434 Citations
Examples are shown of protein crystallization in and data collection from solutions sandwiched between thin polymer films using vapour-diffusion and batch methods The crystallization platform is optimal for both visualization and in situ data collection with the need for traditional harvesting being eliminated In wells constructed from the thinnest plastic and with a minimum of aqueous liquid flash-cooling to K is possible without significant ice formation and without any degradation in crystal quality The approach is simple it utilizes low-cost consumables but yields high-quality data with minimal sample intervention and with the very low levels of background X-ray scatter that ... More
Examples are shown of protein crystallization in, and data collection from, solutions sandwiched between thin polymer films using vapour-diffusion and batch methods. The crystallization platform is optimal for both visualization and in situ data collection, with the need for traditional harvesting being eliminated. In wells constructed from the thinnest plastic and with a minimum of aqueous liquid, flash-cooling to 100 K is possible without significant ice formation and without any degradation in crystal quality. The approach is simple; it utilizes low-cost consumables but yields high-quality data with minimal sample intervention and, with the very low levels of background X-ray scatter that are observed, is optimal for microcrystals. Less
Metal ions and metallocofactors play important roles in a broad range of biochemical reactions Accordingly it has been estimated that as much as of the proteome uses transition metal ions to carry out a variety of essential functions The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties the diversity of which arises as transition metals can adopt different redox states and geometries dictated by the identity of the metal and the protein environment The coupling of a metal ion with an organic framework in metallocofactors such as heme and cobalamin further expands the chemical functionality of ... More
Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25�50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the availability of advanced detectors, and the incorporation of spectroscopic equipment at a number of synchrotron beamlines, have yielded exciting developments in metalloprotein structure determination. Here we will present results on the advantageous uses of metals in metalloprotein crystallography, including using metallocofactors to obtain phasing information, using K-edge X-ray absorption spectroscopy to identify metals coordinated in metalloprotein crystals, and using UV�vis spectroscopy on crystals to probe the enzymatic activity of the crystallized protein. Less
Targeting A has recently been the main objective in Alzheimer s disease therapeutic approaches Passive immunization trials have encountered undesirable side effects but the therapy remains a promising option A -x has not been previously considered as a targetfor AD immunotherapy Tg - mouse model has been recently established and validated as an advantageous research tool in AD The current work deals with A - and explores its potential as atarget while elucidating therapeutic mechanism and crystallizing the NT X Fab in complex with A - aiming to further reveal the structural basis of antibody target affinity In the present ... More
Targeting Aβ has recently been the main objective in Alzheimer�s disease therapeutic approaches. Passive immunization trials have encountered undesirable side effects but the therapy remains a promising option. A�4-x has not been previously considered as a targetfor AD immunotherapy. Tg4-42 mouse model has been recently established and validated as an advantageous research tool in AD. The current work deals with A�4-42 and explores its potential as atarget, while elucidating therapeutic mechanism and crystallizing the NT4X Fab in complex with A�4-19 aiming to further reveal the structural basis of antibody: target affinity. In the present work, the novel monoclonal antibody NT4X specifically reacts with N-truncated A� at position 4 of A�. It binds Ntruncated A� under native and denaturing conditions and rescues invitro toxicity of A�4-42 and that of pyroglutamate A�pE3-42. The Fab fragment of the antibody was also able to prevent the in vitro toxicitycaused by A�4-42 in rat primary cortical neuron cultures. A�4-42 intracerebroventricular injection into wildtype miceinduced a behavioral deficit, shown as a reduction in alteration rate in a Y-Maze, which was prevented using the NT4X. The Fab fragmentof the antibody, at a higher dosage, was also able to prevent the in vivo behavioral deficit in a replicate experiment. The Tg4-42 homozygous mouse model, expressed A�4-42 andallows for is intraneuronal accumulation. At 6 months of age, the model already exhibits 50% neuronal loss in the CA1 region of thehippocampus and severe reference memory deficits in a Morris water maze. Preventative passive immunotherapy with the NT4X antibody and its Fab fragment was able to mitigate neuron loss significantly and rescue spatial memory deficits as compared to an isotype controlgroup. Crystallization of the NT4X Fab in complex with A�4-19 has beensuccessful. Diffraction data has been collected at 2.8 �. Efforts to v resolve the crystal structure of the complex are ongoing. Issues with antibody-target engagement in terms of affinity, species and conformation of A� bound may be dealt with before going to a clinical setting, with the help of information arising from the crystal structure of the NT4X Fab: A�4-19 complex. Less
Staphylococcus pseudintermedius is a leading cause of disease in dogs and zoonosis causes human infections Methicillin-resistant S pseudintermedius strains are emerging resembling the global health threat of S aureus Therefore it is increasingly important to characterize potential targets for intervention against S pseudintermedius Here FhuD an S pseudintermedius surface lipoprotein implicated in iron uptake was characterized It was found that FhuD bound ferrichrome in an iron-dependent manner which increased the thermostability of FhuD by C The crystal structure of ferrichrome-free FhuD was determined via molecular replacement at resolution FhuD exhibits the class III solute-binding protein SBP fold with a ligand-binding ... More
Staphylococcus pseudintermedius is a leading cause of disease in dogs, and zoonosis causes human infections. Methicillin-resistant S. pseudintermedius strains are emerging, resembling the global health threat of S. aureus. Therefore, it is increasingly important to characterize potential targets for intervention against S. pseudintermedius. Here, FhuD, an S. pseudintermedius surface lipoprotein implicated in iron uptake, was characterized. It was found that FhuD bound ferrichrome in an iron-dependent manner, which increased the thermostability of FhuD by >15�C. The crystal structure of ferrichrome-free FhuD was determined via molecular replacement at 1.6 � resolution. FhuD exhibits the class III solute-binding protein (SBP) fold, with a ligand-binding cavity between the N- and C-terminal lobes, which is here occupied by a PEG molecule. The two lobes of FhuD were oriented in a closed conformation. These results provide the first detailed structural characterization of FhuD, a potential therapeutic target of S. pseudintermedius. Less
The characterization of macromolecular samples at synchrotrons has traditionally been restricted to direct exposure to X-rays but beamline automation and diversification of the user community has led to the establishment of complementary characterization facilities off-line The Sample Preparation and Characterization SPC facility at the EMBL PETRA synchrotron provides synchrotron users access to a range of biophysical techniques for preliminary or parallel sample characterization to optimize sample usage at the beamlines Here we describe a sample pipeline from bench to beamline to assist successful structural characterization using small angle X-ray scattering SAXS or macromolecular X-ray crystallography MX The SPC has developed ... More
The characterization of macromolecular samples at synchrotrons has traditionally been restricted to direct exposure to X-rays, but beamline automation and diversification of the user community has led to the establishment of complementary characterization facilities off-line. The Sample Preparation and Characterization (SPC) facility at the EMBL@PETRA3 synchrotron provides synchrotron users access to a range of biophysical techniques for preliminary or parallel sample characterization, to optimize sample usage at the beamlines. Here we describe a sample pipeline from bench to beamline, to assist successful structural characterization using small angle X-ray scattering (SAXS) or macromolecular X-ray crystallography (MX). The SPC has developed a range of quality control protocols to assess incoming samples and to suggest optimization protocols. A high-throughput crystallization platform has been adapted to reach a broader user community, to include chemists and biologists that are not experts in structural biology. The SPC in combination with the beamline and computational facilities at EMBL Hamburg provide a full package of integrated facilities for structural biology and can serve as model for implementation of such resources for other infrastructures. Less
The aggregation of proteins became one of the major challenges in the development of biopharmaceu ticals since the formation of aggregates can affect drug quality and immunogenicity However aggregation mechanisms are highly complex and the investigation requires cost time and material intensive experi mental effort In the present work the predictive power of protein characteristics for the phase behavior of three different proteins which are very similar in size and structure was studied In particular the surface hydrophobicity zeta potential and conformational flexibility of human lysozyme lysozyme from chicken egg white and -lactalbumin at pH and were assessed and examined ... More
The aggregation of proteins became one of the major challenges in the development of biopharmaceuticals since the formation of aggregates can affect drug quality and immunogenicity. However, aggregation mechanisms are highly complex and the investigation requires cost, time, and material intensive experimental effort. In the present work, the predictive power of protein characteristics for the phase behavior of three different proteins which are very similar in size and structure was studied. In particular, the surface hydrophobicity, zeta potential, and conformational flexibility of human lysozyme, lysozyme from chicken egg white, and α-lactalbumin at pH 3, 5, 7, and 9 were assessed and examined for correlation with experimental stability studies focusing on protein phase behavior induced by sodium chloride and ammonium sulfate. The molecular dynamics (MD) simulation based study of the conformational flexibility without precipitants was able to identify highly flexible protein regions which could be associated to the less regular secondary structure elements and random coiled and terminal regions in particular. Conformational flexibility of the entire protein structure and protein surface hydrophobicity could be correlated to differing aggregation propensities among the studied proteins and could be identified to be applicable for prediction of protein phase behavior in aqueous solution without precipitants. For prediction of protein phase behavior and aggregation propensity in aqueous solution with precipitants, protein flexibility was further studied in dependency of salt concentration and species by means of human lysozyme. Even though the results of the salt dependent MD simulations could not be shown to be sufficient for prediction of salt depending phase behavior, this study revealed a more pronounced destabilizing effect of ammonium sulfate in comparison to sodium chloride and thus, was found to be in good agreement with theoretical considerations along the Hofmeister series as well as experimentally evaluated phase behavior. Less
Trimeric autotransporter adhesins TAAs on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins However AtaA a TAA in the nonpathogenic Acinetobacter sp strain Tol shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain TM and the passenger domain contains an N-terminal head N-terminal stalk C-terminal head Chead and C-terminal stalk Cstalk The Chead-Cstalk-TM fragment which is conserved in many Acinetobacter TAAs has by itself the head-stalk-anchor architecture of a complete TAA Here we show ... More
Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three �-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner �-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions. Less
-Carboxyvanillate decarboxylase LigW catalyzes the conversion of -carboxyvanillate to vanillate in the biochemical pathway for the degradation of lignin This enzyme was shown to require Mn for catalytic activity and the kinetic constants for the decarboxylation of -carboxyvanillate by the enzymes from Sphingomonas paucimobilis SYK- kcat s and kcat Km M s and Novosphingobium aromaticivorans kcat s and kcat Km M s were determined The three-dimensional structures of both enzymes were determined in the presence and absence of ligands bound in the active site The structure of LigW from N aromaticivorans bound with the substrate analogue -nitrovanillate Kd nM was ... More
5-Carboxyvanillate decarboxylase (LigW) catalyzes the conversion of 5-carboxyvanillate to vanillate in the biochemical pathway for the degradation of lignin. This enzyme was shown to require Mn2+ for catalytic activity and the kinetic constants for the decarboxylation of 5-carboxyvanillate by the enzymes from Sphingomonas paucimobilis SYK-6 (kcat = 2.2 s�1 and kcat/Km = 4.0 � 104 M�1 s�1) and Novosphingobium aromaticivorans (kcat = 27 s�1 and kcat/Km = 1.1 � 105 M�1 s�1) were determined. The three-dimensional structures of both enzymes were determined in the presence and absence of ligands bound in the active site. The structure of LigW from N. aromaticivorans, bound with the substrate analogue, 5-nitrovanillate (Kd = 5.0 nM), was determined to a resolution of 1.07 �. The structure of this complex shows a remarkable enzyme-induced distortion of the nitro-substituent out of the plane of the phenyl ring by approximately 23�. A chemical reaction mechanism for the decarboxylation of 5-carboxyvanillate by LigW was proposed on the basis of the high resolution X-ray structures determined in the presence ligands bound in the active site, mutation of active site residues, and the magnitude of the product isotope effect determined in a mixture of H2O and D2O. In the proposed reaction mechanism the enzyme facilitates the transfer of a proton to C5 of the substrate prior to the decarboxylation step. Less
Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry Proteins can either be soluble crystalline or precipitated Additionally liquid-liquid phase separation gelation and skin formation can occur A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed For lysozyme from chicken egg white human lysozyme glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values pH and Sodium chloride ammonium sulfate olyethylene glycol and polyethylene glycol were used as precipitants Crystallizing conditions could be found for lysozyme from chicken egg ... More
Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry. Proteins can either be soluble, crystalline or precipitated. Additionally liquid-liquid phase separation, gelation and skin formation can occur. A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed. For lysozyme from chicken egg white, human lysozyme, glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values pH 3, 5, 7 and 9. Sodium chloride, ammonium sulfate, olyethylene glycol 300 and polyethylene glycol 1000 were used as precipitants. Crystallizing conditions could be found for lysozyme from chicken egg white using sodium chloride, for human lysozyme using sodium chloride or ammonium sulfate and glucose isomerase using ammonium sulfate. PEG caused destabilization of human lysozyme and glucose oxidase solutions or a balance of stabilizing and destabilizing effects for glucose isomerase near the isoelectric point. This work presents a systematic generation and extensive study
of phase diagrams of proteins. Thus, it adds to the general understanding of protein behavior in liquid formulation and presents a convenient methodology applicable to any
protein solution.
Less
of phase diagrams of proteins. Thus, it adds to the general understanding of protein behavior in liquid formulation and presents a convenient methodology applicable to any
protein solution.
Less
Zn is an essential nutrient for all known forms of life In the major human pathogen Streptococcus pneumoniae the acquisition of Zn is facilitated by two Zn -specific solute-binding proteins AdcA and AdcAII To date there has been a paucity of structural information on AdcA which has hindered a deeper understanding of the mechanism underlying pneumococcal Zn acquisition Native AdcA consists of two domains an N-terminal ZnuA domain and a C-terminal ZinT domain In this study the ZnuA domain of AdcA was crystallized The initial crystals of the ZnuA-domain protein were obtained using dried seaweed as a heterogeneous nucleating agent ... More
Zn2+ is an essential nutrient for all known forms of life. In the major human pathogen Streptococcus pneumoniae, the acquisition of Zn2+ is facilitated by two Zn2+-specific solute-binding proteins: AdcA and AdcAII. To date, there has been a paucity of structural information on AdcA, which has hindered a deeper understanding of the mechanism underlying pneumococcal Zn2+ acquisition. Native AdcA consists of two domains: an N-terminal ZnuA domain and a C-terminal ZinT domain. In this study, the ZnuA domain of AdcA was crystallized. The initial crystals of the ZnuA-domain protein were obtained using dried seaweed as a heterogeneous nucleating agent. No crystals were obtained in the absence of the heterogeneous nucleating agent. These initial crystals were subsequently used as seeds to produce diffraction-quality crystals. The crystals diffracted to 2.03 � resolution and had the symmetry of space group P1. This study demonstrates the utility of heterogeneous nucleation. The solution of the crystal structures will lead to further understanding of Zn2+ acquisition by S. pneumoniae. Less
Undesired protein aggregation in general and non-native protein aggregation in particular need to be inhibited during bio-pharmaceutical processing to ensure patient safety and to maintain product activity In this work the potency of different additives namely glycerol PEG and glycine to prevent lysozyme aggregation and selectively manipulate lysozyme phase behavior was investigated The results revealed a strong pH dependency of the additive impact on lysozyme phase behavior lysozyme solubility crystal size and morphology This work aims to link this pH dependent impact to a protein-specific parameter the conformational stability of lysozyme At pH the addition of w v glycerol w ... More
Undesired protein aggregation in general and non-native protein aggregation in particular need to be inhibited during bio-pharmaceutical processing to ensure patient safety and to maintain product activity. In this work the potency of different additives, namely glycerol, PEG 1000, and glycine, to prevent lysozyme aggregation and selectively manipulate lysozyme phase behavior was investigated. The results revealed a strong pH dependency of the additive impact on lysozyme phase behavior, lysozyme solubility, crystal size and morphology. This work aims to link this pH dependent impact to a protein-specific parameter, the conformational stability of lysozyme. At pH 3 the addition of 10% (w/v) glycerol, 10% (w/v) PEG 1000, and 1 M glycine stabilized or destabilized lysozymes’ native conformation resulting in a modified size of the crystallization area without influencing lysozyme solubility, crystal size and morphology. Addition of 1 M glycine even promoted non-native aggregation at pH 3 whereas addition of PEG 1000 completely inhibited non-native aggregation. At pH 5 the addition of 10% (w/v) glycerol, 10% (w/v) PEG 1000, and 1 M glycine did not influence lysozymes’ native conformation, but strongly influenced the position of the crystallization area, lysozyme solubility, crystal size and morphology. The observed pH dependent impact of the additives could be linked to a differing lysozyme conformational stability in the binary systems without additives at pH 3 and pH 5. However, in any case lysozyme phase behavior could selectively be manipulated by addition of glycerol, PEG 1000 and glycine. Furthermore, at pH 5 crystal size and morphology could selectively be manipulated. Less
Strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity tissue-tropism and zoonosis Rotaviruses RVs which cause life-threatening gastroenteritis in infants and children display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP domain of the spike protein VP The structural basis of this genotype-dependent glycan specificity particularly in human RVs remains poorly understood Here from crystallographic studies we show how genotypic variations configure a novel binding site in the VP of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type II precursor glycans and ... More
Strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type II precursor glycans, and to restrict type II glycan binding in the bovine counterpart. Such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses. Less
The MERS-CoV is an emerging virus which already infected more than humans with high mortality Here we show that m an exceptionally potent human anti-MERS-CoV antibody is almost germline with only one somatic mutation in the heavy chain The structure of Fab m in complex with the MERS-CoV receptor-binding domain reveals that its IGHV - -derived heavy chain provides more than binding surface and that its epitope almost completely overlaps with the receptor-binding site Analysis of antibodies from healthy humans suggests an important role of the V D J recombination-generated junctional and allele-specific residues for achieving high affinity of binding ... More
The MERS-CoV is an emerging virus, which already infected more than 1,300 humans with high (∼36%) mortality. Here, we show that m336, an exceptionally potent human anti-MERS-CoV antibody, is almost germline with only one somatic mutation in the heavy chain. The structure of Fab m336 in complex with the MERS-CoV receptor-binding domain reveals that its IGHV1-69-derived heavy chain provides more than 85% binding surface and that its epitope almost completely overlaps with the receptor-binding site. Analysis of antibodies from 69 healthy humans suggests an important role of the V(D)J recombination-generated junctional and allele-specific residues for achieving high affinity of binding at such low levels of somatic hypermutation. Our results also have important implications for development of vaccine immunogens based on the newly identified m336 epitope as well as for elucidation of mechanisms of neutralization by m336-like antibodies and their elicitation in vivo. Less
Inosine- '-monophosphate dehydrogenases IMPDHs which are the rate-limiting enzymes in guanosine-nucleotide biosynthesis are important therapeutic targets Despite in-depth functional and structural characterizations of various IMPDHs the role of the Bateman domain containing two CBS motifs remains controversial Their involvement in the allosteric regulation of Pseudomonas aeruginosa IMPDH by Mg-ATP has recently been reported To better understand the function of IMPDH and the importance of the CBS motifs the structure of a variant devoid of these modules CBS was solved at high resolution in the apo form and in complex with IMP In addition a single amino-acid substitution variant D N ... More
Inosine-5'-monophosphate dehydrogenases (IMPDHs), which are the rate-limiting enzymes in guanosine-nucleotide biosynthesis, are important therapeutic targets. Despite in-depth functional and structural characterizations of various IMPDHs, the role of the Bateman domain containing two CBS motifs remains controversial. Their involvement in the allosteric regulation of Pseudomonas aeruginosa IMPDH by Mg-ATP has recently been reported. To better understand the function of IMPDH and the importance of the CBS motifs, the structure of a variant devoid of these modules (ΔCBS) was solved at high resolution in the apo form and in complex with IMP. In addition, a single amino-acid substitution variant, D199N, was also structurally characterized: the mutation corresponds to the autosomal dominant mutant D226N of human IMPDH1, which is responsible for the onset of the retinopathy adRP10. These new structures shed light onto the possible mechanism of regulation of the IMPDH enzymatic activity. In particular, three conserved loops seem to be key players in this regulation as they connect the tetramer-tetramer interface with the active site and show significant modification upon substrate binding. Less
Human cytomegalovirus HCMV poses a significant threat to immunocompromised individuals and neonates infected in utero Glycoprotein B gB the herpesvirus fusion protein is a target for neutralizing antibodies and a vaccine candidate due to its indispensable role in infection Here we show the crystal structure of the HCMV gB ectodomain bound to the Fab fragment of G a neutralizing human monoclonal antibody isolated from a seropositive subject The gB G interaction is dominated by aromatic residues in the G heavy chain CDR protruding into a hydrophobic cleft in the gB antigenic domain AD- Structural analysis and comparison with HSV gB ... More
Human cytomegalovirus (HCMV) poses a significant threat to immunocompromised individuals and neonates infected in utero. Glycoprotein B (gB), the herpesvirus fusion protein, is a target for neutralizing antibodies and a vaccine candidate due to its indispensable role in infection. Here we show the crystal structure of the HCMV gB ectodomain bound to the Fab fragment of 1G2, a neutralizing human monoclonal antibody isolated from a seropositive subject. The gB/1G2 interaction is dominated by aromatic residues in the 1G2 heavy chain CDR3 protruding into a hydrophobic cleft in the gB antigenic domain 5 (AD-5). Structural analysis and comparison with HSV gB suggest the location of additional neutralizing antibody binding sites on HCMV gB. Finally, immunoprecipitation experiments reveal that 1G2 can bind to HCMV virion gB suggesting that its epitope is exposed and accessible on the virus surface. Our data will support the development of vaccines and therapeutic antibodies against HCMV infection. Less
G protein-coupled receptors GPCRs are of particular importance for drug discovery being the targets of many existing drugs and being linked to many diseases where new therapies are required However as integral membrane proteins they are generally unstable when removed from their membrane environment precluding them from the wide range of structural and biophysical techniques which can be applied to soluble proteins such as kinases Through the use of protein engineering methods mutations can be identified which both increase the thermostability of GPCRs when purified in detergent as well as biasing the receptor toward a specific physiologically relevant conformational state ... More
G protein-coupled receptors (GPCRs) are of particular importance for drug discovery, being the targets of many existing drugs, and being linked to many diseases where new therapies are required. However, as integral membrane proteins, they are generally unstable when removed from their membrane environment, precluding them from the wide range of structural and biophysical techniques which can be applied to soluble proteins such as kinases. Through the use of protein engineering methods, mutations can be identified which both increase the thermostability of GPCRs when purified in detergent, as well as biasing the receptor toward a specific physiologically relevant conformational state. The resultant stabilized receptor (known as a StaR) can be purified in multiple-milligram quantities, whilst retaining correct folding, thus enabling the generation of reagents suitable for a broad range of structural and biophysical studies. Example protocols for the purification of StaR proteins for analysis, ligand screening with the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM), surface plasmon resonance (SPR), and crystallization for structural studies are presented. Less
The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases which shape and remodel membranes in diverse cellular processes Dynamin forms predominantly tetramers in the cytosol which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane Previous studies have described the architecture of dynamin dimers but the molecular determinants for dynamin assembly and its regulation have remained unclear Here we present the crystal structure of the human dynamin tetramer in the nucleotide-free state Combining structural data with mutational studies oligomerization measurements and Markov state models of molecular dynamics simulations ... More
The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases, which shape and remodel membranes in diverse cellular processes1. Dynamin forms predominantly tetramers in the cytosol, which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane1. Previous studies have described the architecture of dynamin dimers2,3, but the molecular determinants for dynamin assembly and its regulation have remained unclear. Here we present the crystal structure of the human dynamin tetramer in the nucleotide-free state. Combining structural data with mutational studies, oligomerization measurements and Markov state models of molecular dynamics simulations, we suggest a mechanism by which oligomerization of dynamin is linked to the release of intramolecular autoinhibitory interactions. We elucidate how mutations that interfere with tetramer formation and autoinhibition can lead to the congenital muscle disorders Charcot–Marie–Tooth neuropathy4 and centronuclear myopathy5, respectively. Notably, the bent shape of the tetramer explains how dynamin assembles into a right-handed helical oligomer of defined diameter, which has direct implications for its function in membrane constriction. Less
PRMT is a protein arginine methyltransferase involved in transcriptional regulation human immunodeficiency virus pathogenesis DNA base excision repair and cell cycle progression Like other PRMTs PRMT is overexpressed in several cancer types and is therefore considered as a potential anti-cancer drug target In the present study we described six crystal structures of PRMT from Mus musculus solved and refined at for the highest resolution structure The crystal structures revealed that the folding of the helix X is required to stabilize a productive active site before methylation of the bound peptide can occur In the absence of cofactor metal cations can ... More
PRMT6 is a protein arginine methyltransferase involved in transcriptional regulation, human immunodeficiency virus pathogenesis, DNA base excision repair, and cell cycle progression. Like other PRMTs, PRMT6 is overexpressed in several cancer types and is therefore considered as a potential anti-cancer drug target. In the present study, we described six crystal structures of PRMT6 from Mus musculus, solved and refined at 1.34 Å for the highest resolution structure. The crystal structures revealed that the folding of the helix αX is required to stabilize a productive active site before methylation of the bound peptide can occur. In the absence of cofactor, metal cations can be found in the catalytic pocket at the expected position of the guanidinium moiety of the target arginine substrate. Using mass spectrometry under native conditions, we show that PRMT6 dimer binds two cofactor and a single H4 peptide molecules. Finally, we characterized a new site of in vitro automethylation of mouse PRMT6 at position 7. Less
Molecular machines or macromolecular complexes are supramolecular assemblies of biomolecules that ensure cellular homeostasis Structure determination of those complexes in a purified state is often a tedious undertaking due to the compositional complexity and the related relative structural instability To improve the stability of macromolecular complexes in vitro we present here a generic method that optimizes the stability homogeneity and solubility of macromolecular complexes by sparse-matrix screening of their thermal unfolding behaviour in the presence of various buffers and small molecules The method includes the automated analysis of thermal unfolding curves based on a newly developed biophysical unfolding model for ... More
Molecular machines or macromolecular complexes are supramolecular assemblies of biomolecules that ensure cellular homeostasis. Structure determination of those complexes in a purified state is often a tedious undertaking due to the compositional complexity and the related relative structural instability. To improve the stability of macromolecular complexes in vitro, we present here a generic method that optimizes the stability, homogeneity and solubility of macromolecular complexes by sparse-matrix screening of their thermal unfolding behaviour in the presence of various buffers and small molecules. The method includes the automated analysis of thermal unfolding curves based on a newly developed biophysical unfolding model for complexes. We found that under stabilizing conditions even large multi-component complexes reveal an almost ideal two-state unfolding behaviour. We envisage an improved biochemical understanding of purified macromolecules as well as a substantial boost in successful macromolecular complex structure determination by both X-ray crystallography and Cryo EM. Less
The potentially structured core domain of the intrinsically disordered protein Knr from Saccharomyces cerevisiae comprising residues was expressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method Selenomethionine-containing SeMet protein was also purified and crystallized Crystals of both proteins belonged to space group P with unit-cell parameters a b c for the native protein and a b c for the SeMet protein and diffracted to and resolution respectively There are two molecules in the asymmetric unit related by a twofold axis The anomalous signal of selenium was recorded and yielded an electron-density map of sufficient quality to allow the ... More
The potentially structured core domain of the intrinsically disordered protein Knr4 from Saccharomyces cerevisiae, comprising residues 80–340, was expressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. Selenomethionine-containing (SeMet) protein was also purified and crystallized. Crystals of both proteins belonged to space group P6522, with unit-cell parameters a = b = 112.44, c = 265.21 Å for the native protein and a = b = 112.49, c = 262.21 Å for the SeMet protein, and diffracted to 3.50 and 3.60 Å resolution, respectively. There are two molecules in the asymmetric unit related by a twofold axis. The anomalous signal of selenium was recorded and yielded an electron-density map of sufficient quality to allow the identification of secondary-structure elements. Less
-Xylosidases EC catalyze the hydrolysis of short xylooligosaccharides into xylose which is an essential step in the complete depolymerization of xylan the major hemicellulosic polysaccharide of plant cell walls and has great biotechnological relevance for the production of lignocellulose-based biofuels and the paper industry In this study a GH -xylosidase identified from the bacterium Bacillus licheniformis BlXylA was cloned into the the pET- a bacterial expression vector recombinantly overexpressed in Escherichia coli BL DE cells and purified to homogeneity by metal-affinity and size-exclusion chromatography The protein was crystallized in the presence of the organic solvent -methyl- -pentanediol and a single ... More
β-Xylosidases (EC 3.2.1.37) catalyze the hydrolysis of short xylooligosaccharides into xylose, which is an essential step in the complete depolymerization of xylan, the major hemicellulosic polysaccharide of plant cell walls, and has great biotechnological relevance for the production of lignocellulose-based biofuels and the paper industry. In this study, a GH43 β-xylosidase identified from the bacterium Bacillus licheniformis (BlXylA) was cloned into the the pET-28a bacterial expression vector, recombinantly overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity by metal-affinity and size-exclusion chromatography. The protein was crystallized in the presence of the organic solvent 2-methyl-2,4-pentanediol and a single crystal diffracted to 2.49 Å resolution. The X-ray diffraction data were indexed in the monoclinic space group C2, with unit-cell parameters a = 152.82, b = 41.9, c = 71.79 Å, β = 91.7°. Structural characterization of this enzyme will contribute to a better understanding of the structural requirements for xylooligosaccharide specificity within the GH43 family. Less
The present study was undertaken to evaluate the anti-influenza vasorelaxant and vasoprotective potential of a series of flavan- -ols and proanthocyanidins and to gain insight into the mode of action The first part of this thesis addressed the inhibitory potential of the tested substances against a bacterial V cholera and a viral influenza A California H N neuraminidase NA using a MuNANA -fluorescence-based assay The present study verified that the substrate MuNANA is recognized by both NAs Compared to the positive controls zanamivir und oseltamivir carboxylic acid the studied proanthocyanidin enriched fractions Salix spp Nelia meyeri Cephalophyllum spp Betula spp ... More
The present study was undertaken to evaluate the anti-influenza, vasorelaxant and vasoprotective potential of a series of flavan-3-ols and proanthocyanidins and to gain insight into the mode of action. The first part of this thesis addressed the inhibitory potential of the tested substances against a bacterial (V. cholera) and a viral influenza A (California/04/2009[H1N1]) neuraminidase (NA), using a (MuNANA)-fluorescence-based assay. The present study verified that the substrate MuNANA is recognized by both NAs. Compared to the positive controls zanamivir und oseltamivir carboxylic acid, the studied proanthocyanidin enriched fractions (Salix spp., Nelia meyeri, Cephalophyllum spp., Betula spp., Potentilla erecta, Rhus leptodictya, Diospyros kaki, Pelargonium sidoides) and ellagitannins (Geraniin, Granatin A, Carpinusin, Terchebin, Casuariin, Vescalagin, Paeonianin C) were significantly more effective against the bacterial NA. In contrast, all polyphenolic samples were far less effective inhibitors of the viral NA than the reference samples. Structure-activity relationships indicated that the degree of polymerization, the 2,3-stereochemistry, hydroxylation patterns and the presence of a 3-O-galloyl group largely affected the inhibitory potential of the tested flavan-3-ols and proanthocyanidins. The degree of galloylation appeared crucial in the series of ellagitannins. In addition, the combination of Zanamivir with EPs® 7630 (1:10) showed a synergistic inhibitory effect in studies against V. cholerae NA. The evaluation of co-crystal data of the bacterial respectively viral NA and either Zanamivir or oseltamivir carboxylate showed due to the interactions with the only viral occurring 150-loop both drugs inhibit the viral enzyme more effectively than the bacterial NA. In the second part of this thesis the vasorelaxant potential was investigated using a tissue bath protocol. Porcine coronary arterial rings were contracted with U46619 and relaxed by cumulative addition of the polyphenolic samples. Within the series of flavan-3-ols and proanthocyanidin- enriched fractions, galloylated compounds induced a concentration-dependent vasodilatation. The experiments showed that Diospyros-proanthocyanidins and procyanidin B2-3-O-gallate stimulated phosphoinositide-3-kinase/protein kinase B, endothelial NO synthase, and soluble guanylyl cyclase, respectively. Both samples evoked an endothelium-dependent relaxations via the NO/cGMP pathway. In addition, Na+/K+-ATPase was involved in the relaxant response to these polyphenols. Intracellular ROS formation in response to the proanthocyanidin samples was verified and the mitochondrial respiratory chain identified as a potential source of reactive oxygen species for the procyanidin B2-3-O-gallate. Less
Suppressor of cytokine signalling SOCS is the substrate-binding component of a Cullin-RING E ubiquitin ligase CRL complex that targets phosphorylated hormone receptors for degradation by the ubiquitin-proteasome system As a key regulator of the transcriptional response to growth signals SOCS and its protein complex partners are potential targets for small molecule development We found that crystals of SOCS in complex with its adaptor proteins Elongin C and Elongin B underwent a change in crystallographic parameters when treated with dimethyl sulfoxide during soaking experiments To solve the phase problem for the new crystal form we identified the presence of arsenic atoms ... More
Suppressor of cytokine signalling 2 (SOCS2) is the substrate-binding component of a Cullin-RING E3 ubiquitin ligase (CRL) complex that targets phosphorylated hormone receptors for degradation by the ubiquitin-proteasome system. As a key regulator of the transcriptional response to growth signals, SOCS2 and its protein complex partners are potential targets for small molecule development. We found that crystals of SOCS2 in complex with its adaptor proteins, Elongin C and Elongin B, underwent a change in crystallographic parameters when treated with dimethyl sulfoxide during soaking experiments. To solve the phase problem for the new crystal form we identified the presence of arsenic atoms in the crystals, a result of covalent modification of cysteines by cacodylate, and successfully extracted anomalous signal from these atoms for experimental phasing. The resulting structure provides a means for solving future structures where the crystals must be treated with DMSO for ligand soaking approaches. Additionally, the conformational changes induced in this structure reveal flexibility within SOCS2 that match those postulated by previous molecular dynamics simulations. This conformational flexibility illustrates how SOCS2 can orient its substrates for successful ubiquitination by other elements of the CRL complex. Less
In response to adenosine -diphosphate the P Y receptor P Y R facilitates platelet aggregation and thus serves as an important antithrombotic drug target Here we report the crystal structures of the human P Y R in complex with a nucleotide antagonist MRS at resolution and with a non-nucleotide antagonist BPTU at resolution The structures reveal two distinct ligand binding sites providing atomic details of P Y R s unique ligand binding modes MRS recognizes a binding site within the seven transmembrane bundle of P Y R which however is different in shape and location from the nucleotide binding site ... More
In response to adenosine 5′-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7Å resolution, and with a non-nucleotide antagonist BPTU at 2.2Å resolution. The structures reveal two distinct ligand binding sites, providing atomic details of P2Y1R’s unique ligand binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which, however, is different in shape and location from the nucleotide binding site in previously determined P2Y12R structure. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects. Less
Coxiella burnetii is a highly infectious bacterium and potential agent of bioterrorism However it has not been studied as extensively as other biological agents and very few of its proteins have been structurally characterized To address this situation we undertook a study of critical metabolic enzymes in C burnetii that have great potential as drug targets We used high-throughput techniques to produce novel crystal structures of of these proteins We selected one protein C burnetii dihydrofolate reductase CbDHFR for additional work to demonstrate the value of these structures for structure-based drug design This enzyme's structure reveals a feature in the ... More
Coxiella burnetii is a highly infectious bacterium and potential agent of bioterrorism. However, it has not been studied as extensively as other biological agents, and very few of its proteins have been structurally characterized. To address this situation, we undertook a study of critical metabolic enzymes in�C. burnetii�that have great potential as drug targets. We used high-throughput techniques to produce novel crystal structures of 48 of these proteins. We selected one protein,�C. burnetii�dihydrofolate reductase (CbDHFR), for additional work to demonstrate the value of these structures for structure-based drug design. This enzyme's structure reveals a feature in the substrate binding groove that is different between CbDHFR and human dihydrofolate reductase (hDHFR). We then identified a compound by�in silico�screening that exploits this binding groove difference, and demonstrated that this compound inhibits CbDHFR with at least 25-fold greater potency than hDHFR. Since this binding groove feature is shared by many other prokaryotes, the compound identified could form the basis of a novel antibacterial agent effective against a broad spectrum of pathogenic bacteria. Less
Chlorophyll is one of the most abundant pigments worldwide Every year chlorophyll is not only newly synthesized but big amounts of chlorophyll are degraded during fruit ripening and in senescing leaves The pathway of chlorophyll breakdown the so called PAO phyllobilin pathway has been extensively studied over the past decades Most of the involved genes have been cloned and characterized except for the activity responsible for magnesium removal of chlorophyll and some of the side chain-modifying enzymes Pheophytinase PPH is one of the first enzymes involved in chlorophyll degradation specifically hydrolyzing the ester bond between the porphyrin ring and the ... More
Chlorophyll is one of the most abundant pigments worldwide. Every year, chlorophyll is not only newly synthesized, but big amounts of chlorophyll are degraded during fruit ripening and in senescing leaves. The pathway of chlorophyll breakdown, the so called PAO/phyllobilin pathway, has been extensively studied over the past decades. Most of the involved genes have been cloned and characterized, except for the activity responsible for magnesium removal of chlorophyll and some of the side chain-modifying enzymes. Pheophytinase (PPH) is one of the first enzymes involved in chlorophyll degradation, specifically hydrolyzing the ester bond between the porphyrin ring and the phytol moiety of pheophytin, but not of chlorophyll. In this work, the substrate specificity of PPH was further determined. PPH can be characterized as an esterase with tight specificity for the acid moiety, which is the porphyrin ring. The KM for pheophytin a and for two other accepted substrates with similar molecular structure is in the µM range, indicating high substrate affinities. Chlorophyll is a likely inhibitor of the enzymatic activity. To reveal the substrate-binding mechanism of PPH, a crystallization approach was performed. To this end, one focus of this work was to elucidate a good purification system for PPH. However, purification and tag-separation turned out to be difficult for PPH and first potential crystallization conditions were defined with the uncleaved PPH-MBP fusion. In this work I also demonstrate that PPH is not only involved in leaf senescence, but also in chlorophyll degradation during fruit ripening. For a comparison study Tomato was chosen as a model plant. I could show that tomato PPH is an ortholog of Arabidopsis thaliana PPH and is responsible for phytol cleavage in senescing tomato leaves. The PAO/ phyllobilin pathway is active in ripening fruits and PPH activity was found in chromoplasts. However, the absence of PPH did not impair color break in fruits, indicating that other, so far unknown, hydrolases are active in parallel. A last focus of this work was on the identification of factors responsible for magnesium dechelation from chlorophyll, the first step in the breakdown process. I obtained a first good indication that this is a non-enzymatic process, where changes in the local pH trigger the loss of magnesium. However, additional experiments will be required to corroborate this hypothesis. Less
Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain PD that promotes virulence by detecting the macrophage phagosome PhoQ activity is repressed by divalent cations and induced in environments of acidic pH limited divalent cations and cationic antimicrobial peptides CAMP Previously it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in a-helices and suggesting this region contributes to pH sensing Therefore we engineered a disulfide bond between W C and A C in the ... More
Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain (PD) that promotes virulence by detecting the macrophage phagosome. PhoQ activity is repressed by divalent cations and induced in environments of acidic pH, limited divalent cations, and cationic antimicrobial peptides (CAMP). Previously, it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence. We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in a-helices 4 and 5, suggesting this region contributes to pH sensing. Therefore, we engineered a disulfide bond between W104C and A128C in the PhoQ PD that restrains conformational flexibility in a-helices 4 and 5. PhoQ(W104C-A128C) is responsive to CAMP, but is inhibited for activation by acidic pH and divalent cation limitation. phoQ(W104C-A128C) Salmonella enterica Typhimurium is virulent in mice, indicating that acidic pH and divalent cation sensing by PhoQ are dispensable for virulence. Less
Identifying and then optimizing initial crystallization conditions is a prerequisite for macromolecular structure determination by crystallography Improved technologies enable data collection on crystals that are difficult if not impossible to detect using visible imaging The application of second-order nonlinear imaging of chiral crystals and ultraviolet two-photon excited fluorescence detection is shown to be applicable in a high-throughput manner to rapidly verify the presence of nanocrystals in crystallization screening conditions It is noted that the nanocrystals are rarely seen without also producing microcrystals from other chemical conditions A crystal volume optimization method is described and associated with a phase diagram for ... More
Identifying and then optimizing initial crystallization conditions is a prerequisite for macromolecular structure determination by crystallography. Improved technologies enable data collection on crystals that are difficult if not impossible to detect using visible imaging. The application of second-order nonlinear imaging of chiral crystals and ultraviolet two-photon excited fluorescence detection is shown to be applicable in a high-throughput manner to rapidly verify the presence of nanocrystals in crystallization screening conditions. It is noted that the nanocrystals are rarely seen without also producing microcrystals from other chemical conditions. A crystal volume optimization method is described and associated with a phase diagram for crystallization. Less
The lipid cubic phase LCP continues to grow in popularity as a medium in which to generate crystals of membrane and soluble proteins for high-resolution X-ray crystallographic structure determination To date the PDB includes records attributed to the LCP or in meso method Among the listings are some of the highest profile membrane proteins including the -adrenoreceptor Gs protein complex that figured in the award of the Nobel Prize in Chemistry to Lefkowitz and Kobilka The most successful in meso protocol to date uses glass sandwich crystallization plates Despite their many advantages glass plates are challenging to harvest crystals from ... More
The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the �2-adrenoreceptor�Gs protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 � using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development. Less
Upon binding to its bacterial host receptor the tail tip of phage T perforates by an unknown mechanism the heavily armoured cell wall of the host This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions In the perspective of a structural study of the phage tail we have systematically overproduced eight of the eleven T tail proteins with or without a N- or a C-terminal His -tag The widely used Hi -tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography The presence of a ... More
Upon binding to its bacterial host receptor, the tail tip of phage T5 perforates, by an unknown mechanism, the heavily armoured cell wall of the host. This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions. In the perspective of a structural study of the phage tail, we have systematically overproduced eight of the eleven T5 tail proteins, with or without a N- or a C-terminal His6-tag. The widely used Hi6-tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography. The presence of a tag however is not always innocuous. We combined automated gene cloning and expression tests to rapidly identify the most promising constructs for proteins of phage T5 tail, and performed biochemical and biophysical characterisation and crystallisation screening on available proteins. Automated small-scale purification was adapted for two highly expressed proteins. We obtained structural information for three of the proteins. We showed that the presence of a His6-tag can have drastic effect on protein expression, solubility, oligomerisation propensity and crystal quality. Less
Pmi an enzyme of unknown function from Proteus Mirabilis HI and the amidohydrolase superfamily was cloned purified to homogeneity and functionally characterized The three-dimensional structure of Pmi was determined with zinc and cacodylate bound in the active site PDB id RHG The structure was also determined with manganese and butyrate in the active site PDB id QSF Pmi folds as a distorted a -barrel that is typical for members of the amidohydrolase superfamily and cog The substrate profile for Pmi was determined via a strategy that marshaled the utilization of bioinformatics structural characterization and focused library screening The protein was ... More
Pmi1525, an enzyme of unknown function from Proteus Mirabilis HI4320 and the amidohydrolase superfamily, was cloned, purified to homogeneity, and functionally characterized. The three-dimensional structure of Pmi1525 was determined with zinc and cacodylate bound in the active site (PDB id: 3RHG). The structure was also determined with manganese and butyrate in the active site (PDB id: 4QSF). Pmi1525 folds as a distorted (�/a)8-barrel that is typical for members of the amidohydrolase superfamily and cog1735. The substrate profile for Pmi1525 was determined via a strategy that marshaled the utilization of bioinformatics, structural characterization and focused library screening. The protein was found to efficiently catalyze the hydrolysis of organophosphonate and carboxylate esters. The best substrates identified for Pmi1525 are ethyl 4-nitrophenylmethyl phosphonate (kcat and kcat/Km values of 580 s-1 and 1.2 � 105 M-1 s-1, respectively) and 4-nitrophenyl butyrate (kcat and kcat/Km values of 140 s-1 and 1.4 � 105 M-1 s-1, respectively). Pmi1525 is stereoselective for the hydrolysis of chiral methylphosphonate esters. The enzyme hydrolyzes the (SP)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate 14 times faster than the corresponding (RP)-enantiomer. The catalytic properties of this enzyme make it an attractive template for the evolution of novel enzymes for the detection, destruction, and detoxification of organophosphonate nerve agents. Less
HIV- -neutralizing antibodies develop in most HIV- -infected individuals although highly effective antibodies are generally observed only after years of chronic infection Here we characterize the rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC through longitudinal sampling of peripheral B cell transcripts over years and co-crystal structures of lineage members Next-generation sequencing identified VRC -lineage transcripts which encompassed diverse antibodies organized into distinct phylogenetic clades Prevalent clades maintained characteristic features of antigen recognition though each evolved binding loops and disulfides that formed distinct recognition surfaces Over the course of the study ... More
HIV-1-neutralizing antibodies develop in most HIV-1-infected individuals, although highly effective antibodies are generally observed only after years of chronic infection. Here, we characterize the rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC01 through longitudinal sampling of peripheral B cell transcripts over 15 years and co-crystal structures of lineage members. Next-generation sequencing identified VRC01-lineage transcripts, which encompassed diverse antibodies organized into distinct phylogenetic clades. Prevalent clades maintained characteristic features of antigen recognition, though each evolved binding loops and disulfides that formed distinct recognition surfaces. Over the course of the study period, VRC01-lineage clades showed continuous evolution, with rates of ∼2 substitutions per 100 nucleotides per year, comparable to that of HIV-1 evolution. This high rate of antibody evolution provides a mechanism by which antibody lineages can achieve extraordinary diversity and, over years of chronic infection, develop effective HIV-1 neutralization. Less
POTs proton-dependent oligopeptide transporters are integral membrane proteins and essential for maintaining homeostasis in cells by switching between two major conformations during the transport cycle Di- and tripeptides as well as some small peptide like compounds are recognized and transported across the membrane This fact leads to the pharmacological interest in these transporters for drug delivery In humans two transporters PepT and PepT occur So far only structures of five bacterial homologues are available Eukaryotic POTs exist of transmembrane helices whereas prokaryotic POTs have two additional transmembrane helices It was possible to clone different prokaryotic transporter constructs into pET expression ... More
POTs (proton-dependent oligopeptide transporters) are integral membrane proteins and essential for maintaining homeostasis in cells by switching between two major conformations during the transport cycle. Di- and tripeptides as well as some small peptide like compounds are recognized and transported across the membrane. This fact leads to
the pharmacological interest in these transporters for drug delivery. In humans two transporters, PepT1 and PepT2, occur. So far only structures of five bacterial homologues are available. Eukaryotic POTs exist of 12 transmembrane helices whereas prokaryotic POTs have two additional transmembrane helices. It was possible to clone different prokaryotic transporter constructs into pET expression vectors as well as the PepT-like transporter (CtPOT) of Chaetomium thermophilum, a thermophilic eukaryote, and the human PepT2. Two transporters of Shewanella oneidensis (PepTSo2) modified with the thermostabilized apocytochrome b562RIL as well as the CtPOT and PepT2 were expressed in Escherichia coli cells. These transporters except PepT2 were solubilized and purified. Crystals were obtained of all proteins but it was not possible to solve any structure so far due to limited crystal diffraction. Less
the pharmacological interest in these transporters for drug delivery. In humans two transporters, PepT1 and PepT2, occur. So far only structures of five bacterial homologues are available. Eukaryotic POTs exist of 12 transmembrane helices whereas prokaryotic POTs have two additional transmembrane helices. It was possible to clone different prokaryotic transporter constructs into pET expression vectors as well as the PepT-like transporter (CtPOT) of Chaetomium thermophilum, a thermophilic eukaryote, and the human PepT2. Two transporters of Shewanella oneidensis (PepTSo2) modified with the thermostabilized apocytochrome b562RIL as well as the CtPOT and PepT2 were expressed in Escherichia coli cells. These transporters except PepT2 were solubilized and purified. Crystals were obtained of all proteins but it was not possible to solve any structure so far due to limited crystal diffraction. Less
Synthesis of '- '-oligoadenylates - A by oligoadenylate synthetase OAS is an important innate cellular response that limits viral replication by activating the latent cellular RNase RNase L to degrade single-stranded RNA Some rotaviruses and coronaviruses antagonize the OAS RNase L pathway through the activity of an encoded H phosphoesterase domain that cleaves - A These viral H phosphoesterases are phylogenetically related to the cellular A kinase anchoring protein AKAP and share a core structure and an active site that contains two well-defined HF S T F where F is a hydrophobic residue motifs but their mechanism of substrate binding ... More
Synthesis of 2'-5'-oligoadenylates (2-5A) by oligoadenylate synthetase (OAS) is an important innate cellular response that limits viral replication by activating the latent cellular RNase, RNase L, to degrade single-stranded RNA. Some rotaviruses and coronaviruses antagonize the OAS/RNase L pathway through the activity of an encoded 2H phosphoesterase domain that cleaves 2-5A. These viral 2H phosphoesterases are phylogenetically related to the cellular A kinase anchoring protein 7 (AKAP7) and share a core structure and an active site that contains two well-defined HF(S/T)F (where F is a hydrophobic residue) motifs, but their mechanism of substrate binding is unknown. Here, we report the structures of a viral 2H phosphoesterase, the C-terminal domain (CTD) of the group A rotavirus (RVA) VP3 protein, both alone and in complex with 2-5A. The domain forms a compact fold, with a concave �-sheet that contains the catalytic cleft, but it lacks two a-helical regions and two �-strands observed in AKAP7 and other 2H phosphoesterases. The cocrystal structure shows significant conformational changes in the R loop upon ligand binding. Bioinformatics and biochemical analyses reveal that conserved residues and residues required for catalytic activity and substrate binding comprise the catalytic motifs and a region on one side of the binding cleft. We demonstrate that the VP3 CTD of group B rotavirus, but not that of group G, cleaves 2-5A. These findings suggest that the VP3 CTD is a streamlined version of a 2H phosphoesterase with a ligand-binding mechanism that is shared among 2H phosphodiesterases that cleave 2-5A. Less
The programmable RNA-guided DNA cleavage activity of the bacterial CRISPR-associated endonuclease Cas is the basis of genome editing applications in numerous model organisms and cell types In a binary complex with a dual crRNA tracrRNA guide or single-molecule guide RNA Cas targets double-stranded DNAs harboring sequences complementary to a -nucleotide segment in the guide RNA Recent structural studies of the enzyme have uncovered the molecular mechanism of RNA-guided DNA recognition Here we provide protocols for electrophoretic mobility shift and fluorescence-detection size exclusion chromatography assays used to probe DNA binding by Cas that allowed us to reconstitute and crystallize the enzyme ... More
The programmable RNA-guided DNA cleavage activity of the bacterial CRISPR-associated endonuclease Cas9 is the basis of genome editing applications in numerous model organisms and cell types. In a binary complex with a dual crRNA:tracrRNA guide or single-molecule guide RNA, Cas9 targets double-stranded DNAs harboring sequences complementary to a 20-nucleotide segment in the guide RNA. Recent structural studies of the enzyme have uncovered the molecular mechanism of RNA-guided DNA recognition. Here, we provide protocols for electrophoretic mobility shift and fluorescence-detection size exclusion chromatography assays used to probe DNA binding by Cas9 that allowed us to reconstitute and crystallize the enzyme in a ternary complex with a guide RNA and a bona fide target DNA. The procedures can be used for further mechanistic investigations of the Cas9 endonuclease family and are potentially applicable to other multicomponent protein-nucleic acid complexes. Less
Aminoacyl tRNA synthetases play a critical role in protein synthesis by providing precursor transfer-RNA molecules correctly charged with their cognate amino-acids The essential nature of these enzymes make them attractive targets for designing new drugs against important pathogenic protozoans like Toxoplasma Because no structural data currently exists for a protozoan glutaminyl-tRNA synthetase QRS an understanding of its potential as a drug target and its function in the assembly of the Toxoplasma multi-aminoacyl tRNA MARS complex is therefore lacking Here we describe the optimization of expression and purification conditions that permitted the recovery and crystallization of both domains of the Toxoplasma ... More
Aminoacyl tRNA synthetases play a critical role in protein synthesis by providing precursor transfer-RNA molecules correctly charged with their cognate amino-acids. The essential nature of these enzymes make them attractive targets for designing new drugs against important pathogenic protozoans like Toxoplasma. Because no structural data currently exists for a protozoan glutaminyl-tRNA synthetase (QRS), an understanding of its potential as a drug target and its function in the assembly of the Toxoplasma multi-aminoacyl tRNA (MARS) complex is therefore lacking. Here we describe the optimization of expression and purification conditions that permitted the recovery and crystallization of both domains of the Toxoplasma QRS enzyme from a heterologous Escherichia coli expression system. Expression of full-length QRS was only achieved after the addition of an N-terminal histidine affinity tag and the isolated protein was active on both cellular and in vitro produced Toxoplasma tRNA. Taking advantage of the proteolytic susceptibility of QRS to cleavage into component domains, N-terminal glutathione S-transferase (GST) motif-containing domain fragments were isolated and crystallization conditions discovered. Isolation of the C-terminal catalytic domain was accomplished after subcloning the domain and optimizing expression conditions. Purified catalytic domain survived cryogenic storage and yielded large diffraction-quality crystals over-night after optimization of screening conditions. This work will form the basis of future structural studies into structural–functional relationships of both domains including potential targeted drug-design studies and investigations into the assembly of the Toxoplasma MARS complex. Less
Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry Proteins can either be soluble crystalline or precipitated Additionally liquid liquid phase separation gelation and skin formation can occur A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed For lysozyme from chicken egg white human lysozyme glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values pH and Sodium chloride ammonium sulfate polyethylene glycol and polyethylene glycol were used as precipitants Crystallizing conditions could be found for lysozyme from chicken ... More
Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry. Proteins can either be soluble, crystalline or precipitated. Additionally liquid–liquid phase separation, gelation and skin formation can occur. A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed. For lysozyme from chicken egg white, human lysozyme, glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values – pH 3, 5, 7 and 9. Sodium chloride, ammonium sulfate, polyethylene glycol 300 and polyethylene glycol 1000 were used as precipitants. Crystallizing conditions could be found for lysozyme from chicken egg white using sodium chloride, for human lysozyme using sodium chloride or ammonium sulfate and glucose isomerase using ammonium sulfate. PEG caused destabilization of human lysozyme and glucose oxidase solutions or a balance of stabilizing and destabilizing effects for glucose isomerase near the isoelectric point. This work presents a systematic generation and extensive study of phase diagrams of proteins. Thus, it adds to the general understanding of protein behavior in liquid formulation and presents a convenient methodology applicable to any protein solution. Less
Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP which are universal second messengers Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain CNBD of these channels is thought to cause a conformational change that promotes channel opening The C-linker domain which connects the channel pore to this CNBD plays an important role in coupling ligand binding to channel opening Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker CNBD from hyperpolarization-activated cyclic nucleotide-modulated HCN channels However these structures reveal little to no conformational changes ... More
Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the Kv1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels. Less
Xyloglucan-specific endo- - -glucanases Xegs EC exhibit high catalytic specificity for - linkages of xyloglucan a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species In nature GH Xegs are not associated with carbohydrate-binding modules CBMs and here we have investigated the effect of the fusion of the xyloglucan-specific CBM on the structure and function of a GH Xeg from Aspergillus niveus XegA This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA An increased k cat chimera s - XegA s - and reduced KM chimera mg mL - ... More
Xyloglucan-specific endo-β-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for β-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s(-1); XegA, 144.31 s(-1)) and reduced KM (chimera, 1.30 mg mL(-1); XegA, 1.50 mg mL(-1)) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera. Less
The identification of suitable conditions for crystallization is a rate-limiting step in protein structure determination The pH of an experiment is an important parameter and has the potential to be used in data-mining studies to help reduce the number of crystallization trials required However the pH is usually recorded as that of the buffer solution which can be highly inaccurate
Modern high-throughput structural biology laboratories produce vast amounts of raw experimental data The traditional method of data reduction is very simple results are summarized in peer-reviewed publications which are hopefully published in high-impact journals By their nature publications include only the most important results derived from experiments that may have been performed over the course of many years The main content of the published paper is a concise compilation of these data an interpretation of the experimental results and a comparison of these results with those obtained by other scientists Due to an avalanche of structural biology manuscripts submitted to ... More
Modern high-throughput structural biology laboratories produce vast amounts of raw experimental data. The traditional method of data reduction is very simple�results are summarized in peer-reviewed publications, which are hopefully published in high-impact journals. By their nature, publications include only the most important results derived from experiments that may have been performed over the course of many years. The main content of the published paper is a concise compilation of these data, an interpretation of the experimental results, and a comparison of these results with those obtained by other scientists.
Due to an avalanche of structural biology manuscripts submitted to scientific journals, in many recent cases descriptions of experimental methodology (and sometimes even experimental results) are pushed to supplementary materials that are only published online and sometimes may not be reviewed as thoroughly as the main body of a manuscript. Trouble may arise when experimental results are contradicting the results obtained by other scientists, which requires (in the best case) the reexamination of the original raw data or independent repetition of the experiment according to the published description of the experiment. There are reports that a significant fraction of experiments obtained in academic laboratories cannot be repeated in an industrial environment (Begley CG & Ellis LM, Nature 483(7391):531�3, 2012). This is not an indication of scientific fraud but rather reflects the inadequate description of experiments performed on different equipment and on biological samples that were produced with disparate methods. For that reason the goal of a modern data management system is not only the simple replacement of the laboratory notebook by an electronic one but also the creation of a sophisticated, internally consistent, scalable data management system that will combine data obtained by a variety of experiments performed by various individuals on diverse equipment. All data should be stored in a core database that can be used by custom applications to prepare internal reports, statistics, and perform other functions that are specific to the research that is pursued in a particular laboratory.
This chapter presents a general overview of the methods of data management and analysis used by structural genomics (SG) programs. In addition to a review of the existing literature on the subject, also presented is experience in the development of two SG data management systems, UniTrack and LabDB. The description is targeted to a general audience, as some technical details have been (or will be) published elsewhere. The focus is on �data management,� meaning the process of gathering, organizing, and storing data, but also briefly discussed is �data mining,� the process of analysis ideally leading to an understanding of the data. In other words, data mining is the conversion of data into information. Clearly, effective data management is a precondition for any useful data mining. If done properly, gathering details on millions of experiments on thousands of proteins and making them publicly available for analysis�even after the projects themselves have ended�may turn out to be one of the most important benefits of SG programs. Less
Due to an avalanche of structural biology manuscripts submitted to scientific journals, in many recent cases descriptions of experimental methodology (and sometimes even experimental results) are pushed to supplementary materials that are only published online and sometimes may not be reviewed as thoroughly as the main body of a manuscript. Trouble may arise when experimental results are contradicting the results obtained by other scientists, which requires (in the best case) the reexamination of the original raw data or independent repetition of the experiment according to the published description of the experiment. There are reports that a significant fraction of experiments obtained in academic laboratories cannot be repeated in an industrial environment (Begley CG & Ellis LM, Nature 483(7391):531�3, 2012). This is not an indication of scientific fraud but rather reflects the inadequate description of experiments performed on different equipment and on biological samples that were produced with disparate methods. For that reason the goal of a modern data management system is not only the simple replacement of the laboratory notebook by an electronic one but also the creation of a sophisticated, internally consistent, scalable data management system that will combine data obtained by a variety of experiments performed by various individuals on diverse equipment. All data should be stored in a core database that can be used by custom applications to prepare internal reports, statistics, and perform other functions that are specific to the research that is pursued in a particular laboratory.
This chapter presents a general overview of the methods of data management and analysis used by structural genomics (SG) programs. In addition to a review of the existing literature on the subject, also presented is experience in the development of two SG data management systems, UniTrack and LabDB. The description is targeted to a general audience, as some technical details have been (or will be) published elsewhere. The focus is on �data management,� meaning the process of gathering, organizing, and storing data, but also briefly discussed is �data mining,� the process of analysis ideally leading to an understanding of the data. In other words, data mining is the conversion of data into information. Clearly, effective data management is a precondition for any useful data mining. If done properly, gathering details on millions of experiments on thousands of proteins and making them publicly available for analysis�even after the projects themselves have ended�may turn out to be one of the most important benefits of SG programs. Less
The catalytic domain of the trimeric human -enoyl-CoA isomerase type HsECI has the typical crotonase fold In the active site of this fold two main chain NH groups form an oxyanion hole for binding the thioester oxygen of the E- or Z-enoyl-CoA substrate molecules A catalytic glutamate is essential for the proton transfer between the substrate C and C atoms for forming the product E-enoyl-CoA which is a key intermediate in the -oxidation pathway The active site is covered by the C-terminal helix- In HsECI the isomerase domain is extended at its N terminus by an acyl-CoA binding protein ACBP ... More
The catalytic domain of the trimeric human ?3,?2-enoyl-CoA isomerase, type 2 (HsECI2), has the typical crotonase fold. In the active site of this fold two main chain NH groups form an oxyanion hole for binding the thioester oxygen of the 3E- or 3Z-enoyl-CoA substrate molecules. A catalytic glutamate is essential for the proton transfer between the substrate C2 and C4 atoms for forming the product 2E-enoyl-CoA, which is a key intermediate in the �-oxidation pathway. The active site is covered by the C-terminal helix-10. In HsECI2, the isomerase domain is extended at its N terminus by an acyl-CoA binding protein (ACBP) domain. Small angle X-ray scattering analysis of HsECI2 shows that the ACBP domain protrudes out of the central isomerase trimer. X-ray crystallography of the isomerase domain trimer identifies the active site geometry. A tunnel, shaped by loop-2 and extending from the catalytic site to bulk solvent, suggests a likely mode of binding of the fatty acyl chains. Calorimetry data show that the separately expressed ACBP and isomerase domains bind tightly to fatty acyl-CoA molecules. The truncated isomerase variant (without ACBP domain) has significant enoyl-CoA isomerase activity; however, the full-length isomerase is more efficient. Structural enzymological studies of helix-10 variants show the importance of this helix for efficient catalysis. Its hydrophobic side chains, together with residues from loop-2 and loop-4, complete a hydrophobic cluster that covers the active site, thereby fixing the thioester moiety in a mode of binding competent for efficient catalysis. Less
The modified base -formylcytosine fC was recently identified in mammalian DNA and might be considered to be the 'seventh' base of the genome This nucleotide has been implicated in active demethylation mediated by the base excision repair enzyme thymine DNA glycosylase Genomics and proteomics studies have suggested an additional role for fC in transcription regulation through chromatin remodeling Here we propose that fC might affect these processes through its effect on DNA conformation Biophysical and structural analysis revealed that fC alters the structure of the DNA double helix and leads to a conformation unique among known DNA structures including those ... More
The modified base 5-formylcytosine (5fC) was recently identified in mammalian DNA and might be considered to be the 'seventh' base of the genome. This nucleotide has been implicated in active demethylation mediated by the base excision repair enzyme thymine DNA glycosylase. Genomics and proteomics studies have suggested an additional role for 5fC in transcription regulation through chromatin remodeling. Here we propose that 5fC might affect these processes through its effect on DNA conformation. Biophysical and structural analysis revealed that 5fC alters the structure of the DNA double helix and leads to a conformation unique among known DNA structures including those comprising other cytosine modifications. The 1.4-�-resolution X-ray crystal structure of a DNA dodecamer comprising three 5fCpG sites shows how 5fC changes the geometry of the grooves and base pairs associated with the modified base, leading to helical underwinding. Less
For the successful application of protein crystallization as a downstream step a profound knowledge of protein phase behavior in solutions is needed Therefore a systematic screening was conducted to analyze the influence of macromolecular precipitants in the form of polyethylene glycol PEG First the influence of molecular weight and concentration of PEG at different pH-values were investigated and analyzed in three-dimensional -D phase diagrams to find appropriate conditions in terms of a fast kinetic and crystal size for downstream processing In comparison to the use of salts as precipitant PEG was more suitable to obtain compact -D crystals over a ... More
For the successful application of protein crystallization as a downstream step, a profound knowledge of protein phase behavior in solutions is needed. Therefore, a systematic screening was conducted to analyze the influence of macromolecular precipitants in the form of polyethylene glycol (PEG). First, the influence of molecular weight and concentration of PEG at different pH-values were investigated and analyzed in three-dimensional (3-D) phase diagrams to find appropriate conditions in terms of a fast kinetic and crystal size for downstream processing. In comparison to the use of salts as precipitant, PEG was more suitable to obtain compact 3-D crystals over a broad range of conditions, whereby the molecular weight of PEG is, besides the pH-value, the most important parameter. Second, osmotic second virial coefficients as parameters for protein interactions are experimentally determined with static light scattering to gain a deep insight view in the phase behavior on a molecular basis. The PEG-protein solutions were analyzed as a pseudo-one-compartment system. As the precipitant is also a macromolecule, the new approach of analyzing cross-interactions between the protein and the macromolecule PEG in form of the osmotic second cross-virial coefficient (B23) was applied. Both parameters help to understand the protein phase behavior. However, a predictive description of protein phase behavior for systems consisting of monoclonal antibodies and PEG as precipitant is not possible, as kinetic phenomena and concentration dependencies were not taken into account. Less
Wax esters WEs are esters of fatty acids and fatty alcohols They can cover a broad range of physical properties which makes them especially interesting for industrial applications including additives in cosmetics and high class lubricants Biosynthesis of WEs is a widespread feature in nature and is carried out by two essential classes of enzymes One of them is the class of fatty acyl reductases FARs which reduce acyl-CoAs or acyl-acyl carrier proteins acyl-ACPs to the corresponding fatty alcohols The second one is the class of wax synthases WSs which esterify fatty alcohols with acyl CoAs yielding WEs The majority ... More
Wax esters (WEs) are esters of fatty acids and fatty alcohols. They can cover a broad range of physical properties, which makes them especially interesting for industrial applications, including additives in cosmetics and high class lubricants. Biosynthesis of WEs is a widespread feature in nature and is carried out by two essential classes of enzymes. One of them is the class of fatty acyl reductases (FARs), which reduce acyl-CoAs or acyl-acyl carrier proteins (acyl-ACPs) to the corresponding fatty alcohols. The second one is the class of wax synthases (WSs), which esterify fatty alcohols with acyl CoAs, yielding WEs. The majority of FARs and WSs described to date exhibit a broad substrate range, resulting in wax blends of heterogeneous compositions. With respect to a commercial production of WEs in genetically modified plants, a defined WE blend is desired rather than a mixture of WEs. Enzymes with improved substrate specificities, tailored for the production of individual WE blends, are one way to overcome these issues. However, the required knowledge about structure-function relationships in FARs and WSs for the construction of respective enzymes is lacking to date. The aim of the present thesis was thus to elucidate structural determinants of substrate specificity in FARs and WSs. To date, crystal structures are neither available from FARs nor from WSs. Hence, four FARs (DmFAR1 from Drosophila melanogaster, MaFAR1 from Marinobacter aquaeolei, MmFAR1 from Mus musculus and TcFAR1 from Tribolium castaneum), two WSs (AbWSD1 from Acinetobacter baylyi and MmAWAT2 from Mus musculus) and a soluble diacylglycerol O-acyltransferase (AtDGAT3 from Arabidopsis thaliana) were studied in order to obtain structural insights. The activity of DmFAR1 was first described in the present work. It produces tetracosanol and hexacosanol upon expression in yeast, while expression in E. coli yielded tetradecanol, hexadecanol, hexadecenol and octadecenol. DmFAR1, MaFAR1, MmFAR1, AbWSD1, MmAWAT2 and AtDGAT3 were expressed in and purified from E. coli. Although this resulted in aggregated proteins in most cases, AbWSD1 was obtained in a quaternary structure corresponding to a trimer, when expressed as a fusion protein with the bacterial trigger factor. The respective fusion protein reproducibly formed crystals, which diffracted to 2.1 Å. Attempts to soak the crystals with iodine led to disruption of the crystals. Hence, the respective selenoprotein was produced and applied to crystallisation screens, which were ongoing at the end of this thesis. In order to elucidate substrate specificity determining structures in MmAWAT2, comparative studies of MmAWAT2 and MmDGAT2, which share ~ 70 % homology, were carried out. Both enzymes are acyltransferases and are capable of synthesising both, TAGs and WEs. Furthermore, both enzymes show distinct substrate specificities with respect to WE synthesis. Two predicted, neighboured hairpin forming transmembrane (TM) domains were identified to have an influence on the substrate specificity of MmAWAT2. Chimeric enzyme variants of MmAWAT2 carrying the respective section of the MmDGAT2 sequence showed a severely altered acyl chain incorporation pattern into WEs as compared to both, MmAWAT2 and MmDGAT2. Furthermore, respective variants showed an altered ratio of produced WEs and TAGs. This phenotype was also exhibited by the MmAWAT2 single amino acid exchange variant N36R, carrying a mutation in the part of the sequence which encodes the two predicted TM domains. Thus, the predicted TM domains of MmAWAT2 seem to have a role in substrate specificity determination of the enzyme. This work provides further insights into structure function relationships concerning substrate specificity in DGAT2-type acyltransferases. Furthermore, the successful crystallisation of a WS might pave the way for an extensive comprehension of this class of enzymes. Less
The Gram-positive bacterium Staphylococcus pseudintermedius is a leading cause of canine bacterial pyoderma resulting in worldwide morbidity in dogs S pseudintermedius also causes life-threatening human infections Furthermore methicillin-resistant S pseudintermedius is emerging resembling the human health threat of methicillin-resistant Staphylococcus aureus Therefore it is increasingly important to characterize targets for intervention strategies to counteract S pseudintermedius infections Here we used biophysical methods mutagenesis and X-ray crystallography to define the ligand-binding properties and structure of SitA an S pseudintermedius surface lipoprotein SitA was strongly and specifically stabilized by Mn and Zn ions Crystal structures of SitA complexed with Mn and Zn ... More
The Gram-positive bacterium Staphylococcus pseudintermedius is a leading cause of canine bacterial pyoderma, resulting in worldwide morbidity in dogs. S. pseudintermedius also causes life-threatening human infections. Furthermore, methicillin-resistant S. pseudintermedius is emerging, resembling the human health threat of methicillin-resistant Staphylococcus aureus. Therefore it is increasingly important to characterize targets for intervention strategies to counteract S. pseudintermedius infections. Here we used biophysical methods, mutagenesis, and X-ray crystallography, to define the ligand-binding properties and structure of SitA, an S. pseudintermedius surface lipoprotein. SitA was strongly and specifically stabilized by Mn2+ and Zn2+ ions. Crystal structures of SitA complexed with Mn2+ and Zn2+ revealed a canonical class III solute-binding protein with the metal cation bound in a cavity between N- and C-terminal lobes. Unexpectedly, one crystal contained both apo- and holo-forms of SitA, revealing a large side-chain reorientation of His64, and associated structural differences accompanying ligand binding. Such conformational changes may regulate fruitful engagement of the cognate ABC (ATP-binding cassette) transporter system (SitBC) required for metal uptake. These results provide the first detailed characterization and mechanistic insights for a potential therapeutic target of the major canine pathogen S. pseudintermedius, and also shed light on homologous structures in related staphylococcal pathogens afflicting humans. Less
Hydrocarbon stapling can restore bioactive -helical structure to natural peptides yielding research tools and prototype therapeutics to dissect and target protein interactions Here we explore the capacity of peptide stapling to generate high-fidelity protease-resistant mimics of antigenic structures for vaccine development HIV- has been refractory to vaccine technologies thus far although select human antibodies can broadly neutralize HIV- by targeting sequences of the gp juxtamembrane fusion apparatus To develop candidate HIV- immunogens we generated and characterized stabilized -helices of the membrane-proximal external region SAH-MPER of gp SAH-MPER peptides were remarkably protease resistant and bound to the broadly neutralizing E and ... More
Hydrocarbon stapling can restore bioactive α-helical structure to natural peptides, yielding research tools and prototype therapeutics to dissect and target protein interactions. Here we explore the capacity of peptide stapling to generate high-fidelity, protease-resistant mimics of antigenic structures for vaccine development. HIV-1 has been refractory to vaccine technologies thus far, although select human antibodies can broadly neutralize HIV-1 by targeting sequences of the gp41 juxtamembrane fusion apparatus. To develop candidate HIV-1 immunogens, we generated and characterized stabilized α-helices of the membrane-proximal external region (SAH-MPER) of gp41. SAH-MPER peptides were remarkably protease resistant and bound to the broadly neutralizing 4E10 and 10E8 antibodies with high affinity, recapitulating the structure of the MPER epitope when differentially engaged by the two anti-HIV Fabs. Thus, stapled peptides may provide a new opportunity to develop chemically stabilized antigens for vaccination. Less
Secondary structure refolding is a key event in biology as it modulates the conformation of many proteins in the cell generating functional or aberrant states The crystal structures of mannosyltransferase PimA reveal an exceptional flexibility of the protein along the catalytic cycle including -strand to -helix and -helix to -strand transitions These structural changes modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane
The visual inspection of crystallization experiments is an important yet time-consuming and subjective step in X-ray crystallo graphy Previously published studies have focused on automatically classifying crystallization droplets into distinct but ultimately arbitrary experiment outcomes here a method is described that instead ranks droplets by their likelihood of containing crystals or microcrystals thereby prioritizing for visual inspection those images that are most likely to contain useful information The use of textons is introduced to describe crystallization droplets objectively allowing them to be scored with the posterior probability of a random forest classifier trained against droplets manually annotated for the presence ... More
The visual inspection of crystallization experiments is an important yet time-consuming and subjective step in X-ray crystallo�graphy. Previously published studies have focused on automatically classifying crystallization droplets into distinct but ultimately arbitrary experiment outcomes; here, a method is described that instead ranks droplets by their likelihood of containing crystals or microcrystals, thereby prioritizing for visual inspection those images that are most likely to contain useful information. The use of textons is introduced to describe crystallization droplets objectively, allowing them to be scored with the posterior probability of a random forest classifier trained against droplets manually annotated for the presence or absence of crystals or microcrystals. Unlike multi-class classification, this two-class system lends itself naturally to unidirectional ranking, which is most useful for assisting sequential viewing because images can be arranged simply by using these scores: this places droplets with probable crystalline behaviour early in the viewing order. Using this approach, the top ten wells included at least one human-annotated crystal or microcrystal for 94% of the plates in a data set of 196 plates imaged with a Minstrel HT system. The algorithm is robustly transferable to at least one other imaging system: when the parameters trained from Minstrel HT images are applied to a data set imaged by the Rock Imager system, human-annotated crystals ranked in the top ten wells for 90% of the plates. Because rearranging images is fundamental to the approach, a custom viewer was written to seamlessly support such ranked viewing, along with another important output of the algorithm, namely the shape of the curve of scores, which is itself a useful overview of the behaviour of the plate; additional features with known usefulness were adopted from existing viewers. Evidence is presented that such ranked viewing of images allows faster but more accurate evaluation of drops, in particular for the identification of microcrystals. Less
The inner membrane ring of the bacterial type III secretion system TTSS is composed of two proteins In Chlamydia trachomatis this ring is formed by CdsD gene name CT and CdsJ gene name CTA CdsD consists of amino acids The last amino acids at its C-terminal end relate it to the type III secretion system YscD HrpQ protein family The C-terminal domain consisting of amino acids of C trachomatis CdsD was overexpressed in Escherichia coli and purified using immobilized metal-affinity chromatography IMAC and size-exclusion chromatography The protein was crystallized using the vapour-diffusion method A data set was collected to resolution ... More
The inner membrane ring of the bacterial type III secretion system (TTSS) is composed of two proteins. In Chlamydia trachomatis this ring is formed by CdsD (gene name CT_664) and CdsJ (gene name CTA_0609). CdsD consists of 829 amino acids. The last 400 amino acids at its C-terminal end relate it to the type III secretion system YscD/HrpQ protein family. The C-terminal domain, consisting of amino acids 558–771, of C. trachomatis CdsD was overexpressed in Escherichia coli and purified using immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography. The protein was crystallized using the vapour-diffusion method. A data set was collected to 2.26 Å resolution. The crystals have the symmetry of space group C2, with unit-cell parameters a = 106.60, b = 23.91, c = 118.65 Å, β = 104.95°. According to the data analysis there is expected to be one molecule in the asymmetric unit, with a Matthews coefficient of 3.0 Å3 Da−1. Less