434 Citations
Peroxide sensing is essential for bacterial survival during aerobic metabolism and host infection Peroxide stress regulators PerRs are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites PerR binding to gene promoters is controlled by the presence of iron in the regulatory site and iron-catalyzed oxidation of PerR by H O leads to the dissociation of PerR from DNA In addition to a regulatory metal most PerRs require a structural metal for proper dimeric assembly We present here a structural and functional characterization of the PerR from the pathogenic spirochete Leptospira interrogans a rare example ... More
Peroxide sensing is essential for bacterial survival during aerobic metabolism and host infection. Peroxide stress regulators (PerRs) are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites. PerR binding to gene promoters is controlled by the presence of iron in the regulatory site, and iron-catalyzed oxidation of PerR by H2O2 leads to the dissociation of PerR from DNA. In addition to a regulatory metal, most PerRs require a structural metal for proper dimeric assembly. We present here a structural and functional characterization of the PerR from the pathogenic spirochete Leptospira interrogans, a rare example of PerR lacking a structural metal-binding site. In vivo studies showed that the leptospiral PerR belongs to the peroxide stimulon in pathogenic species and is involved in controlling resistance to peroxide. Moreover, a perR mutant had decreased fitness in other host-related stress conditions, including at 37 °C or in the presence of superoxide anion. In vitro, leptospiral PerR could bind to the perR promoter region in a metal-dependent manner. The crystal structure of the leptospiral PerR revealed an asymmetric homodimer, with one monomer displaying complete regulatory metal coordination in the characteristic caliper-like DNA-binding conformation and the second monomer exhibiting disrupted regulatory metal coordination in an open non-DNA–binding conformation. This structure showed that leptospiral PerR assembles into a dimer in which a metal-induced conformational switch can occur independently in the two monomers. Our study demonstrates that structural metal binding is not compulsory for PerR dimeric assembly and for regulating peroxide stress. Less
Antimicrobial peptides as part of the mammalian innate immune system target and remove major bacterial pathogens often through irreversible damage of their cellular membranes To explore the mechanism by which the important cathelicidin peptide LL- of the human innate immune system interacts with membranes we performed biochemical biophysical and structural studies The crystal structure of LL- displays dimers of anti-parallel helices and the formation of amphipathic surfaces Peptide-detergent interactions introduce remodeling of this structure after occupation of defined hydrophobic sites at the dimer interface Furthermore hydrophobic nests are shaped between dimer structures providing another scaffold enclosing detergents Both scaffolds underline ... More
Antimicrobial peptides as part of the mammalian innate immune system target and remove major bacterial pathogens, often through irreversible damage of their cellular membranes. To explore the mechanism by which the important cathelicidin peptide LL-37 of the human innate immune system interacts with membranes, we performed biochemical, biophysical and structural studies. The crystal structure of LL-37 displays dimers of anti-parallel helices and the formation of amphipathic surfaces. Peptide-detergent interactions introduce remodeling of this structure after occupation of defined hydrophobic sites at the dimer interface. Furthermore, hydrophobic nests are shaped between dimer structures providing another scaffold enclosing detergents. Both scaffolds underline the potential of LL-37 to form defined peptide-lipid complexes in vivo. After adopting the activated peptide conformation LL-37 can polymerize and selectively extract bacterial lipids whereby the membrane is destabilized. The supramolecular fibril-like architectures formed in crystals can be reproduced in a peptide-lipid system after nanogold-labelled LL-37 interacted with lipid vesicles as followed by electron microscopy. We suggest that these supramolecular structures represent the LL-37-membrane active state. Collectively, our study provides new insights into the fascinating plasticity of LL-37 demonstrated at atomic resolution and opens the venue for LL-37-based molecules as novel antibiotics. Less
The present work contributes to the eld of process development and optimization for the manufacturing of recombinant proteins Recombinant proteins are biological macromolecules which are produced using genetically modi ed organisms GMOs In addition to the food industry and for the synthesis of organic compounds industrially produced proteins are mainly used in the eld of medicine There they make a decisive contribution to the diagnosis prevention and treatment of various human diseases The production of protein-based drugs biopharmaceuticals and their provision in a stable and bioavailable dosage form formulation are a challenge due to the size and complexity of these ... More
The present work contributes to the eld of process development and optimization for the manufacturing of recombinant proteins. Recombinant proteins are biological macromolecules, which are produced using genetically modi ed organisms (GMOs). In addition to the food industry and for the synthesis of organic compounds, industrially produced proteins are mainly used in the eld of medicine. There, they make a decisive contribution to the diagnosis, prevention and treatment of various human diseases. The production of protein-based drugs (biopharmaceuticals) and their provision in a stable and bioavailable dosage form (formulation) are a challenge due to the size and complexity of these molecules. On the way to the nal drug product, the target protein undergoes a long and complex processing chain consisting of GMO cultivation (upstream processing), puri - cation operations (downstream processing) and formulation steps. The reaction medium for the entire manufacturing process is mainly based on aqueous solutions. Some of the process steps used require extreme conditions such as unphysiological salt concentrations (e.g. in chromatographic puri cation) or acidic pH values (e.g. virus inactivation); all of which are potential stress factors for the protein integrity. Resulting irreversible changes in protein structure and physical instabilities, e.g. aggregation can a ect both drug safety and ecacy. In order to ensure the product quality during the entire manufacturing process, the development of strategies for the stabilization of proteins in aqueous solutions is of paramount importance. From an economic perspective, short development times are desirable in order to reduce the `time to market' of a drug product. Standardized procedures are of great interest for shortening the development times of stabilized protein products and are therefore the subject of this dissertation. Less
Multiprotein assemblies play major roles in most pathways involved in cell regulation and signaling Weak binary interactions are transformed co-operatively into very specific systems which achieve sensitivity specificity and temporal control Due to the complexity and transience of these regulatory and signaling systems a combination of in vivo cell biochemical biophysical and structural approaches is needed to investigate their structures and dynamics Here we describe the architecture and spatial organisation of the complexes mediating Non-Homologous End Joining NHEJ one of the two major pathways involved in DNA double-strand break repair Our example illustrates the experimental challenges and conceptual questions that ... More
Multiprotein assemblies play major roles in most pathways involved in cell regulation and signaling. Weak binary interactions are transformed co-operatively into very specific systems, which achieve sensitivity, specificity and temporal control. Due to the complexity and transience of these regulatory and signaling systems, a combination of in vivo, cell, biochemical, biophysical, and structural approaches is needed to investigate their structures and dynamics. Here we describe the architecture and spatial organisation of the complexes mediating Non-Homologous End Joining (NHEJ), one of the two major pathways involved in DNA double-strand break repair. Our example illustrates the experimental challenges and conceptual questions that are raised by studying such complex systems. We discuss the potential of using knowledge of the spatial and temporal organization of multiprotein systems not only to give insights into the mechanisms of pathway regulation but also to help in the design of chemical tools and ultimately new therapeutic agents. Less
Solution stability attributes are one of the key parameters within the production and launching phase of new biopharmaceuticals Instabilities of active biological compounds can reduce the yield of biopharmaceutical productions and may induce undesired reactions in patients such as immunogenic rejections Protein solution stability thus needs to be engineered and monitored throughout production and storage In contrast to the gold standard of long-term storage experiments applied in industry novel experimental and in silico molecular dynamics tools for predicting protein solution stability can be applied within several minutes or hours Here a rheological approach in combination with molecular dynamics simulations are ... More
Solution stability attributes are one of the key parameters within the production and launching phase of new biopharmaceuticals. Instabilities of active biological compounds can reduce the yield of biopharmaceutical productions, and may induce undesired reactions in patients, such as immunogenic rejections. Protein solution stability thus needs to be engineered and monitored throughout production and storage. In contrast to the gold standard of long-term storage experiments applied in industry, novel experimental and in silico molecular dynamics tools for predicting protein solution stability can be applied within several minutes or hours. Here, a rheological approach in combination with molecular dynamics simulations are presented, for determining and predicting long-term phase behavior of highly concentrated protein solutions. A diversity of liquid phase conditions, including salt type, ionic strength, pH and protein concentration are tested in a Glutathione-S-Transferase (GST) case study, in combination with the enzyme with and without solubility-enhancing Cherry-Tag™. The rheological characterization of GST and Cherry-GST solutions enabled a fast and efficient prediction of protein instabilities without the need of long-term protein phase diagrams. Finally, the strong solubility enhancing properties of the Cherry-Tag™ were revealed by investigating protein surface properties in MD simulations. The tag highly altered the overall surface charge and hydrophobicity of GST, making it less accessible to alteration by the chemical surrounding. Less
Automated sample changers are now standard equipment for modern macromolecular crystallography synchrotron beamlines Nevertheless most are only compatible with a single type of sample holder and puck Recent work aimed at reducing sample-handling efforts and crystal-alignment times at beamlines has resulted in a new generation of compact and precise sample holders for cryocrystallography miniSPINE and NewPin see the companion paper by Papp et al Acta Cryst D With full data collection now possible within seconds at most advanced beamlines and future fourth-generation synchrotron sources promising to extract data in a few tens of milliseconds the time taken to mount and ... More
Automated sample changers are now standard equipment for modern macromolecular crystallography synchrotron beamlines. Nevertheless, most are only compatible with a single type of sample holder and puck. Recent work aimed at reducing sample-handling efforts and crystal-alignment times at beamlines has resulted in a new generation of compact and precise sample holders for cryocrystallography: miniSPINE and NewPin [see the companion paper by Papp et al. (2017, Acta Cryst., D73, 829�840)]. With full data collection now possible within seconds at most advanced beamlines, and future fourth-generation synchrotron sources promising to extract data in a few tens of milliseconds, the time taken to mount and centre a sample is rate-limiting. In this context, a versatile and fast sample changer, FlexED8, has been developed that is compatible with the highly successful SPINE sample holder and with the miniSPINE and NewPin sample holders. Based on a six-axis industrial robot, FlexED8 is equipped with a tool changer and includes a novel open sample-storage dewar with a built-in ice-filtering system. With seven versatile puck slots, it can hold up to 112 SPINE sample holders in uni-pucks, or 252 miniSPINE or NewPin sample holders, with 36 samples per puck. Additionally, a double gripper, compatible with the SPINE sample holders and uni-pucks, allows a reduction in the sample-exchange time from 40 s, the typical time with a standard single gripper, to less than 5 s. Computer vision-based sample-transfer monitoring, sophisticated error handling and automatic error-recovery procedures ensure high reliability. The FlexED8 sample changer has been successfully tested under real conditions on a beamline. Less
Tepsin is currently the only accessory trafficking protein identified in adaptor-related protein AP coated vesicles originating at the trans-Golgi network TGN The molecular basis for interactions between AP subunits and motifs in the tepsin C-terminus have been characterized but the biological role of tepsin remains unknown We determined X-ray crystal structures of the tepsin ENTH and VHS ENTH-like domains Our data reveal unexpected structural features that suggest key functional differences between these and similar domains in other trafficking proteins The tepsin ENTH domain lacks helix helix and a lipid binding pocket found in epsin These results explain why tepsin requires ... More
Tepsin is currently the only accessory trafficking protein identified in adaptor-related protein 4 (AP4) coated vesicles originating at the trans-Golgi network (TGN). The molecular basis for interactions between AP4 subunits and motifs in the tepsin C-terminus have been characterized, but the biological role of tepsin remains unknown. We determined X-ray crystal structures of the tepsin ENTH and VHS/ENTH-like domains. Our data reveal unexpected structural features that suggest key functional differences between these and similar domains in other trafficking proteins. The tepsin ENTH domain lacks helix0, helix8, and a lipid binding pocket found in epsin1/2/3. These results explain why tepsin requires AP4 for its membrane recruitment and further suggest ENTH domains cannot be defined solely as lipid binding modules. The VHS domain lacks helix8 and thus contains fewer helices than other VHS domains. Structural data explain biochemical and biophysical evidence that tepsin VHS does not mediate known VHS functions, including recognition of dileucine-based cargo motifs or ubiquitin. Structural comparisons indicate the domains are very similar to each other, and phylogenetic analysis reveals their evolutionary pattern within the domain superfamily. Phylogenetics and comparative genomics further show tepsin within a monophyletic clade that diverged away from epsins early in evolutionary history (~1,500 million years ago). Together, these data provide the first detailed molecular view of tepsin and suggest tepsin structure and function diverged away from other epsins. More broadly, these data highlight the challenges inherent in classifying and understanding protein function based only on sequence and structure. Less
American trypanosomiasis or Chagas Disease is caused by the protozoan Trypanosoma cruzi The treatment is based on drugs that present serious side effects and are inefficient against the chronic phase of the disease The research in new metabolic targets may lead to the development of safer and more efficient drugs Malic enzyme ME is considered a promising target due to its ability to produce NADPH reducing agent that participates in a number of biosynthetic pathways and in detoxication of reactive oxygen species produced from endogenous metabolic reactions or from exogenous immune insults generated by mammalian host cells T cruzi expresses ... More
American trypanosomiasis, or Chagas Disease, is caused by the protozoan
Trypanosoma cruzi. The treatment is based on drugs that present serious side effects
and are inefficient against the chronic phase of the disease. The research in new
metabolic targets may lead to the development of safer and more efficient drugs. Malic
enzyme (ME) is considered a promising target due to its ability to produce NADPH,
reducing agent that participates in a number of biosynthetic pathways and in
detoxication of reactive oxygen species produced from endogenous metabolic
reactions or from exogenous immune insults generated by mammalian host cells. T.
cruzi expresses two isoforms of the enzyme, one mitochondrial (TcMEm) and other
cytosolic (TcMEc). In this thesis, the first characterized inhibitors for these enzymes
are reported, identified by a biochemical high-throughput screening (HTS) assay
against a library of 30 thousand compounds. IC50 values of 262 molecules were
determined for both TcMEs as well as for three human ME isoforms, with the inhibitors
clustered into six groups according to their chemical similarity. The most potent hits
belonged to group ATR3, comprised of 218 aryl-sulfonamides that specifically target
TcMEc. Moreover, several selected inhibitors of both TcMEs showed trypanocidal
effect against the replicative forms of T. cruzi. Also, in this work the first crystallographic
structures of both TcMEs are shown, revealing different features from the human
homologues. In addition, the complex of TcMEc with 6 different ATR3 molecules were
determined, unravelling the inhibition site at the dimer interface. In conclusion, the HTS
results demonstrate that TcMEs are druggable, being modulated by small hydrophobic
molecules, which is an essential requirement for a drug target. Moreover, the identified
compounds can be used as chemical probes in the validation of the enzymes. The
enzyme structures are important tools which may be employed to design new inhibitors
or analogues. Less
Trypanosoma cruzi. The treatment is based on drugs that present serious side effects
and are inefficient against the chronic phase of the disease. The research in new
metabolic targets may lead to the development of safer and more efficient drugs. Malic
enzyme (ME) is considered a promising target due to its ability to produce NADPH,
reducing agent that participates in a number of biosynthetic pathways and in
detoxication of reactive oxygen species produced from endogenous metabolic
reactions or from exogenous immune insults generated by mammalian host cells. T.
cruzi expresses two isoforms of the enzyme, one mitochondrial (TcMEm) and other
cytosolic (TcMEc). In this thesis, the first characterized inhibitors for these enzymes
are reported, identified by a biochemical high-throughput screening (HTS) assay
against a library of 30 thousand compounds. IC50 values of 262 molecules were
determined for both TcMEs as well as for three human ME isoforms, with the inhibitors
clustered into six groups according to their chemical similarity. The most potent hits
belonged to group ATR3, comprised of 218 aryl-sulfonamides that specifically target
TcMEc. Moreover, several selected inhibitors of both TcMEs showed trypanocidal
effect against the replicative forms of T. cruzi. Also, in this work the first crystallographic
structures of both TcMEs are shown, revealing different features from the human
homologues. In addition, the complex of TcMEc with 6 different ATR3 molecules were
determined, unravelling the inhibition site at the dimer interface. In conclusion, the HTS
results demonstrate that TcMEs are druggable, being modulated by small hydrophobic
molecules, which is an essential requirement for a drug target. Moreover, the identified
compounds can be used as chemical probes in the validation of the enzymes. The
enzyme structures are important tools which may be employed to design new inhibitors
or analogues. Less
DHHC enzymes catalyze palmitoylation a major post-translational modification that regulates a number of key cellular processes There are up to DHHCs in mammals and hundreds of substrate proteins that get palmitoylated However how DHHC enzymes engage with their substrates is still poorly understood There is currently no structural information about the interaction between any DHHC enzyme and protein substrates In this study we have investigated the structural and thermodynamic bases of interaction between the ankyrin repeat domain of Human DHHC ANK and Snap b We solved a high-resolution crystal structure of the complex between ANK and a peptide fragment of ... More
DHHC enzymes catalyze palmitoylation, a major post-translational modification that regulates a number of key cellular processes. There are up to 24 DHHCs in mammals and hundreds of substrate proteins that get palmitoylated. However, how DHHC enzymes engage with their substrates is still poorly understood. There is currently no structural information about the interaction between any DHHC enzyme and protein substrates. In this study we have investigated the structural and thermodynamic bases of interaction between the ankyrin repeat domain of Human DHHC17 (ANK17) and Snap25b. We solved a high-resolution crystal structure of the complex between ANK17 and a peptide fragment of Snap25b. Through structure-guided mutagenesis, we discovered key residues in DHHC17 that are critically important for interaction with Snap25b. We further extended our finding by showing that the same residues are also crucial for the interaction of DHHC17 with Huntingtin, one of its most relevant substrates. Less
Vancomycin is known to bind to Zn II and can induce a zinc starvation response in bacteria Here we identify a novel polymerization of vancomycin dimers by structural analysis of vancomycin-Zn II crystals and fibre X-ray diffraction Bioassays indicate that this structure is associated with an increased antibiotic activity against bacterial strains possessing high level vancomycin resistance mediated by the reprogramming of peptidoglycan biosynthesis to use precursors terminating in D-Ala-D-Lac in place of D-Ala-D-Ala Polymerization occurs via interaction of Zn II with the N-terminal methylleucine group of vancomycin and we show that the activity of other glycopeptide antibiotics with this ... More
Vancomycin is known to bind to Zn(II) and can induce a zinc starvation response in bacteria. Here we identify a novel polymerization of vancomycin dimers by structural analysis of vancomycin-Zn(II) crystals and fibre X-ray diffraction. Bioassays indicate that this structure is associated with an increased antibiotic activity against bacterial strains possessing high level vancomycin resistance mediated by the reprogramming of peptidoglycan biosynthesis to use precursors terminating in D-Ala-D-Lac in place of D-Ala-D-Ala. Polymerization occurs via interaction of Zn(II) with the N-terminal methylleucine group of vancomycin, and we show that the activity of other glycopeptide antibiotics with this feature can also be similarly augmented by Zn(II). Construction and analysis of a model strain predominantly using D-Ala-D-Lac precursors for peptidoglycan biosynthesis during normal growth supports the hypothesis that Zn(II) mediated vancomycin polymerization enhances the binding affinity towards these precursors. Less
Lipoproteins serve essential roles in the bacterial cell envelope The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes All are potential targets for the development of new antibiotics Here we report the crystal structure of the last enzyme in the pathway apolipoprotein N-acyltransferase Lnt responsible for adding a third acyl chain to the lipoprotein s invariant diacylated N-terminal cysteine Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved they are remarkably similar Both consist of a membrane domain on which sits a globular periplasmic domain The active site resides above the membrane interface where the ... More
Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein�s invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects. Less
High protein titers are gaining importance in biopharmaceutical industry A major challenge in the development of highly concentrated mAb solutions is their long-term stability and often incalculable viscosity The complexity of the molecule itself as well as the various molecular interactions make it difficult to describe their solution behavior To study the formulation stability long- and short-range interactions and the formation of complex network structures have to be taken into account For a better understanding of highly concentrated solutions we combined established and novel analytical tools to characterize the effect of solution properties on the stability of highly concentrated mAb ... More
High protein titers are gaining importance in biopharmaceutical industry. A major challenge in the development of highly concentrated mAb solutions is their long-term stability and often incalculable viscosity. The complexity of the molecule itself, as well as the various molecular interactions, make it difficult to describe their solution behavior. To study the formulation stability, long- and short-range interactions and the formation of complex network structures have to be taken into account. For a better understanding of highly concentrated solutions, we combined established and novel analytical tools to characterize the effect of solution properties on the stability of highly concentrated mAb formulations. In this study, monoclonal antibody solutions in a concentration range of 50–200 mg/ml at pH 5–9 with and without glycine, PEG4000, and Na2SO4 were analyzed. To determine the monomer content, analytical size-exclusion chromatography runs were performed. ζ-potential measurements were conducted to analyze the electrophoretic properties in different solutions. The melting and aggregation temperatures were determined with the help of fluorescence and static light scattering measurements. Additionally, rheological measurements were conducted to study the solution viscosity and viscoelastic behavior of the mAb solutions. The so-determined analytical parameters were scored and merged in an analytical toolbox. The resulting scoring was then successfully correlated with long-term storage (40 d of incubation) experiments. Our results indicate that the sensitivity of complex rheological measurements, in combination with the applied techniques, allows reliable statements to be made with respect to the effect of solution properties, such as protein concentration, ionic strength, and pH shift, on the strength of protein-protein interaction and solution colloidal stability. Less
The human glucagon receptor GCGR belongs to the class B G protein-coupled receptor GPCR family and plays a key role in glucose homeostasis and the pathophysiology of type diabetes Here we report the crystal structure of full-length GCGR containing both extracellular domain ECD and transmembrane domain TMD in an inactive conformation The two domains are connected by a -residue segment termed the stalk which adopts a -strand conformation instead of forming an -helix as observed in the previously solved structure of GCGR-TMD The first extracellular loop ECL exhibits a -hairpin conformation and interacts with the stalk to form a compact ... More
The human glucagon receptor (GCGR) belongs to the class B G protein-coupled receptor (GPCR) family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both extracellular domain (ECD) and transmembrane domain (TMD) in an inactive conformation. The two domains are connected by a 12-residue segment termed the ‘stalk’, which adopts a β-strand conformation, instead of forming an α-helix as observed in the previously solved structure of GCGR-TMD. The first extracellular loop (ECL1) exhibits a β-hairpin conformation and interacts with the stalk to form a compact β-sheet structure. Hydrogen/deuterium exchange, disulfide cross-linking and molecular dynamics studies suggest that the stalk and ECL1 play critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding about the signaling mechanisms of class B GPCRs. Less
Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis the most common chronic inflammatory disease in humans worldwide Its comorbidity with systemic diseases such as type diabetes oral cancers and cardiovascular diseases continues to generate considerable interest Surprisingly these two microorganisms do not ferment carbohydrates rather they use proteinaceous substrates as carbon and energy sources However the underlying biochemical mechanisms of their energy metabolism remain unknown Here we show that dipeptidyl peptidase DPP a central metabolic enzyme in these bacteria undergoes a conformational change upon peptide binding to distinguish substrates from end products It binds substrates through an ... More
Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme (“entropy reservoirs”). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors. Less
Schistosoma mansoni is the parasite responsible for schistosomiasis a disease that affects about million people worldwide Currently both direct treatment and disease control initiatives rely on chemotherapy using a single drug praziquantel Concerns over the possibility of resistance developing to praziquantel have stimulated efforts to develop new drugs for the treatment of schistosomiasis Schistosomes do not have the de novo purine biosynthetic pathway and instead depend entirely on the purine salvage pathway to supply its need for purines The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis Adenylosuccinate lyase SmADSL is an ... More
Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 218 million people worldwide. Currently, both direct treatment and disease control initiatives rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, have stimulated efforts to develop new drugs for the treatment of schistosomiasis. Schistosomes do not have the de novo purine biosynthetic pathway, and instead depend entirely on the purine salvage pathway to supply its need for purines. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenylosuccinate lyase (SmADSL) is an enzyme in this pathway, which cleaves adenylosuccinate (ADS) into adenosine 5′-monophosphate (AMP) and fumarate. SmADSL kinetic characterization was performed by isothermal titration calorimetry (ITC) using both ADS and SAICAR as substrates. Structures of SmADSL in Apo form and in complex with AMP were elucidated by x-ray crystallography revealing a highly conserved tetrameric structure required for their function since the active sites are formed from residues of three different subunits. The active sites are also highly conserved between species and it is difficult to identify a potent species-specific inhibitor for the development of new therapeutic agents. In contrast, several mutagenesis studies have demonstrated the importance of dimeric interface residues in the stability of the quaternary structure of the enzyme. The lower conservation of these residues between SmADSL and human ADSL could be used to lead the development of anti-schistosomiasis drugs based on disruption of subunit interfaces. These structures and kinetics data add another layer of information to Schistosoma mansoni purine salvage pathway. Less
Neisserial heparin-binding antigen NHBA is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero the crystal structures of two fragment antigen-binding domains Fabs isolated from human monoclonal antibodies targeting NHBA were determined Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs these structures provide broad insights into the NHBA epitopes recognized by the human immune system As expected these Fabs also show remarkable structural conservation ... More
Neisserial heparin-binding antigen (NHBA) is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero. As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero, the crystal structures of two fragment antigen-binding domains (Fabs) isolated from human monoclonal antibodies targeting NHBA were determined. Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs, these structures provide broad insights into the NHBA epitopes recognized by the human immune system. As expected, these Fabs also show remarkable structural conservation, as shown by a structural comparison of 15 structures of apo Fab 10C3 which were obtained from crystals grown in different crystallization conditions and were solved while searching for a complex with a bound NHBA fragment or epitope peptide. This study also provides indirect evidence for the intrinsically disordered nature of two N-terminal regions of NHBA. Less
Structures of enzyme-substrate product complexes have been studied for over four decades but have been limited to either before or after a chemical reaction Recently using in crystallo catalysis combined with X-ray diffraction we have discovered that many enzymatic reactions in nucleic-acid metabolism require additional metal-ion cofactors that are not present in the substrate or product state By controlling metal ions essential for catalysis the in crystallo approach has revealed unprecedented details of reaction intermediates Here we present protocols used for successful studies of Mg -dependent DNA polymerases and ribonucleases that are applicable to analyses of a variety of metal ... More
Structures of enzyme-substrate/product complexes have been studied for over four decades but have been limited to either before or after a chemical reaction. Recently using in crystallo catalysis combined with X-ray diffraction, we have discovered that many enzymatic reactions in nucleic-acid metabolism require additional metal-ion cofactors that are not present in the substrate or product state. By controlling metal ions essential for catalysis, the in crystallo approach has revealed unprecedented details of reaction intermediates. Here we present protocols used for successful studies of Mg2+-dependent DNA polymerases and ribonucleases that are applicable to analyses of a variety of metal ion-dependent reactions. Less
Bacteria sense and adapt to environmental changes using two-component systems These signaling pathways are formed by a histidine kinase that phosphorylates a response regulator RR which finally modulates the transcription of target genes The bacterium Brucella abortus codes for a two-component system formed by the histidine kinase NtrY and the RR NtrX that participates in sensing low oxygen tension and generating an adaptive response NtrX is a modular protein with REC AAA and DNA-binding domains an architecture that classifies it among the NtrC subfamily of RRs However it lacks the signature GAFTGA motif that is essential for activating transcription by ... More
Bacteria sense and adapt to environmental changes using two-component systems. These signaling pathways are formed by a histidine kinase that phosphorylates a response regulator (RR), which finally modulates the transcription of target genes. The bacterium Brucella abortus codes for a two-component system formed by the histidine kinase NtrY and the RR NtrX that participates in sensing low oxygen tension and generating an adaptive response. NtrX is a modular protein with REC, AAA +, and DNA-binding domains, an architecture that classifies it among the NtrC subfamily of RRs. However, it lacks the signature GAFTGA motif that is essential for activating transcription by the mechanism proposed for canonical members of this subfamily. In this article, we present the first crystal structure of full-length NtrX, which is also the first structure of a full-length NtrC-like RR with all the domains solved, showing that the protein is structurally similar to other members of the subfamily. We also report that NtrX binds nucleotides and the structures of the protein bound to ATP and ADP. Despite binding ATP, NtrX does not have ATPase activity and does not form oligomers in response to phosphorylation or nucleotide binding. We also identify a nucleotide sequence recognized by NtrX that allows it to bind to a promoter region that regulates its own transcription and to establish a negative feedback mechanism to modulate its expression. Overall, this article provides a detailed description of the NtrX RR and supports that it functions by a mechanism different to classical NtrC-like RRs. Less
Autophagy is a conserved cellular process involved in the elimination of proteins and organelles It is also used to combat infection with pathogenic microbes The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg LC proteins coupled to phosphatidylethanolamine PE on autophagosomal membranes To understand how RavZ recognizes and deconjugates LC -PE we prepared semisynthetic LC proteins and elucidated the structures of the RavZ LC interaction Semisynthetic LC proteins allowed the analysis of structure-function relationships RavZ extracts LC -PE from the membrane before deconjugation RavZ initially recognizes the LC molecule on membranes via its ... More
Autophagy is a conserved cellular process involved in the elimination of proteins and organelles. It is also used to combat infection with pathogenic microbes. The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3 proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes. To understand how RavZ recognizes and deconjugates LC3-PE, we prepared semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3 interaction. Semisynthetic LC3 proteins allowed the analysis of structure-function relationships. RavZ extracts LC3-PE from the membrane before deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and docking of the acyl chains into the lipid-binding site of RavZ that is related in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has evolved a novel mechanism to specifically evade host autophagy. Less
Dehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids Given the insolubility of the natural C C fatty acyl substrate of the PpsC dehydratase DH domain we investigated its structure function relationships in the presence of shorter surrogate substrates Since most enzymes belonging to the R -specific enoyl hydratase hydroxyacyl dehydratase family conduct the reverse hydration reaction ... More
Dehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine. The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids. Given the insolubility of the natural C28?C30 fatty acyl substrate of the PpsC dehydratase (DH) domain, we investigated its structure?function relationships in the presence of shorter surrogate substrates. Since most enzymes belonging to the (R)-specific enoyl hydratase/hydroxyacyl dehydratase family conduct the reverse hydration reaction in vitro, we have determined the X-ray structures of the PpsC DH domain, both unliganded (apo) and in complex with trans-but-2-enoyl-CoA or trans-dodec-2-enoyl-CoA derivatives. This study provides for the first time a snapshot of dehydratase?ligand interactions following a hydration reaction. Our structural analysis allowed us to identify residues essential for substrate binding and activity. The structural comparison of the two complexes also sheds light on the need for long acyl chains for this dehydratase to carry out its function, consistent with both its in vitro catalytic behavior and the physiological role of the PpsC enzyme. Less
Insulin-regulated aminopeptidase IRAP is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates although the mechanism behind this wide specificity is not clearly understood We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at resolution In the presence of this inhibitor the enzyme adopts a novel conformation in which domains II and IV are juxtaposed forming a hollow structure that excludes external solvent access to the catalytic center A loop adjacent to the enzyme s GAMEN motif undergoes structural reconfiguration allowing the ... More
Insulin-regulated aminopeptidase (IRAP) is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates, although the mechanism behind this wide specificity is not clearly understood. We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at 2.53 � resolution. In the presence of this inhibitor, the enzyme adopts a novel conformation in which domains II and IV are juxtaposed, forming a hollow structure that excludes external solvent access to the catalytic center. A loop adjacent to the enzyme�s GAMEN motif undergoes structural reconfiguration, allowing the accommodation of bulky inhibitor side chains. Atomic interactions between the inhibitor and IRAP that are unique to this conformation can explain the strong selectivity compared to homologous aminopeptidases ERAP1 and ERAP2. This conformation provides insight on IRAP�s catalytic cycle and reveals significant active-site plasticity that may underlie its substrate permissiveness. Less
This multidisciplinary project begins with one overarching aim to elucidate the role of the rigidity of the lipid tail on the phase transitions of lipidic mesophases Previous studies have demonstrated that the position and the number of cis double bonds in monoacylglycerols determine the chain splay of the molecule establishing how this parameter was essential in influencing the phase behavior Following on from this novel lipids which are inspired by naturally occuring cyclopropanated lipids have been synthesized and their phase behavior elucidated The chain rigidity has been systematically varied by locking the cis configuration of the double bond on the ... More
This multidisciplinary project begins with one overarching aim: to elucidate the role of the
rigidity of the lipid tail on the phase transitions of lipidic mesophases. Previous studies have
demonstrated that the position and the number of cis double bonds in monoacylglycerols
determine the chain splay of the molecule, establishing how this parameter was essential in
influencing the phase behavior. Following on from this, novel lipids which are inspired by
naturally occuring cyclopropanated lipids have been synthesized, and their phase behavior
elucidated. The chain rigidity has been systematically varied by locking the cis configuration
of the double bond on the alkyl chain in a confined geometry. To understand the relationship
between chain rigidity and phase behavior a library of new lipids has been synthesized replacing
the cis double bond by a geometrically confined cyclopropyl ring. The replacement of the
double bond with a chemically analogous cyclopropyl group was designed in order to maintain
a similar chain splay and CPP parameter. The insertion of an additional carbon into the lipidic
chain doesn�t significantly change the length or the curvature of the chain but varies
substantially the packing frustration and the lateral stress of the lipid.
The phase behavior of these novel lipids with identical head group and different alkyl chains
has been investigated with utmost care. Small angle X-ray scattering (SAXS) measurements at
different hydration level and at different temperatures have been used to study the thermal
behavior of these lipid and the effect of this novel motif on the lipidic packing, with particular
attention to low temperature effects.
Since cyclopropanated lipids are present in several dairy products, and since lipidic
nanoparticles have been proved to be excellent drug delivery systems, digestion studies of
cubosomes and hexosomes formed by the novel synthesized cyclopropanated lipids have been
performed. Time resolved synchrotron SAXS has been used to monitor the phase changes
during the enzymatic reaction.
In order to test the utility of the cyclopropanated lipidic systems for membrane protein
crystallization the novel lipidic cubic phase (LCP) matrices have been employed in
crystallization studies with the membrane protein model system bacteriorhodopsin (bR).
IV
Finally, the successful crystallization attempts for membrane protein structural studies of the
chloride channels EcClC and Rm1ClC, as well as the lipopolysaccharide transporter LptD-LptE
show the broad applicability of the LCP crystallization method and the utility of tuning
crystallization conditions, including a screening of different lipids, to optimize crystal growt. Less
rigidity of the lipid tail on the phase transitions of lipidic mesophases. Previous studies have
demonstrated that the position and the number of cis double bonds in monoacylglycerols
determine the chain splay of the molecule, establishing how this parameter was essential in
influencing the phase behavior. Following on from this, novel lipids which are inspired by
naturally occuring cyclopropanated lipids have been synthesized, and their phase behavior
elucidated. The chain rigidity has been systematically varied by locking the cis configuration
of the double bond on the alkyl chain in a confined geometry. To understand the relationship
between chain rigidity and phase behavior a library of new lipids has been synthesized replacing
the cis double bond by a geometrically confined cyclopropyl ring. The replacement of the
double bond with a chemically analogous cyclopropyl group was designed in order to maintain
a similar chain splay and CPP parameter. The insertion of an additional carbon into the lipidic
chain doesn�t significantly change the length or the curvature of the chain but varies
substantially the packing frustration and the lateral stress of the lipid.
The phase behavior of these novel lipids with identical head group and different alkyl chains
has been investigated with utmost care. Small angle X-ray scattering (SAXS) measurements at
different hydration level and at different temperatures have been used to study the thermal
behavior of these lipid and the effect of this novel motif on the lipidic packing, with particular
attention to low temperature effects.
Since cyclopropanated lipids are present in several dairy products, and since lipidic
nanoparticles have been proved to be excellent drug delivery systems, digestion studies of
cubosomes and hexosomes formed by the novel synthesized cyclopropanated lipids have been
performed. Time resolved synchrotron SAXS has been used to monitor the phase changes
during the enzymatic reaction.
In order to test the utility of the cyclopropanated lipidic systems for membrane protein
crystallization the novel lipidic cubic phase (LCP) matrices have been employed in
crystallization studies with the membrane protein model system bacteriorhodopsin (bR).
IV
Finally, the successful crystallization attempts for membrane protein structural studies of the
chloride channels EcClC and Rm1ClC, as well as the lipopolysaccharide transporter LptD-LptE
show the broad applicability of the LCP crystallization method and the utility of tuning
crystallization conditions, including a screening of different lipids, to optimize crystal growt. Less
Effect of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme
During production purification formulation and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability Such harmful conditions include extreme pH changes high ionic strengths or elevated temperatures The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development In this context PEGylation i e the covalent attachment of polyethylene glycol PEG to proteins represents a valuable strategy to improve the physico-chemical properties of proteins In this work the influence ... More
During production, purification, formulation, and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability. Such harmful conditions include extreme pH changes, high ionic strengths or elevated temperatures. The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation, as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development. In this context PEGylation, i.e. the covalent attachment of polyethylene glycol (PEG) to proteins, represents a valuable strategy to improve the physico-chemical properties of proteins. In this work, the influence of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme is investigated. Specifically, conformational and colloidal properties were studied by means of high-throughput melting point determination and automated generation of protein phase diagrams, respectively. Lysozyme from chicken egg-white as a model protein was randomly conjugated to 2 kDa, 5 kDa and 10 kDa mPEG-aldehyde and resulting PEGamer species were purified by chromatographic separation. Besides protein stability assessment, residual enzyme activities were evaluated employing a Micrococcus lysodeikticus based activity assay. PEG molecules with lower molecular weights and lower PEGylation degrees resulted in higher residual activities. Changes in enzyme activities upon PEGylation have shown to result from a combination of steric hindrance and molecular flexibility. In contrast, higher PEG molecular weights and PEGylation degrees enhanced conformational and colloidal stability. By PEGylating lysozyme an increase of the protein solubility by more than 11-fold was achieved. Less
Eps epidermal growth factor receptor pathway substrate -homology domain containing proteins EHDs comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking EHD proteins consist of a dynamin-related GTPase domain a helical domain and a C-terminal Eps -homology EH domain Previous studies have revealed the structure of the EHD dimer Furthermore the N terminal region of EHD was demonstrated to bind to a hydrophobic groove of the GTPase domain and to switch into the membrane in the presence of liposome suggesting an autoinhibitory role However the molecular mechanisms of membrane binding oligomerization and nucleotide hydrolysis have remained obscure ... More
Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. EHD proteins consist of a dynamin-related GTPase domain, a helical domain and a C-terminal Eps15-homology (EH) domain,. Previous studies have revealed the structure of the EHD2 dimer. Furthermore, the N terminal region of EHD2 was demonstrated to bind to a hydrophobic groove of the GTPase domain and to switch into the membrane in the presence of liposome, suggesting an autoinhibitory role However, the molecular mechanisms of membrane binding, oligomerization and nucleotide hydrolysis have remained obscure. To understand the mechanism of membrane recruitment, the crystal structure of an aminoterminally truncated EHD4 dimer in complex with ATPγS and ADP were determined in this thesis. Compared with the EHD2 structure, the helical domains assume an open conformation featuring a 50° rotation relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), it was shown that the opening aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop region in the GTPase domain undergoes a large rearrangement and creates a new interface that allows oligomerization on membranes. These results suggest that the EHD4 structures represent the active EHD conformation, whereas the EHD2 structure is autoinhibited. A model for the activation and oligomerization of EHD proteins was proposed in which a series of domain rearrangements control membrane recruitment and remodeling in the EHD family. A comparison with other peripheral membrane proteins elucidated common and specific features of this activation mechanism. Less
Eps epidermal growth factor receptor pathway substrate -homology domain containing proteins EHDs are molecular machines that use the energy of ATP binding and ATP hydrolysis to remodel shallow membranes into highly curved membrane tubules This activity is required in many cellular membrane trafficking pathways In this work we have determined a high-resolution structure of an EHD machine in the active state The structure indicates how EHDs assemble at the membrane surface into ring-like scaffolds that deform the underlying membrane By comparing this active state with a previously determined autoinhibited conformation we can deduce the mechanistic details how recruitment of EHDs ... More
Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) are molecular machines that use the energy of ATP binding and ATP hydrolysis to remodel shallow membranes into highly curved membrane tubules. This activity is required in many cellular membrane trafficking pathways. In this work, we have determined a high-resolution structure of an EHD machine in the active state. The structure indicates how EHDs assemble at the membrane surface into ring-like scaffolds that deform the underlying membrane. By comparing this active state with a previously determined autoinhibited conformation, we can deduce the mechanistic details how recruitment of EHDs to membranes is regulated. A comparison with other membrane-associated molecular machines reveals commonalities and differences in the activation mechanism. Less
Type phosphatidic acid phosphatases PAP s can be either soluble or integral membrane enzymes In bacteria integral membrane PAP s play major roles in the metabolisms of glycerophospholipids undecaprenyl-phosphate C -P lipid carrier and lipopolysaccharides By in vivo functional experiments and biochemical characterization we show that the membrane PAP coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate PGP phosphatase of B subtilis We also confirm that this enzyme renamed bsPgpB has a weaker activity on C -PP Moreover we solved the crystal structure of bsPgpB at resolution with tungstate a phosphate analog in the active site ... More
Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s. Less
Current influenza vaccines are mostly formulated as liquids which requires a continuous cold chain to maintain the stability of the antigen For development of vaccines with an increased stability at ambient temperatures manifold parameters and their influences on the colloidal stability and activity of the antigen have to be understood This work presents a strategy to examine both the colloidal stability and the remaining biological activity of H N influenza viruses under various conditions after an incubation of days H N phase diagrams were generated for several pH values and different initial H N and NaCl concentrations It was shown ... More
Current influenza vaccines are mostly formulated as liquids which requires a continuous cold chain to maintain the stability of the antigen. For development of vaccines with an increased stability at ambient temperatures, manifold parameters and their influences on the colloidal stability and activity of the antigen have to be understood. This work presents a strategy to examine both, the colloidal stability and the remaining biological activity of H1N1 influenza viruses under various conditions after an incubation of 40 days. H1N1 phase diagrams were generated for several pH values and different initial H1N1 and NaCl concentrations. It was shown that the highest H1N1 recoveries were obtained for pH 6 and that moderate amounts of NaCl are favorable for increased recoveries. In contrast to colloidal stability, the highest remaining HA activity was observed at pH 9. The electrostatic and hydrophobic surface properties of H1N1 were investigated to reveal the mechanisms accounting for the decrease in stability. Secondly, the capability of virus precipitation by polyethylene glycol in combination with determination of surface hydrophobicity was proven to be useful as a predictive tool to rank stability under different conditions. This methodology enables the rapid assessment of aggregation propensity of H1N1 formulations and the influence on the activity of the virus particles and might become a standard tool during the development of vaccine formulations. Less
Oxidation of halides and thiocyanate by heme peroxidases to antimicrobial oxidants is an important cornerstone in the innate immune system of mammals Interestingly phylogenetic and physiological studies suggest that homologous peroxidases are already present in mycetozoan eukaryotes such as Dictyostelium discoideum This social amoeba kills bacteria via phagocytosis for nutrient acquisition at its single-cell stage and for antibacterial defense at its multicellular stages Here we demonstrate that peroxidase A from D discoideum DdPoxA is a stable monomeric glycosylated and secreted heme peroxidase with homology to mammalian peroxidases The first crystal structure resolution of a mycetozoan peroxidase of this superfamily shows ... More
Oxidation of halides and thiocyanate by heme peroxidases to antimicrobial oxidants is an important cornerstone in the innate immune system of mammals. Interestingly, phylogenetic and physiological studies suggest that homologous peroxidases are already present in mycetozoan eukaryotes such as Dictyostelium discoideum. This social amoeba kills bacteria via phagocytosis for nutrient acquisition at its single-cell stage and for antibacterial defense at its multicellular stages. Here, we demonstrate that peroxidase A from D. discoideum (DdPoxA) is a stable, monomeric, glycosylated, and secreted heme peroxidase with homology to mammalian peroxidases. The first crystal structure (2.5 Å resolution) of a mycetozoan peroxidase of this superfamily shows the presence of a post-translationally-modified heme with one single covalent ester bond between the 1-methyl heme substituent and Glu-236. The metalloprotein follows the halogenation cycle, whereby compound I oxidizes iodide and thiocyanate at high rates (>108 m−1 s−1) and bromide at very low rates. It is demonstrated that DdPoxA is up-regulated and likely secreted at late multicellular development stages of D. discoideum when migrating slugs differentiate into fruiting bodies that contain persistent spores on top of a cellular stalk. Expression of DdPoxA is shown to restrict bacterial contamination of fruiting bodies. Structure and function of DdPoxA are compared with evolutionary-related mammalian peroxidases in the context of non-specific immune defense. Less
By interacting with hundreds of protein partners - - proteins coordinate vital cellular processes Phosphorylation of the small heat shock protein HSPB within its intrinsically disordered N-terminal domain activates its interaction with - - ultimately triggering smooth muscle relaxation After analyzing the binding of an HSPB -derived phosphopeptide to - - using isothermal calorimetry and X-ray crystallography we have determined the crystal structure of the complete assembly consisting of the - - dimer and full-length HSPB dimer and further characterized this complex in solution using fluorescence spectroscopy small-angle X-ray scattering and limited proteolysis We show that selected intrinsically disordered regions ... More
By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein HSPB6 within its intrinsically disordered N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first-ever atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants. Less
Studies of the intermediate filament IF structure are a prerequisite of understanding their function In addition the structural information is indispensable if one wishes to gain a mechanistic view on the disease-related mutations in the IFs Over the years considerable progress has been made on the atomic structure of the elementary building block of all IFs the coiled-coil dimer Here we discuss the approaches methods and practices that have contributed to this advance With abundant genetic information on hand bioinformatics approaches give important insights into the dimer structure including the head and tail regions poorly assessable experimentally At the same ... More
Studies of the intermediate filament (IF) structure are a prerequisite of understanding their function. In addition, the structural information is indispensable if one wishes to gain a mechanistic view on the disease-related mutations in the IFs. Over the years, considerable progress has been made on the atomic structure of the elementary building block of all IFs, the coiled-coil dimer. Here, we discuss the approaches, methods and practices that have contributed to this advance. With abundant genetic information on hand, bioinformatics approaches give important insights into the dimer structure, including the head and tail regions poorly assessable experimentally. At the same time, the most important contribution has been provided by X-ray crystallography. Following the “divide-and-conquer” approach, many fragments from several IF proteins could be crystallized and resolved to atomic resolution. We will systematically cover the main procedures of these crystallographic studies, suggest ways to maximize their efficiency, and also discuss the possible pitfalls and limitations. In addition, electron paramagnetic resonance with site-directed spin labeling was another method providing a major impact toward the understanding of the IF structure. Upon placing the spin labels into specific positions within the full-length protein, one can evaluate the proximity of the labels and their mobility. This makes it possible to make conclusions about the dimer structure in the coiled-coil region and beyond, as well as to explore the dimer–dimer contacts. Less
In concentrated protein solutions attractive protein interactions may not only cause the formation of undesired aggregates but also of gel-like networks with elevated viscosity To guarantee stable biopharmaceutical processes and safe formulations both phenomenons have to be avoided as these may hinder regular processing steps This work screens the impact of additives on both phase behavior and viscosity of concentrated protein solutions For this purpose additives known for stabilizing proteins in solution or modulating the dynamic viscosity were selected These additives were PEG PEG glycerol glycine NaCl and ArgHCl Concentrated lysozyme and glucose oxidase solutions at pH and served as ... More
In concentrated protein solutions attractive protein interactions may not only cause the formation of undesired aggregates but also of gel-like networks with elevated viscosity. To guarantee stable biopharmaceutical processes and safe formulations, both phenomenons have to be avoided as these may hinder regular processing steps. This work screens the impact of additives on both phase behavior and viscosity of concentrated protein solutions. For this purpose, additives known for stabilizing proteins in solution or modulating the dynamic viscosity were selected. These additives were PEG 300, PEG 1000, glycerol, glycine, NaCl and ArgHCl. Concentrated lysozyme and glucose oxidase solutions at pH 3 and 9 served as model systems. Fourier-transformed-infrared spectroscopy was chosen to determine the conformational stability of selected protein samples. Influencing protein interactions, the impact of additives was strongly dependent on pH. Of all additives investigated, glycine was the only one that maintained protein conformational and colloidal stability while decreasing the dynamic viscosity. Low concentrations of NaCl showed the same effect, but increasing concentrations resulted in visible protein aggregates. Less
PRMT is the less-characterized member of the protein arginine methyltransferase family in terms of structure activity and cellular functions PRMT is a modular protein containing a catalytic Ado-Met-binding domain and unique Src homology domain that binds proteins with proline-rich motifs PRMT is involved in a variety of cellular processes and has diverse roles in transcriptional regulation through different mechanisms depending on its binding partners PRMT has been demonstrated to have weak methyltransferase activity on a histone H substrate but its optimal substrates have not yet been identified To obtain insights into the function and activity of PRMT we solve several ... More
PRMT2 is the less-characterized member of the protein arginine methyltransferase family in terms of structure, activity, and cellular functions. PRMT2 is a modular protein containing a catalytic Ado-Met-binding domain and unique Src homology 3 domain that binds proteins with proline-rich motifs. PRMT2 is involved in a variety of cellular processes and has diverse roles in transcriptional regulation through different mechanisms depending on its binding partners. PRMT2 has been demonstrated to have weak methyltransferase activity on a histone H4 substrate, but its optimal substrates have not yet been identified. To obtain insights into the function and activity of PRMT2, we solve several crystal structures of PRMT2 from two homologs (zebrafish and mouse) in complex with either the methylation product S-adenosyl-L-homocysteine or other compounds including the first synthetic PRMT2 inhibitor (Cp1) studied so far. We reveal that the N-terminal-containing SH3 module is disordered in the full-length crystal structures, and highlights idiosyncratic features of the PRMT2 active site. We identify a new nonhistone protein substrate belonging to the serine-/arginine-rich protein family which interacts with PRMT2 and we characterize six methylation sites by mass spectrometry. To better understand structural basis for Cp1 binding, we also solve the structure of the complex PRMT4:Cp1. We compare the inhibitor–protein interactions occurring in the PRMT2 and PRMT4 complex crystal structures and show that this compound inhibits efficiently PRMT2. These results are a first step toward a better understanding of PRMT2 substrate recognition and may accelerate the development of structure-based drug design of PRMT2 inhibitors. Less
Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators Here we report the generation of a quantitative interaction network directly linking human proteins to the AAA ATPase p an essential hexameric protein with multiple cellular functions We show that the high-affinity interacting protein ASPL efficiently promotes p hexamer disassembly resulting in the formation of stable p ASPL heterotetramers High-resolution structural and biochemical studies indicate that an extended UBX domain eUBX in ASPL is critical for p hexamer disassembly and facilitates the assembly of p ASPL heterotetramers This spontaneous process is accompanied by ... More
Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity. Less
Tn and Tn -like transposons are complex elements found in disparate environments and are responsible for mobilizing a wide variety of genes and forming pathogenicity fitness islands They are novel in their ability to recognize both a single site in the chromosome and specifically target transposition into mobile plasmids via dedicated TnsD and TnsE targeting proteins TnsE recognizes mobile plasmids through an association with the processivity clamp and a ' recessed DNA end during conjugal replication However the mechanism for the specific recognition of ' recessed DNA ends remains unclear Structural analyses of the C-terminal domain of TnsE identified a ... More
Tn7 and Tn7-like transposons are complex elements found in disparate environments and are responsible for mobilizing a wide variety of genes and forming pathogenicity/fitness islands. They are novel in their ability to recognize both a single site in the chromosome and specifically target transposition into mobile plasmids via dedicated TnsD and TnsE targeting proteins. TnsE recognizes mobile plasmids through an association with the processivity clamp and a 3' recessed DNA end during conjugal replication. However, the mechanism for the specific recognition of 3' recessed DNA ends remains unclear. Structural analyses of the C-terminal domain of TnsE identified a novel protein fold
including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-function variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structurebased analysis of a series of TnsE variants relates transposition to DNA binding stability. Follow up studies of full length TnsE bound to DNA are in progress. Less
including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-function variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structurebased analysis of a series of TnsE variants relates transposition to DNA binding stability. Follow up studies of full length TnsE bound to DNA are in progress. Less
Deamination of choline catalyzed by the glycyl radical enzyme choline trimethylamine-lyase CutC has emerged as an important route for the production of trimethylamine a microbial metabolite associated with both human disease and biological methane production Here we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline Within an unexpectedly polar active site CutC orients choline through hydrogen bonding with a putative general base and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety These structural data along with ... More
Deamination of choline catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as an important route for the production of trimethylamine, a microbial metabolite associated with both human disease and biological methane production. Here, we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline. Within an unexpectedly polar active site, CutC orients choline through hydrogen bonding with a putative general base, and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety. These structural data, along with biochemical analysis of active site mutants, support a mechanism that involves direct elimination of trimethylamine. This work broadens our understanding of radical-based enzyme catalysis and will aid in the rational design of inhibitors of bacterial trimethylamine production. Less
The colloidal stability of a protein solution during downstream processing formulation and storage is a key issue for the biopharmaceutical production process Thus knowledge about colloidal solution characteristics such as the tendency to form aggregates or high viscosity at various processing conditions is of interest This work correlates changes in the apparent diffusion coefficient as a parameter of protein interactions with observed protein aggregation and dynamic viscosity of the respective protein samples For this purpose the diffusion coefficient the protein phase behavior and the dynamic viscosity in various systems containing the model proteins -lactalbumin lysozyme and glucose oxidase were studied ... More
The colloidal stability of a protein solution during downstream processing, formulation, and storage is a key issue for the biopharmaceutical production process. Thus, knowledge about colloidal solution characteristics, such as the tendency to form aggregates or high viscosity, at various processing conditions is of interest. This work correlates changes in the apparent diffusion coefficient as a parameter of protein interactions with observed protein aggregation and dynamic viscosity of the respective protein samples. For this purpose, the diffusion coefficient, the protein phase behavior, and the dynamic viscosity in various systems containing the model proteins α-lactalbumin, lysozyme, and glucose oxidase were studied. Each of these experiments revealed a wide range of variations in protein interactions depending on protein type, protein concentration, pH, and the NaCl concentration. All these variations showed to be mirrored by changes in the apparent diffusion coefficient in the respective samples. Whereas stable samples with relatively low viscosity showed an almost linear dependence, the deviation from the concentration-dependent linearity indicated both an increase in the sample viscosity and probability of protein aggregation. This deviation of the apparent diffusion coefficient from concentration-dependent linearity was independent of protein type and solution properties for this study. Thus, this single parameter shows the potential to act as a prognostic tool for colloidal stability of protein solutions. Less
In the biopharmaceutical industry it is mandatory to know and ensure the correct protein phase state as a critical quality attribute in every process step Unwanted protein precipitation or crystallization can lead to column pipe or filter blocking In formulation the formation of aggregates can even be lethal when injected into the patient The typical methodology to illustrate protein phase states is the generation of protein phase diagrams Commonly protein phase behavior is shown in dependence of protein and precipitant concentration Despite using high-throughput methods for the generation of phase diagrams the time necessary to reach equilibrium is the bottleneck ... More
In the biopharmaceutical industry it is mandatory to know and ensure the correct protein phase state as a critical quality attribute in every process step. Unwanted protein precipitation or crystallization can lead to column, pipe or filter blocking. In formulation, the formation of aggregates can even be lethal when injected into the patient. The typical methodology to illustrate protein phase states is the generation of protein phase diagrams. Commonly, protein phase behavior is shown in dependence of protein and precipitant concentration. Despite using high-throughput methods for the generation of phase diagrams, the time necessary to reach equilibrium is the bottleneck. Faster methods to predict protein phase behavior are desirable. In this study, hydrophobic interaction chromatography retention times were correlated to crystal size and form. High-throughput thermal stability measurements (melting and aggregation temperatures), using an Optim®2 system, were successfully correlated to glucose isomerase stability. By using hydrophobic interaction chromatography and thermal stability determinations, glucose isomerase conformational and colloidal stability were successfully predicted for different salts in a specific pH range. Less
Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation However alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease Glycogen phosphorylase GP a key enzyme in glycogen metabolism catalyzes the rate-limiting step of glycogen mobilization Moreover the allosteric regulation of the three GP isozymes muscle liver and brain by metabolites and phosphorylation in response to hormonal signaling fine-tunes glycogenolysis to fulfill energetic and metabolic requirements Whereas the structures of muscle and liver GPs have been known for decades the structure of brain GP ... More
Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 �) and in complex with its allosteric activator AMP (3.4 �). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen. Less
Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures and most VacA activities are dependent on its capacity to oligomerize The -kDa secreted VacA protein can undergo limited proteolysis to yield two domains designated p and p The p domain is required for membrane channel formation and intracellular toxic activities and the p domain has an important role in mediating VacA binding to cells Previous studies showed that the p domain has a predominantly -helical structure ... More
Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly �-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori. The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the �-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the �-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly. Less
Clostridium perfringens spores employ two peptidoglycan lysins to degrade the spore cortex during germination SleC initiates cortex hydrolysis to generate cortical fragments that are degraded further by the muramidase SleM Here we present the crystal structure of the C perfringens S SleM protein at SleM comprises an N-terminal catalytic domain that adopts an irregular -barrel fold that is common to GH family lysozymes plus a C-terminal fibronectin type III domain The latter is involved in forming the SleM dimer that is evident in both the crystal structure and in solution A truncated form of SleM that lacks the FnIII domain ... More
Clostridium perfringens spores employ two peptidoglycan lysins to degrade the spore cortex during germination. SleC initiates cortex hydrolysis to generate cortical fragments that are degraded further by the muramidase SleM. Here, we present the crystal structure of the�C. perfringens�S40 SleM protein at 1.8 �. SleM comprises an N-terminal catalytic domain that adopts an irregular ?/?-barrel fold that is common to GH25 family lysozymes, plus a C-terminal fibronectin type III domain. The latter is involved in forming the SleM dimer that is evident in both the crystal structure and in solution. A truncated form of SleM that lacks the FnIII domain shows reduced activity against spore sacculi indicating that this domain may have a role in facilitating the position of substrate with respect to the enzyme's active site. Less
Extraordinary antibodies capable of near pan-neutralization of HIV- have been identified One of the broadest is antibody E which recognizes the membrane-proximal external region MPER of the HIV- envelope and neutralizes of circulating HIV- strains If delivered passively E might serve to prevent or treat HIV- infection Antibody E however is markedly less soluble than other antibodies Here we describe the use of both structural biology and somatic variation to develop optimized versions of E with increased solubility From the structure of E we identified a prominent hydrophobic patch reversion of four hydrophobic residues in this patch to their hydrophilic ... More
Extraordinary antibodies capable of near pan-neutralization of HIV-1 have been identified. One of the broadest is antibody 10E8, which recognizes the membrane-proximal external region (MPER) of the HIV-1 envelope and neutralizes >95% of circulating HIV-1 strains. If delivered passively, 10E8 might serve to prevent or treat HIV-1 infection. Antibody 10E8, however, is markedly less soluble than other antibodies. Here, we describe the use of both structural biology and somatic variation to develop optimized versions of 10E8 with increased solubility. From the structure of 10E8, we identified a prominent hydrophobic patch; reversion of four hydrophobic residues in this patch to their hydrophilic germ line counterparts resulted in an ~10-fold decrease in turbidity. We also used somatic variants of 10E8, identified previously by next-generation sequencing, to optimize heavy and light chains; this process yielded several improved variants. Of these, variant 10E8v4 with 26 changes versus the parent 10E8 was the most soluble, with a paratope we showed crystallographically to be virtually identical to that of 10E8, a potency on a panel of 200 HIV-1 isolates also similar to that of 10E8, and a half-life in rhesus macaques of ~10 days. An anomaly in 10E8v4 size exclusion chromatography that appeared to be related to conformational isomerization was resolved by engineering an interchain disulfide. Thus, by combining a structure-based approach with natural variation in potency and solubility from the 10E8 lineage, we successfully created variants of 10E8 which retained the potency and extraordinary neutralization breadth of the parent 10E8 but with substantially increased solubility. Less
A detailed understanding of chemical and biological function and the mechanisms underlying the molecular activities ultimately requires atomic-resolution structural data Diffraction-based techniques such as single-crystal X-ray crystallography electron microscopy and neutron diffraction are well established and they have paved the road to the stunning successes of modern-day structural biology The major advances achieved in the last years in all aspects of structural research including sample preparation crystallization the construction of synchrotron and spallation sources phasing approaches and high-speed computing and visualization now provide specialists and nonspecialists alike with a steady flow of molecular images of unprecedented detail The present unit ... More
A detailed understanding of chemical and biological function and the mechanisms underlying the molecular activities ultimately requires atomic-resolution structural data. Diffraction-based techniques such as single-crystal X-ray crystallography, electron microscopy, and neutron diffraction are well established and they have paved the road to the stunning successes of modern-day structural biology. The major advances achieved in the last 20 years in all aspects of structural research, including sample preparation, crystallization, the construction of synchrotron and spallation sources, phasing approaches, and high-speed computing and visualization, now provide specialists and nonspecialists alike with a steady flow of molecular images of unprecedented detail. The present unit combines a general overview of diffraction methods with a detailed description of the process of a single-crystal X-ray structure determination experiment, from chemical synthesis or expression to phasing and refinement, analysis, and quality control. For novices it may serve as a stepping-stone to more in-depth treatises of the individual topics. Readers relying on structural information for interpreting functional data may find it a useful consumer guide. Less
Magnetotactic bacteria are Gram-negative bacteria that navigate along geomagnetic fields using the magnetosome an organelle that consists of a membrane-enveloped magnetic nanoparticle Magnetite formation and its properties are controlled by a specific set of proteins MamC is a small magnetosome-membrane protein that is known to be active in iron biomineralization but its mechanism has yet to be clarified Here we studied the relationship between the MamC magnetite-interaction loop MIL structure and its magnetite interaction using an inert biomineralization protein-MamC chimera Our determined structure shows an alpha-helical fold for MamC-MIL with highly charged surfaces Additionally the MamC-MIL induces the formation of ... More
Magnetotactic bacteria are Gram-negative bacteria that navigate along geomagnetic fields using the magnetosome, an organelle that consists of a membrane-enveloped magnetic nanoparticle. Magnetite formation and its properties are controlled by a specific set of proteins. MamC is a small magnetosome-membrane protein that is known to be active in iron biomineralization but its mechanism has yet to be clarified. Here, we studied the relationship between the MamC magnetite-interaction loop (MIL) structure and its magnetite interaction using an inert biomineralization protein-MamC chimera. Our determined structure shows an alpha-helical fold for MamC-MIL with highly charged surfaces. Additionally, the MamC-MIL induces the formation of larger magnetite crystals compared to protein-free and inert biomineralization protein control experiments. We suggest that the connection between the MamC-MIL structure and the protein’s charged surfaces is crucial for magnetite binding and thus for the size control of the magnetite nanoparticles. Less
Haemophilus influenzae is an obligate human commensal pathogen that requires haem for survival and can acquire it from several host haemoproteins including haemopexin The haem transport system from haem-haemopexin consists of HxuC a haem receptor and the two-partner-secretion system HxuB HxuA HxuA which is exposed at the cell surface is strictly required for haem acquisition from haemopexin HxuA forms complexes with haem-haemopexin leading to haem release and its capture by HxuC The key question is how HxuA liberates haem from haemopexin Here we solve crystal structures of HxuA alone and HxuA in complex with the N-terminal domain of haemopexin A ... More
Haemophilus influenzae is an obligate human commensal/pathogen that requires haem for survival and can acquire it from several host haemoproteins, including haemopexin. The haem transport system from haem-haemopexin consists of HxuC, a haem receptor, and the two-partner-secretion system HxuB/HxuA. HxuA, which is exposed at the cell surface, is strictly required for haem acquisition from haemopexin. HxuA forms complexes with haem-haemopexin, leading to haem release and its capture by HxuC. The key question is how HxuA liberates haem from haemopexin. Here, we solve crystal structures of HxuA alone, and HxuA in complex with the N-terminal domain of haemopexin. A rational basis for the release of haem from haem-haemopexin is derived from both in vivo and in vitro studies. HxuA acts as a wedge that destabilizes the two-domains structure of haemopexin with a mobile loop on HxuA that favours haem ejection by redirecting key residues in the haem-binding pocket of haemopexin. Less
The biopharmaceutical industry is at a turning point moving toward a more customized and patient-oriented medicine precision medicine Straightforward routines such as the antibody platform process are extended to production processes for a new portfolio of molecules As a consequence individual and tailored productions require generic approaches for a fast and dedicated purification process development In this article different effective strategies in biopharmaceutical purification process development are reviewed that can analogously be used for the new generation of antibodies Conventional approaches based on heuristics and high-throughput process development are discussed and compared to modern technologies such as multivariate calibration and ... More
The biopharmaceutical industry is at a turning point moving toward a more customized and patient-oriented medicine (precision medicine). Straightforward routines such as the antibody platform process are extended to production processes for a new portfolio of molecules. As a consequence, individual and tailored productions require generic approaches for a fast and dedicated purification process development. In this article, different effective strategies in biopharmaceutical purification process development are reviewed that can analogously be used for the new generation of antibodies. Conventional approaches based on heuristics and high-throughput process development are discussed and compared to modern technologies such as multivariate calibration and mechanistic modeling tools. Such approaches constitute a good foundation for fast and effective process development for new products and processes, but their full potential becomes obvious in a correlated combination. Thus, different combinatorial approaches are presented, which might become future directions in the biopharmaceutical industry. Less
LUX ARRHYTHMO LUX is a Myb-domain transcription factor that plays an important role in regulating the circadian clock Lux mutations cause severe clock defects and arrhythmia in constant light and dark In order to examine the molecular mechanisms underlying the function of LUX the DNA-binding Myb domain was cloned expressed and purified The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays EMSAs demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity In order to investigate the specificity determinants of protein DNA interactions the protein was co-crystallized with a -mer ... More
LUX ARRHYTHMO (LUX) is a Myb-domain transcription factor that plays an important role in regulating the circadian clock. Lux mutations cause severe clock defects and arrhythmia in constant light and dark. In order to examine the molecular mechanisms underlying the function of LUX, the DNA-binding Myb domain was cloned, expressed and purified. The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays (EMSAs), demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity. In order to investigate the specificity determinants of protein�DNA interactions, the protein was co-crystallized with a 10-mer cognate DNA. Initial crystallization results for the selenomethionine-derivatized protein and data-set collection statistics are reported. Data collection was performed using the MeshAndCollect workflow available at the ESRF. Less
A-kinase anchoring proteins AKAPs interact with the dimerization docking D D domains of regulatory subunits of the ubiquitous protein kinase A PKA AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling Here we elucidated the structure of an extended PKA-binding domain of AKAP bound to the D D domain of the regulatory RIIa subunits of PKA We identified three hydrophilic anchor points in AKAP outside the core PKA-binding domain which mediate contacts with the D D domain Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA We ... More
A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18� bound to the D/D domain of the regulatory RIIa subunits of PKA. We identified three hydrophilic anchor points in AKAP18� outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions. Less
The SCP2-thiolase-like protein (SLP) of Trypanosoma brucei is an enzyme involved in lipid metabolism
Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP -thiolase-like protein SLP which is characterized by having the YDCF thiolase sequence fingerprint of the C -C loop SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals including human Deletion of the Trypanosoma brucei SLP gene TbSLP increases the doubling time of procyclic T brucei and causes a -fold reduction of de novo sterol biosynthesis from glucose- and acetate-derived acetyl-CoA Fluorescence analyses of EGFP-tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion The crystal structure ... More
Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2-thiolase-like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cβ2-Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T. brucei and causes a 5-fold reduction of de novo sterol biosynthesis from glucose- and acetate-derived acetyl-CoA. Fluorescence analyses of EGFP-tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion. The crystal structure of TbSLP (refined at 1.75 Å resolution) confirms that TbSLP has the canonical dimeric thiolase fold. In addition, the structures of the TbSLP-acetoacetyl-CoA (1.90 Å) and TbSLP-malonyl-CoA (2.30 Å) complexes reveal that the two oxyanion holes of the thiolase active site are preserved. TbSLP binds malonyl-CoA tightly (Kd 90 µM), acetoacetyl-CoA moderately (Kd 0.9 mM) and acetyl-CoA and CoA very weakly. TbSLP possesses low malonyl-CoA decarboxylase activity. Altogether, the data show that TbSLP is a mitochondrial enzyme involved in lipid metabolism. Less
Fish and human cytochrome P P A catalyze both steroid a-hydroxylation and a -lyase reactions Fish P A catalyzes only a-hydroxylation Both enzymes are microsomal-type P s integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment the signal anchor sequence The presence of this N-terminal region renders expression of full-length proteins impossible or challenging For some proteins variable truncation of the signal anchor sequence precludes expression or results in poor expression levels To crystallize P A and A in order to gain insight into their different activities we used an alternative N-terminal sequence to boost expression ... More
Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17a-hydroxylation and 17a,20-lyase reactions. Fish P450 17A2 catalyzes only 17a-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins impossible or challenging. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins. Less
Serogroup B Neisseria meningitidis MenB is the cause of an acute potentially severe infection known as invasive meningococcal disease IMD with two peaks in disease incidence occurring among adolescents and young adults to years of age Bexsero is the first genome-derived vaccine against MenB and it has recently been approved in countries worldwide Neisserial adhesin A NadA a meningococcal trimeric autotransporter adhesin TAA that acts in adhesion to and invasion of host epithelial cells is one of the three protein antigens included in Bexsero The main aim of this work was to obtain detailed insights into the structure of NadA ... More
Serogroup B Neisseria meningitidis (MenB) is the cause of an acute, potentially severe infection, known as invasive meningococcal disease (IMD) with two peaks in disease incidence occurring among adolescents and young adults 16 to 21 years of age. Bexsero is the first genome-derived vaccine against MenB, and it has recently been approved in >35 countries worldwide. Neisserial adhesin A (NadA), a meningococcal trimeric autotransporter adhesin (TAA) that acts in adhesion to, and invasion of, host epithelial cells, is one of the three protein antigens included in Bexsero. The main aim of this work was to obtain detailed insights into the structure of NadA variant 3 (NadAv3), the vaccine variant, and into the molecular mechanisms governing its transcriptional regulation by NadR (Neisseria adhesin A Regulator). The amount of NadA exposed on the meningococcal surface influences the antibody-mediated serum bactericidal response measured in vitro, which in turn correlates with protection in immunized subjects. A deep understanding of nadA expression is therefore important, otherwise the contribution of NadA to vaccine-induced protection against meningococcal disease may be underestimated. The abundance of surface-exposed NadA is regulated by the ligand-responsive transcriptional repressor NadR. The functional, biochemical and high-resolution structural characterization of NadR is presented in the first part of the thesis (Part One). These studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. These findings shed light on the regulation of a key virulence factor and vaccine antigen of this important human pathogen. In the second part of the thesis (Part Two), strategies involving both protein engineering and crystal manipulation to increase the likelihood of solving the crystal structure of NadAv3 are described. The first approach was the rational design of new constructs of NadAv3, based on the recently solved crystal structure of a close sequence variant (NadAv5). Then, a comprehensive set of biochemical, biophysical and structural techniques were applied to investigate all the generated NadAv3 constructs, aiming to faithfully represent its natural trimeric status, essential for reliable structural, functional and epitope mapping studies. The well-characterized trimeric NadAv3 constructs represented a set of high quality reagents which were validated as probes for functional studies and as a platform for continued attempts for protein crystallization. Mutagenesis studies and screenings to identify a new crystal form of NadAv3 were performed to improve crystal quality, ultimately allowing the collection of several high quality X-ray diffraction data sets; structure determination is ongoing. The atomic resolution structure of NadAv3 will help to understand its biological role as both an adhesin and a vaccine antigen. For example, the high resolution structure will enable epitope mapping studies using human antibodies and thus permit a 7 deeper understanding of the molecular determinants of antibody binding and protective epitopes. In addition, it will help to understand the molecular basis of host-pathogen interactions mediated by specific human cell receptors. Less