Publications of the research group structure and functions of Proteins(SFPR): Recent submissions
Now showing items 1-20 of 54
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Structural basis of ergothioneine biosynthesis.Ergothioneine is a sulfur-containing histidine derivative synthesized by many bacteria and most fungi but it also finds its way into human tissue by way of specific absorption from the diet. The precise role of ergothioneine is not yet known but there is growing evidence that it plays a role as an antioxidant protecting human cells from oxidative stress and pathogenic bacteria from host defenses. In this review we highlight recent advances in understanding the structural basis of ergothioneine biosynthesis. In addition to unusual carbon–sulfur bond forming enzymology this research has revealed that ergothioneine biosynthesis has emerged at least three times by independent molecular evolution.
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NAD(H)-mediated tetramerization controls the activity of phospholipase PlaB.The virulence factor PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires' disease. It is a highly active phospholipase exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal α/β-hydrolase domain expanded by two noncanonical two-stranded β-sheets, β-6/β-7 and β-9/β-10. The C-terminal domain reveals a fold displaying a bilobed β-sandwich with a hook structure required for dimer formation and structural complementation of the enzymatic domain in the neighboring monomer. This highlights the dimer as the active form. Δβ-9/β-10 mutants showed a decrease in the tetrameric fraction and altered activity profiles. The variant also revealed restricted binding to membranes resulting in mislocalization and bacterial lysis. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetramer and concomitantly reduced activity. Together, these data reveal structural elements and an unprecedented NAD(H)-mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The allosteric regulatory process identified here is suited to fine tune PlaB in a way that protects Legionella pneumophila from self-inflicted lysis while ensuring its activity at the pathogen-host interface.
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Reproducible and Easy Production of Mammalian Proteins by Transient Gene Expression in High Five Insect Cells.he expression of mammalian recombinant proteins in insect cell lines using transient-plasmid-based gene expression enables the production of high-quality protein samples. Here, the procedure for virus-free transient gene expression (TGE) in High Five insect cells is described in detail. The parameters that determine the efficiency and reproducibility of the method are presented in a robust protocol for easy implementation and set-up of the method. The applicability of the TGE method in High Five cells for proteomic, structural, and functional analysis of the expressed proteins is shown.
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Antimicrobial resistance dynamics and the one-health strategy: a reviewAntimicrobial resistance is a global threat that kills at least 75,000 people every year worldwide and causes extended hospital stays. In the coming 10 years, antimicrobial resistance is projected to have huge health and economic burden on countries, and the scarcity of available antibiotics further worsens the situation. Antimicrobial resistance results mainly from indiscriminate antibiotic usage in humans, animals and agriculture, and from the rapid emergence and dissemination of resistant pathogens. This issue is challenging for antibiotic stewardship, strict regulations on antibiotics usage, large-scale surveillance and responsible public behavior. This demands international cooperation and integrated efforts under the ‘one-health’ strategy. Here, we review antimicrobial resistance and the one-health strategy. We discuss the historical issue of using antibiotics. We highlight the effectiveness of hygiene in livestock rearing, careful antibiotic usage and large-scale surveillance of animals, humans and environment domains. We present strategies for mitigation of antimicrobial resistance, exemplified by the successful ban of triclosan which induced a significant decline of resistant pathogens. We emphasize the benefits of the global antibiotic resistance partnership and of the one-health participation of stakeholders from public, healthcare professionals and government to mitigate antimicrobial resistance.
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A New PqsR Inverse Agonist Potentiates Tobramycin Efficacy to Eradicate Pseudomonas aeruginosa BiofilmsPseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) – a crucial transcriptional regulator serving major functions in PA virulence – can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry‐driven hit‐to‐lead optimization and in‐depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter‐gene with IC50 values as low as 200 and 11 × 10−9 m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI‐tobramycin (Tob) combination against PA biofilms using a tailor‐made squalene‐derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32‐fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker‐mediated therapy against PA infections opening up avenues for preclinical development.
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Immunotherapy with antibodies: Tumor development, immune defense and therapeutic antibodiesTumor development is based on mutations of genes involved in cell growth (e.g. transcription factors, growth receptors or intracellular signal molecules) or in suppressor genes (e.g. p53). During tumor growth cell clones are selected, which contain driver genes, leading to uncontrolled growth of these cell clones. During all phases of tumor development (immunosurveillance, equilibrium phase, escape of the tumor from the immune system) the interaction between the immune system and the tumor cells and the development of a chronic inflammation in the tumor microenvironment play a crucial role. The aim of cancer immunotherapy is to activate the immune system. A promising immunotherapy is based on antibodies that activate immune cells, inhibit tumor growth or lead to destruction of tumor cells. Applied are recombinant IgG antibodies or genetically engineered antibody fragments against tumor-associated antigens (TAA’s). They are applied singularly or in combination with chemo- or radiotherapy. Promising are checkpoint antibodies, which abrogate blocking of cytotoxic CD8+ T cells and CD4+ T cells by tumor cells and/or dendritic cells. Other successfully applied antibodies are bispecific antibodies (recognize T‑cell and tumor cell), chimeric antigen receptors (CARs) for T cell therapy, immunocytokines (cytokines fused to antibodies) and immunotoxins (toxins fused to antibodies). In addition intracellular antibodies successfully tested in xenograft tumor mouse models have promising therapeutic potential.
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Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation.ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.
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Crystal structure of bacterial cytotoxic necrotizing factor CNFy reveals molecular building blocks for intoxication.Cytotoxic necrotizing factors (CNFs) are bacterial single-chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three-dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full-length Yersinia pseudotuberculosis CNFY . CNFY consists of five domains (D1-D5), and by integrating structural and functional data, we demonstrate that D1-3 act as export and translocation module for the catalytic unit (D4-5) and for a fused β-lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP-ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4-5 fragment. This liberates D5 from a semi-blocked conformation in full-length CNFY , leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad-specificity protein delivery tool.
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CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering.CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16a-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternate binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that water entrance to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5a-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.
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Expression, purification and crystal structure determination of a ferredoxin reductase from the actinobacterium Thermobifida fusca.he ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (FNR) family, catalyses electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a putative three-component cytochrome P450 monooxygenase system in T. fusca comprising CYP222A1 and the [3Fe-4S]-cluster ferredoxin Fdx8 as well as FdR9. Here, FdR9 was overexpressed and purified and its crystal structure was determined at 1.9 Å resolution. The overall structure of FdR9 is similar to those of other members of the FNR family and is composed of an FAD-binding domain, an NAD-binding domain and a C-terminal domain. Activity measurements with FdR9 confirmed a strong preference for NADH as the cofactor. Comparison of the FAD- and NAD-binding domains of FdR9 with those of other ferredoxin reductases revealed the presence of conserved sequence motifs in the FAD-binding domain as well as several highly conserved residues involved in FAD and NAD cofactor binding. Moreover, the NAD-binding site of FdR9 contains a modified Rossmann-fold motif, GxSxxS, instead of the classical GxGxxG motif.
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Gastrointestinal stress as innate defence against microbial attack.A comparison of the metabolic response of Escherichia coli BL21 (DE3) towards the production of human basic fibroblast growth factor (hFGF-2) or towards carbon overfeeding revealed similarities which point to constraints in anabolic pathways. Contrary to expectations, neither energy generation (e.g., ATP) nor provision of precursor molecules for nucleotides (e.g., uracil) and amino acids (e.g., pyruvate, glutamate) limit host cell and plasmid-encoded functions. Growth inhibition is assumed to occur when hampered anabolic capacities do not match with the ongoing and overwhelming carbon catabolism. Excessive carbon uptake leads to by-product secretion, for example, pyruvate, acetate, glutamate, and energy spillage, for example, accumulation and degradation of adenine nucleotides with concomitant accumulation of extracellular hypoxanthine. The cellular response towards compromised anabolic capacities involves downregulation of cAMP formation, presumably responsible for subsequently better-controlled glucose uptake and resultant accumulation of glucose in the culture medium. Growth inhibition is neglectable under conditions of reduced carbon availability when hampered anabolic capacities also match with catabolic carbon processing. The growth inhibitory effect with accompanying energy spillage, respectively, hypoxanthine secretion and cessation of cAMP formation is not unique to the production of hFGF-2 but observed during the production of other proteins and also during overexpression of genes without transcript translation.
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The crystal structure of the heme d biosynthesis-associated small c-type cytochrome NirC reveals mixed oligomeric states in crystallo.Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Å (spherical resolution of 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain-swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC.
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Structure of heme d-free cd nitrite reductase NirS.A key step in anaerobic nitrate respiration is the reduction of nitrite to nitric oxide, which is catalysed by the cd1 nitrite reductase NirS in, for example, the Gram-negative opportunistic pathogen Pseudomonas aeruginosa. Each subunit of this homodimeric enzyme consists of a cytochrome c domain and an eight-bladed β-propeller that binds the uncommon isobacteriochlorin heme d1 as an essential part of its active site. Although NirS has been well studied mechanistically and structurally, the focus of previous studies has been on the active heme d1-bound form. The heme d1-free form of NirS reported here, which represents a premature state of the reductase, adopts an open conformation with the cytochrome c domains moved away from each other with respect to the active enzyme. Further, the movement of a loop around Trp498 seems to be related to a widening of the propeller, allowing easier access to the heme d1-binding side. Finally, a possible link between the open conformation of NirS and flagella formation in P. aeruginosa is discussed.
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Biocatalysts from Biosynthetic Pathways: Enabling Stereoselective, Enzymatic Cycloether Formation on a Gram ScaleBiosynthetic pathways of natural products contain many enzymes that contribute to the rapid assembly of molecular complexity. Enzymes that form complex structural elements with multiple stereocenters, like chiral saturated oxygen heterocycles (CSOH), are of particular interest for a synthetic application, as their use promises to significantly simplify access to these elements. Here, the biocatalytic characterization of AmbDH3, an enzyme that catalyzes intramolecular oxa-Michael addition (IMOMA) is reported. This reaction essentially gives access to various types of CSOH with adjacent stereocenters, but it is not yet part of the repertoire of preparative biocatalysis. An in-depth study on the synthetic utility of AmbDH3 was performed, which made extensive use of complex synthetic precursor surrogates. The enzyme exhibited stability and broad substrate tolerance in in vitro experiments, which was in agreement with the results of molecular modeling. Its selectivity profile enabled kinetic resolution of chiral tetrahydropyrans (THPs) under control of up to four stereocenters. A systematic optimization of the reaction conditions enabled gram-scale conversions yielding preparative amounts of chiral THP. The synthetic utility of AmbDH3 was finally demonstrated by its successful application in the key step of a chemoenzymatic total synthesis to the THP-containing phenylheptanoid (−)-centrolobine. These results highlight the synthetic potential of AmbDH3 and related IMOMA cyclases as a biocatalytic alternative that further develops the available chemical-synthetic IMOMA methodology.
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Crystal structure of NirF: insights into its role in heme d biosynthesis.Certain facultative anaerobes such as the opportunistic human pathogen Pseudomonas aeruginosa can respire on nitrate, a process generally known as denitrification. This enables denitrifying bacteria to survive in anoxic environments and contributes, for example, to the formation of biofilm, hence increasing difficulties in eradicating P. aeruginosa infections. A central step in denitrification is the reduction of nitrite to nitric oxide by nitrite reductase NirS, an enzyme that requires the unique cofactor heme d1 . While heme d1 biosynthesis is mostly understood, the role of the essential periplasmatic protein NirF in this pathway remains unclear. Here, we have determined crystal structures of NirF and its complex with dihydroheme d1 , the last intermediate of heme d1 biosynthesis. We found that NirF forms a bottom-to-bottom β-propeller homodimer and confirmed this by multi-angle light and small-angle X-ray scattering. The N termini are adjacent to each other and project away from the core structure, which hints at simultaneous membrane anchoring via both N termini. Further, the complex with dihydroheme d1 allowed us to probe the importance of specific residues in the vicinity of the ligand binding site, revealing residues not required for binding or stability of NirF but essential for denitrification in experiments with complemented mutants of a ΔnirF strain of P. aeruginosa. Together, these data suggest that NirF possesses a yet unknown enzymatic activity and is not simply a binding protein of heme d1 derivatives. DATABASE: Structural data are available in PDB database under the accession numbers 6TV2 and 6TV9.
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Molecular Mechanisms of Vaspin Action - From Adipose Tissue to Skin and Bone, from Blood Vessels to the Brain.Visceral adipose tissue-derived serine protease inhibitor (vaspin) or SERPINA12 according to the serpin nomenclature was identified together with other genes and gene products that were specifically expressed or overexpressed in the intra-abdominal or visceral adipose tissue (AT) of the Otsuka Long-Evans Tokushima fatty rat. These rats spontaneously develop visceral obesity, insulin resistance, hyperinsulinemia and -glycemia, as well as hypertension and thus represent a well suited animal model of obesity and related metabolic disorders such as type 2 diabetes.The follow-up study reporting the cloning, expression and functional characterization of vaspin suggested the great and promising potential of this molecule to counteract obesity induced insulin resistance and inflammation and has since initiated over 300 publications, clinical and experimental, that have contributed to uncover the multifaceted functions and molecular mechanisms of vaspin action not only in the adipose, but in many different cells, tissues and organs. This review will give an update on mechanistic and structural aspects of vaspin with a focus on its serpin function, the physiology and regulation of vaspin expression, and will summarize the latest on vaspin function in various tissues such as the different adipose tissue depots as well as the vasculature, skin, bone and the brain.
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The Alkylquinolone Repertoire of Pseudomonas aeruginosa is Linked to Structural Flexibility of the FabH-like 2-Heptyl-3-hydroxy-4(1H)-quinolone (PQS) Biosynthesis Enzyme PqsBC.Pseudomonas aeruginosa is a bacterial pathogen that causes life-threatening infections in immunocompromised patients. It produces a large armory of saturated and mono-unsaturated 2-alkyl-4(1H)-quinolones (AQs) and AQ N-oxides (AQNOs) that serve as signaling molecules to control the production of virulence factors and that are involved in membrane vesicle formation and iron chelation; furthermore, they also have, for example, antibiotic properties. It has been shown that the β-ketoacyl-acyl-carrier protein synthase III (FabH)-like heterodimeric enzyme PqsBC catalyzes the last step in the biosynthesis of the most abundant AQ congener, 2-heptyl-4(1H)-quinolone (HHQ), by condensing octanoyl-coenzyme A (CoA) with 2-aminobenzoylacetate (2-ABA), but the basis for the large number of other AQs/AQNOs produced by P. aeruginosa is not known. Here, we demonstrate that PqsBC uses different medium-chain acyl-CoAs to produce various saturated AQs/AQNOs and that it also biosynthesizes mono-unsaturated congeners. Further, we determined the structures of PqsBC in four different crystal forms at 1.5 to 2.7 Å resolution. Together with a previous report, the data reveal that PqsBC adopts open, intermediate, and closed conformations that alter the shape of the acyl-binding cavity and explain the promiscuity of PqsBC. The different conformations also allow us to propose a model for structural transitions that accompany the catalytic cycle of PqsBC that might have broader implications for other FabH-enzymes, for which such structural transitions have been postulated but have never been observed.
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Insights into the Cnx1E catalyzed MPT-AMP hydrolysis.Molybdenum insertases (Mo-insertases) catalyze the final step of molybdenum cofactor (Moco) biosynthesis, an evolutionary old and highly conserved multi-step pathway. In the first step of the pathway, GTP serves as substrate for the formation of cyclic pyranopterin monophosphate, which is subsequently converted into molybdopterin (MPT) in the second pathway step. In the following synthesis steps, MPT is adenylated yielding MPT-AMP that is subsequently used as substrate for enzyme catalyzed molybdate insertion. Molybdate insertion and MPT-AMP hydrolysis are catalyzed by the Mo-insertase E-domain. Earlier work reported a highly conserved aspartate residue to be essential for Mo-insertase functionality. In this work, we confirmed the mechanistic relevance of this residue for the Arabidopsis thaliana Mo-insertase Cnx1E. We found that the conservative substitution of Cnx1E residue Asp274 by Glu (D274E) leads to an arrest of MPT-AMP hydrolysis and hence to the accumulation of MPT-AMP. We further showed that the MPT-AMP accumulation goes in hand with the accumulation of molybdate. By crystallization and structure determination of the Cnx1E variant D274E, we identified the potential reason for the missing hydrolysis activity in the disorder of the region spanning amino acids 269 to 274. We reasoned that this is caused by the inability of a glutamate in position 274 to coordinate the octahedral Mg2+-water complex in the Cnx1E active site.
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Flexible Fragment Growing Boosts Potency of Quorum Sensing Inhibitors against Pseudomonas aeruginosa Virulence.Hit-to-lead optimization is a critical phase in drug discovery. Herein, we report on the fragment-based discovery and optimization of 2-amino pyridine derivatives as a novel lead-like structure for the treatment of the dangerous opportunistic pathogen Pseudomonas aeruginosa . We pursue an innovative treatment strategy by interfering with the Pseudomonas Quinolone Signal (PQS) Quorum Sensing (QS) system leading to an abolishment of bacterial pathogenicity. Our compounds act on the PQS receptor (PqsR), a key transcription factor controlling the expression of various pathogenicity determinants. In this target-driven approach, we made use of biophysical screening via surface plasmon resonance (SPR) followed by isothermal titration calorimetry (ITC)-enabled enthalpic efficiency (EE) evaluation. Hit optimization then involved growth vector identification and exploitation. Astonishingly, the latter was successfully achieved by introducing flexible linkers rather than rigid motifs leading to a boost in activity on the target receptor and anti-virulence potency.