces between GS subunits using molecular modeling approaches based on available experimental data like cross-linking, mutations and NMR structure of C-terminal fragment of PS1 and transmembrane part of APP. The obtained interfaces of GS subunits may explain its catalysis mechanism which can be exploited for novel lead design. Due to lack of crystal/NMR structure of the GS subunits except the PS1 CTF, it is not possible to predict the effect of mutations in terms of APP cleavage. So I also developed a sequence based approach based on machine learning using support vector machine to predict the effect of PS1 CTF L383 mutations in terms of Aβ40/Aβ42 ratio with 88% accuracy. Mutational data derived from the Molgen database of Presenilin 1 mutations was using for training.
GPCRs (also called 7TM receptors) form a large superfamily of membrane proteins, which can be activated by small molecules, lipids, hormones, peptides, light, pain, taste and smell etc. Although 50% of the drugs in market target GPCRs , only few are targeted therapeutically. Such wide range of targets is due to involvement of GPCRs in signaling pathways related to many diseases i.e. dementia (like Alzheimer's disease), metabolic (like diabetes) including endocrinological disorders, immunological including viral infections, cardiovascular, inflammatory, senses disorders, pain and cancer.
Cannabinoid and adrenergic receptors belong to the class A (similar to rhodopsin) GPCRs. Docking of agonists and antagonists to CB1 and CB2 cannabinoid receptors revealed the importance of a centrally located rotamer toggle switch, and its possible role in the mechanism of agonist/antagonist recognition. The switch is composed of two residues, F3.36 and W6.48, located on opposite transmembrane helices TM3 and TM6 in the central part of the membranous domain of cannabinoid receptors. The CB1 and CB2 receptor models were constructed based on the adenosine A2A receptor template. The two best scored conformations of each receptor were used for the docking procedure. In all poses (ligand-receptor conformations) characterized by the lowest ligand-receptor intermolecular energy and free energy of binding the ligand type matched the state of the rotamer toggle switch: antagonists maintained an inactive state of the switch, whereas agonists changed it. In case of agonists of β2AR, the (R,R) and (S,S) stereoisomers of fenoterol, the molecular dynamics simulations provided evidence of different binding modes while preserving the same average position of ligands in the binding site. The (S,S) isomer was much more labile in the binding site and only one stable hydrogen bond was created. Such dynamical binding modes may also be valid for ligands of cannabinoid receptors because of the hydrophobic nature of their ligand-receptor interactions. However, only very long molecular dynamics simulations could verify the validity of such binding modes and how they affect the process of activation.
Human N-formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) involved in many physiological processes, including host defense against bacterial infection and resolving inflammation. The three human FPRs (FPR1, FPR2 and FPR3) share significant sequence homology and perform their action via coupling to Gi protein. Activation of FPRs induces a variety of responses, which are dependent on the agonist, cell type, receptor subtype, and also species involved. FPRs are expressed mainly by phagocytic leukocytes. Together, these receptors bind a large number of structurally diverse groups of agonistic ligands, including N-formyl and nonformyl peptides of different composition, that chemoattract and activate phagocytes. For example, N-formyl-Met-Leu-Phe (fMLF), an FPR1 agonist, activates human phagocyte inflammatory responses, such as intracellular calcium mobilization, production of cytokines, generation of reactive oxygen species, and chemotaxis. This ligand can efficiently activate the major bactericidal neutrophil functions and it was one of the first characterized bacterial chemotactic peptides. Whereas fMLF is by far the most frequently used chemotactic peptide in studies of neutrophil functions, atomistic descriptions for fMLF-FPR1 binding mode are still scarce mainly because of the absence of a crystal structure of this receptor. Elucidating the binding modes may contribute to designing novel and more efficient non-peptide FPR1 drug candidates. Molecular modeling of FPR1, on the other hand, can provide an efficient way to reveal details of ligand binding and activation of the receptor. However, recent modelings of FPRs were confined only to bovine rhodopsin as a template.
To locate specific ligand-receptor interactions based on a more appropriate template than rhodopsin we generated the homology models of FPR1 using the crystal structure of the chemokine receptor CXCR4, which shares over 30% sequence identity with FPR1 and is located in the same γ branch of phylogenetic tree of GPCRs (rhodopsin is located in α branch). Docking and model refinement procedures were pursued afterward. Finally, 40 ns full-atom MD simulations were conducted for the Apo form as well as for complexes of fMLF (agonist) and tBocMLF (antagonist) with FPR1 in the membrane. Based on locations of the N- and C-termini of the ligand the FPR1 extracellular pocket can be divided into two zones, namely, the anchor and activation regions. The formylated M1 residue of fMLF bound to the activation region led to a series of conformational changes of conserved residues. Internal water molecules participating in extended hydrogen bond networks were found to play a crucial role in transmitting the agonist-receptor interactions. A mechanism of initial steps of the activation concurrent with ligand binding is proposed.
I accurately predicted the structure and ligand binding pose of dopamine receptor 3 (RMSD to the crystal structure: 2.13 Å) and chemokine receptor 4 (CXCR4, RMSD to the crystal structure 3.21 Å) in GPCR-Dock 2010 competition. The homology model of the dopamine receptor 3 was 8 th best overall in the competition.
Small molecule drug discovery is strongly supported by biophysical data. In the reach of this thesis, cell free protein expression was used to produce human target proteins for ligand binding assays using Surface Plasmon Resonance spectroscopy (SPR). In the second step the binding and interaction characteristics of small molecules and fragments were analyzed using Nuclear Magnetic Resonance spectroscopy (NMR).
The first target protein was the human acid sensing channel 1 (ASIC1a). ASIC1a was expressed in a cell free expression system based on E.coli lysate. To optimize the expression, several parameters including fusion tags, ion concentrations and different hydrophobic environments were tested.
The adaption of the folding environment for ASIC1a needed more optimization, because it is a very challenging target to express in an in vitro system. Three different expression modes were employed to find a suitable folding environment.
SPR binding studies with ASIC1a were performed with chicken ASIC1a expressed in insect cells. The immobilization of cASIC1a and the used buffer conditions were tested using Psalmotoxin 1, a naturally occurring peptide venom which binds strong to the trimeric form of ASIC1a. Compound characterization experiments were performed with a variety of different ligands including amiloride, a general blocker of the whole ENaC protein family. None of the used ligands showed titration curves that would match a simple 1:1 binding model. The experiments either show no binding signal or signal that could be interpreted as unspecific binding. Even amiloride that should be binding the protein shows no signals that fit a simple binding model.
Another target protein that was investigated is the soluble prolyl cis/trans isomerase Cyclophilin D (or peptidyl prolyl isomerase F – PPIF). This protein is involved in the regulation of the mitochondrial permeability transition pore and therefore a potential drug target to treat neurodegenerative diseases. Small molecule binding was tested with CypD using SPR. Following the kinetic analysis of small molecule ligands, the binding position of different binding fragments was analyzed. These fragments originated from a SPR based fragment screen and gave no co-crystal structures with CypD. Therefore NMR was used to investigate the binding position of these fragments. An analysis of the chemical shift perturbations upon ligand addition revealed that the NMR analysis was in line with the results gathered by x-ray crystallography. The fragments with unknown binding position however, all bind to a specific patch slightly outside the binding pocket.
The ligand CL1 showed a special behavior in the NMR experiments. Upon addition to CypD, it produced large shifts on many signals of the protein, accompanied by a severe line broadening. The shift perturbations were so numerous and large that the spectrum had to be reassigned in complex with the ligand. Triple selective labeling was applied to allow a fast and nearly complete signal assignment. The possibility to use highly sophisticated labeling schemes, is one of the advantages of cell free protein expression. After the assignment of the complex spectrum, the chemical shift perturbations were analyzed and quantified. The residues showing the strongest CSPs are also identified in the crystal structure to be involved in the binding of CL1, giving a consistent picture. The numerous and large shift perturbations, produced by CL1 led to the assumption, that the ligand induces a conformational change in CypD, which is not represented in the co-crystal structure. This conformational change was characterized by a NMR based structure determination. CypD apo yielded a defined bundle, whose folded regions overlap well with the corresponding crystal structure.
For the calculation of the CypD-CL1 complex structure, the sidechain resonances were assigned using an automated assignment approach with the software FLYA. The calculation of the CypD-CL1 complex structure did not result in a defined bundle. While parts of the protein converge in a well folded state, the region around the active site shows no defined folding. Careful analysis of the structure calculation suggests that the problems during structure calculation did not originate from an incorrect resonance assignment, but rather from a lack of NOE crosspeaks. This might be due to a broadening of the corresponding NOE crosspeaks or the coexistence of many different conformations. This leads to the conclusion, that the protein conformation is not defined by the NMR data and could be in a dynamic interchange between multiple structures.
This hypothesis is supported by other observations. The line broadening of the signals in the complex is pronounced in the area around the active site and the substrate binding pocket, hinting to a connection between catalytic activity and protein dynamics. In addition many NMR signals are sensitive to changes in the measurement field strength and the temperature. This field dependent signal splitting suggests dynamic conformational changes in the protein between at least two different conformations on a millisecond timescale.
The current working model is that CL1 binds to CypD and induces the catalytic cycle and the connected conformational changes in CypD. As a result the proline like moiety in CL1 is constantly switching between the cis and the trans conformation. Due to the high affinity of CL1, the inhibitor does not leave the binding pocket after successful catalysis, but stays bound in the pocket stimulating further catalytic cycles. These findings as well as the working model are well in line with data published for Cyclophilin A, another member of the cyclophilin family, thereby supporting the model.