1052 Citations
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
X-ray transparent Microfluidics for Protein Crystallization and Biomineralization A dissertation presented to the Faculty of the Graduate School of Arts and Sciences of Brandeis University Waltham Massachusetts by Achini Opathalage Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics We ... More
X-ray transparent Microfluidics for Protein Crystallization and Biomineralization A dissertation presented to the Faculty of the Graduate School of Arts and Sciences of Brandeis University, Waltham, Massachusetts by Achini Opathalage Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal. Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions.In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics. We have developed an emulsion-based serial crystallographic technology in nanolitre-sized droplets of protein solution encapsulated in to nucleate one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different un-oriented crystals. As proof of concept, the structure of Glucose Isomerase was solved to 2.1 �. We have developed a suite of X-ray semi-transparent micrfluidic devices which enables; controlled evaporation as a method of increasing supersaturation and manipulating the phase space of proteins and small molecules. We exploited the inherently high water permeability of the thin X-ray semi-transparent devices as a mean of increasing the supersaturation by controlling the evaporation. We fabricated the X-ray semi-transparent version of the PhaseChip with a thin PDMS membrane by which the storage and the reservoir layers are separated, and studies the phase transition of amorphous CaCO3. 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.
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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
In the past decade advances in structure determination with electron microscopy of organic beam sensitive materials have been significant The newly developed techniques triggered by new microscope systems and new cameras made it possible to acquire D structural information from these samples to a resolution which was impossible to achieve before Knowledge is required to improve structure solution and every aspect of the process involved from treatment of radiation sensitive materials sample preparation TEM imaging and diffraction systems all the way to how data must be interpreted In this thesis I explained multiple new techniques and methods developed by us ... More
In the past decade, advances in structure determination with electron microscopy of organic, beam sensitive, materials have been significant. The newly developed techniques, triggered by new microscope systems and new cameras, made it possible to acquire 3D structural information from these samples to a resolution which was impossible to achieve before. Knowledge is required to improve structure solution and every aspect of the process involved, from treatment of radiation sensitive materials, sample preparation, TEM imaging and diffraction systems all the way to how data must be interpreted. In this thesis I explained multiple new techniques and methods developed by us, using both new microscopes as well as a new type of detector: Timepix. I describe how these tools can help to overcome (what were) the most important problems and bottlenecks in detection of very low dose electron diffraction. Less
Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought P aeruginosa disulfide-bond protein A PaDsbA plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target As part of a fragment-based lead discovery approach to PaDsbA inhibitor development the identification of a crystal form of PaDsbA that was more suitable for fragment-soaking experiments was sought A previously identified crystallization condition for this protein was unsuitable as in this crystal form of PaDsbA the active-site surface loops are engaged in the crystal ... More
Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discovery approach to PaDsbA1 inhibitor development, the identification of a crystal form of PaDsbA1 that was more suitable for fragment-soaking experiments was sought. A previously identified crystallization condition for this protein was unsuitable, as in this crystal form of PaDsbA1 the active-site surface loops are engaged in the crystal packing, occluding access to the target site. A single residue involved in crystal-packing interactions was substituted with an amino acid commonly found at this position in closely related enzymes, and this variant was successfully used to generate a new crystal form of PaDsbA1 in which the active-site surface is more accessible for soaking experiments. The PaDsbA1 variant displays identical redox character and in vitro activity to wild-type PaDsbA1 and is structurally highly similar. Two crystal structures of the PaDsbA1 variant were determined in complex with small molecules bound to the protein active site. These small molecules (MES, glycerol and ethylene glycol) were derived from the crystallization or cryoprotectant solutions and provide a proof of principle that the reported crystal form will be amenable to co-crystallization and soaking with small molecules designed to target the protein active-site surface. Less
Catechol-O-methyltransferase COMT plays an important role in the deactivation of catecholamine neurotransmitters and hormones Inhibitors of COMT such as tolcapone and entacapone are used clinically in the treatment of Parkinson s disease Discovery of novel inhibitors has been hampered by a lack of suitable assays for high-throughput screening HTS Although assays using esculetin have been developed these are affected by fluorescence a common property of catechol-type compounds We have therefore evaluated a new homogenous time-resolved fluorescence HTRF based assay from CisBio Codolet France which measures the production of S-adenosyl-L-homocysteine SAH The assay has been run in both HTS and medium-throughput ... More
Catechol-O-methyltransferase (COMT) plays an important role in the deactivation of catecholamine neurotransmitters and hormones. Inhibitors of COMT, such as tolcapone and entacapone, are used clinically in the treatment of Parkinson’s disease. Discovery of novel inhibitors has been hampered by a lack of suitable assays for high-throughput screening (HTS). Although assays using esculetin have been developed, these are affected by fluorescence, a common property of catechol-type compounds. We have therefore evaluated a new homogenous time-resolved fluorescence (HTRF)–based assay from CisBio (Codolet, France), which measures the production of S-adenosyl-L-homocysteine (SAH). The assay has been run in both HTS and medium-throughput screening (MTS) modes. The assay was established using membranes expressing human membrane-bound COMT and was optimized for protein and time to give an acceptable signal window, good potency for tolcapone, and a high degree of translation between data in fluorescence ratio and data in terms of [SAH] produced. pIC50 values for the hits from the HTS mode were determined in the MTS mode. The assay also proved suitable for kinetic studies such as Km,app determination. 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
A method is described for using custom snap-on lids to protect chemicals in microtiter plates from evaporation and contamination The lids contain apertures diameter or mm through which the chemical building blocks can be transferred The lid with mm apertures was tested using a noncontact acoustic liquid handler the and mm lids were tested using two tip-based liquid handlers All of the lids reduced the rate at which solvents evaporated to room air and greatly reduced the rate of contamination by water and oxygen from room air In steady-state measurements the lids reduced the rate of evaporation of methanol -hexene ... More
A method is described for using custom snap-on lids to protect chemicals in microtiter plates from evaporation and contamination. The lids contain apertures (diameter 1.5, 1.0, or 0.5 mm) through which the chemical building blocks can be transferred. The lid with 0.5 mm apertures was tested using a noncontact acoustic liquid handler; the 1.0 and 1.5 mm lids were tested using two tip-based liquid handlers. All of the lids reduced the rate at which solvents evaporated to room air, and greatly reduced the rate of contamination by water and oxygen from room air. In steady-state measurements, the lids reduced the rate of evaporation of methanol, 1-hexene, and water by 33% to 248%. In cycled experiments, the contamination of aqueous solvent with oxygen was reduced below detectability and the rate at which DMSO engorged atmospheric water was reduced by 81%. Our results demonstrate that the lids preserve the integrity of air-sensitive reagents during the time needed for different types of liquid handlers to perform dispensations. Controlling degradation and evaporation of chemical building blocks exposed to the atmosphere is increasingly useful as the reagent volume is reduced by advances in liquid handling technology, such as acoustic droplet ejection. Less
Understanding the mechanism by which ligands impact receptor conformational equilibria is key in accelerating membrane protein structural biology In the case of G protein-coupled receptors GPCRs we currently pursue a brute force approach for identifying ligands that stabilize receptors and facilitate crystallogenesis The nociceptin orphanin FQ peptide receptor NOP is a member of the opioid receptor subfamily of GPCRs for which many structurally diverse ligands are available for screening We observed that antagonist potency is correlated with a ligand s ability to induce receptor stability Tm and crystallogenesis Using this screening strategy we solved two structures of NOP in complex ... More
Understanding the mechanism by which ligands impact receptor conformational equilibria is key in accelerating membrane protein structural biology. In the case of G protein-coupled receptors (GPCRs) we currently pursue a brute force approach for identifying ligands that stabilize receptors and facilitate crystallogenesis. The nociceptin/orphanin FQ peptide receptor (NOP) is a member of the opioid receptor subfamily of GPCRs for which many structurally diverse ligands are available for screening. We observed that antagonist potency is correlated with a ligand’s ability to induce receptor stability (Tm) and crystallogenesis. Using this screening strategy, we solved two structures of NOP in complex with top candidate ligands SB-612111 and C-35. Docking studies indicate that while potent, stabilizing antagonists strongly favor a single binding orientation, less potent ligands can adopt multiple binding modes, contributing to their low Tm values. These results suggest a mechanism for ligand-aided crystallogenesis whereby potent antagonists stabilize a single ligand-receptor conformational pair. 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
Angiotensin II type receptor AT R is the primary blood pressure regulator AT R blockers ARBs have been widely used in clinical settings as anti-hypertensive drugs and share a similar chemical scaffold although even minor variations can lead to distinct therapeutic efficacies toward cardiovascular etiologies The structural basis for AT R modulation by different peptide and non-peptide ligands has remained elusive Here we report the crystal structure of the human AT R in complex with an inverse agonist olmesartan BenicarTM a highly potent anti-hypertensive drug Olmesartan is anchored to the receptor primarily by the residues Tyr- Trp- and Arg- ECL ... More
Angiotensin II type 1 receptor (AT1R) is the primary blood pressure regulator. AT1R blockers (ARBs) have been widely used in clinical settings as anti-hypertensive drugs and share a similar chemical scaffold, although even minor variations can lead to distinct therapeutic efficacies toward cardiovascular etiologies. The structural basis for AT1R modulation by different peptide and non-peptide ligands has remained elusive. Here, we report the crystal structure of the human AT1R in complex with an inverse agonist olmesartan (BenicarTM), a highly potent anti-hypertensive drug. Olmesartan is anchored to the receptor primarily by the residues Tyr-351.39, Trp-842.60, and Arg-167ECL2, similar to the antagonist ZD7155, corroborating a common binding mode of different ARBs. Using docking simulations and site-directed mutagenesis, we identified specific interactions between AT1R and different ARBs, including olmesartan derivatives with inverse agonist, neutral antagonist, or agonist activities. We further observed that the mutation N1113.35A in the putative sodium-binding site affects binding of the endogenous peptide agonist angiotensin II but not the β-arrestin-biased peptide TRV120027. Less
A number of Gram-positive bacteria produce a class of bacteriocins called lantibiotics These lantibiotics are ribosomally synthesized peptides that possess high antimicrobial activity against Gram-positive bacteria including clinically challenging pathogens and are therefore potential alternatives to antibiotics All lantibiotic producer strains and some Gram-positive non producer strains express protein systems to circumvent a suicidal effect or to become resistant respectively Two-component systems consisting of a response regulator and a histidine kinase upregulate the expression of these proteins One of the best-characterized lantibiotics is nisin which is produced by Lactococcus lactis and possesses bactericidal activity against various Gram-positive bacteria including some ... More
A number of Gram-positive bacteria produce a class of bacteriocins called ‘lantibiotics’. These lantibiotics are ribosomally synthesized peptides that possess high antimicrobial activity against Gram-positive bacteria, including clinically challenging pathogens, and are therefore potential alternatives to antibiotics. All lantibiotic producer strains and some Gram-positive nonproducer strains express protein systems to circumvent a suicidal effect or to become resistant, respectively. Two-component systems consisting of a response regulator and a histidine kinase upregulate the expression of these proteins. One of the best-characterized lantibiotics is nisin, which is produced by Lactococcus lactis and possesses bactericidal activity against various Gram-positive bacteria, including some human pathogenic strains. Within many human pathogenic bacterial strains inherently resistant to nisin, a response regulator, NsrR, has been identified which regulates the expression of proteins involved in nisin resistance. In the present study, an expression and purification protocol was established for the NsrR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method, resulting in crystals that diffracted X-rays to 1.4 Å resolution. 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
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
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
RNA binding proteins RBPs are involved in many cellular functions To facilitate functional characterization of RBPs we generated an RNA interference RNAi library for Drosophila cell-based screens comprising reagents targeting known or putative RBPs To test the quality of the library and provide a baseline analysis of the effects of the RNAi reagents on viability we screened the library using a total ATP assay and high-throughput imaging in Drosophila S R cultured cells The results are consistent with production of a high-quality library that will be useful for functional genomics studies using other assays Altogether we provide resources in the ... More
RNA binding proteins (RBPs) are involved in many cellular functions. To facilitate functional characterization of RBPs, we generated an RNA interference (RNAi) library for Drosophila cell-based screens comprising reagents targeting known or putative RBPs. To test the quality of the library and provide a baseline analysis of the effects of the RNAi reagents on viability, we screened the library using a total ATP assay and high-throughput imaging in Drosophila S2R+ cultured cells. The results are consistent with production of a high-quality library that will be useful for functional genomics studies using other assays. Altogether, we provide resources in the form of an initial curated list of Drosophila RBPs; an RNAi screening library we expect to be used with additional assays that address more specific biological questions; and total ATP and image data useful for comparison of those additional assay results with fundamental information such as effects of a given reagent in the library on cell viability. Importantly, we make the baseline data, including more than 200,000 images, easily accessible online. 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
Organophosphorus OP pesticides and nerve agents have been designed to inhibit the hydrolysis of the neurotransmitter acetylcholine by covalently binding to the active site serine of acetylcholinesterase while Alzheimer drugs and prophylactics such as tacrine are characterized by reversible binding Historically the guinea pig has been believed to be the best non-primate model for OP toxicology and medical countermeasure development because similarly to humans guinea pigs have low amounts of circulating OP metabolizing carboxylesterase To explore the hypothesis that guinea pigs are the appropriate responder species for OP toxicology and medical countermeasure development guinea pig acetylcholinesterase gpAChE was cloned into ... More
Organophosphorus (OP) pesticides and nerve agents have been designed to inhibit the hydrolysis of the neurotransmitter acetylcholine by covalently binding to the active site serine of acetylcholinesterase while Alzheimer drugs and prophylactics, such as tacrine, are characterized by reversible binding. Historically, the guinea pig has been believed to be the best non-primate model for OP toxicology and medical countermeasure development because, similarly to humans, guinea pigs have low amounts of circulating OP metabolizing carboxylesterase. To explore the hypothesis that guinea pigs are the appropriate responder species for OP toxicology and medical countermeasure development, guinea pig acetylcholinesterase (gpAChE) was cloned into pENTR/D-TOPO, recombined into pT-Rex-DEST30 and expressed in Human Embryonic Kidney 293 cells. Recombinant gpAChE was purified to a specific activity of 800 U/mg using size exclusion and immobilized nickel affinity chromatography, with purity confirmed by gel electrophoresis. Ellman’s assay was used to enzymatically characterize gpAChE, identifying a KM of 154±18.7 µmol L-1 and a kcat of 4.79x104±5.26x102 /sec. Apparent gpAChE IC50’s for diisopropylfluorophosphate, dicrotophos, paraoxon, and an Alzheimer’s drug, tacrine, were found to be 10.1±1.98, 337±108, 1.02±0.29 and 0.30±0.01 µmol L-1, respectively. Apparent gpAChE inhibition constants for diisopropylfluorophosphate, dicrotophos, paraoxon, and tacrine were found to be 8.40±0.60, 4.50±0.30, 0.29±0.01 and 0.42±0.07 µmol L-1, respectively. Lineweaver-Burk plots confirmed tacrine as a mixed inhibitor and paraoxon, dicrotophos and diisopropylfluorophosphate as irreversible non-competitive inhibitors. gpAChE bimolecular rate constants for diisopropylfluorophosphate, dicrotophos and paraoxon were found to be 1.44±0.33x104, 1.56±0.12x103 and 4.57± 0.23x105 L µmol-1 min-1, respectively. Although the blood levels of OP metabolizing carboxylesterases in the guinea pig are similar to the low levels in human blood, the gpAChE is different in its enzymology. Therefore, medical countermeasures against OP intoxication should be tested for efficacy with the recombinant form of gpAChE prior to initiating animal studies. 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
Crystallization of biological macromolecules such as proteins implies several prerequisites for example the presence of one or more initial nuclei sufficient amounts of the crystallizing substance and the chemical potential to provide the free energy needed to force the process The initiation of a crystallization process itself is a stochastic event forming symmetrically assembled nuclei over kinetically preferred protein-dense liquid clusters The presence of a spatial repetitive orientation of macromolecules in the early stages of the crystallization process has so far proved undetectable However early identification of the occurrences of unit cells is the key to nanocrystal detection The optical ... More
Crystallization of biological macromolecules such as proteins implies several prerequisites, for example, the presence of one or more initial nuclei, sufficient amounts of the crystallizing substance and the chemical potential to provide the free energy needed to force the process. The initiation of a crystallization process itself is a stochastic event, forming symmetrically assembled nuclei over kinetically preferred protein-dense liquid clusters. The presence of a spatial repetitive orientation of macromolecules in the early stages of the crystallization process has so far proved undetectable. However, early identification of the occurrences of unit cells is the key to nanocrystal detection. The optical properties of a crystal lattice offer a potential signal with which to detect whether a transition from disordered to ordered particles occurs, one that has so far not been tested in nanocrystalline applications. The ability of a lattice to depolarize laser light depends on the different refractive indices along different crystal axes. In this study a unique experimental setup is used to detect nanocrystal formation by application of depolarized scattered light. The results demonstrate the successful detection of nano-sized protein crystals at early stages of crystal growth, allowing an effective differentiation between protein-dense liquid cluster formation and ordered nanocrystals. The results are further verified by complementary methods like X-ray powder diffraction, second harmonic generation, ultraviolet two-photon excited fluorescence and scanning electron microscopy. 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
G protein-coupled receptors GPCRs signal primarily through G proteins or arrestins Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin determined by serial femtosecond X-ray laser crystallography Together with extensive biochemical and mutagenesis data the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements including TM and Helix to recruit arrestin Correspondingly arrestin adopts the pre-activated conformation with a rotation between the ... More
G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly, in which rhodopsin uses distinct structural elements, including TM7 and Helix 8 to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the N- and C- domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signaling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology. Less
Macromolecular X-ray crystallography has been the primary methodology for determining the three-dimensional structures of proteins nucleic acids and viruses Structural information has paved the way for structure-guided drug discovery and laid the foundations for structural bioinformatics However X-ray crystallography still has a few fundamental limitations some of which may be overcome and complemented using emerging methods and technologies in other areas of structural biology
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
The second-harmonic generation SHG activity of protein crystals was found to be enhanced by up to -fold by the intercalation of SHG phores within the crystal lattice Unlike the intercalation of fluorophores the SHG phores produced no significant background SHG from solvated dye or from dye intercalated into amorphous aggregates The polarization-dependent SHG is consistent with the chromophores adopting the symmetry of the crystal lattice In addition the degree of enhancement for different symmetries of dyes is consistent with theoretical predictions based on the molecular nonlinear optical response Kinetics studies indicate that intercalation arises over a timeframe of several minutes ... More
The second-harmonic generation (SHG) activity of protein crystals was found to be enhanced by up to ~1000-fold by the intercalation of SHG phores within the crystal lattice. Unlike the intercalation of fluorophores, the SHG phores produced no significant background SHG from solvated dye or from dye intercalated into amorphous aggregates. The polarization-dependent SHG is consistent with the chromophores adopting the symmetry of the crystal lattice. In addition, the degree of enhancement for different symmetries of dyes is consistent with theoretical predictions based on the molecular nonlinear optical response. Kinetics studies indicate that intercalation arises over a timeframe of several minutes in lysozyme, with detectable enhancements within seconds. These results provide a potential means to increase the overall diversity of protein crystals and crystal sizes amenable to characterization by SHG microscopy. 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
Rhodopsin is a membrane protein from the G protein-coupled receptor family Together with its ligand retinal it forms the visual pigment responsible for night vision In order to perform ultrafast dynamics studies a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin In such an approach microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser XFEL after a precise photoactivation delay Here a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols i screening the low-salt crystallization conditions preferred for ... More
Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup. 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
Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology Bacteriorhodopsin from Halobium salinarum bR is a striking example of the difficulties in membrane protein overexpression We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression This complementary protein approach allowed us to increase bR expression by two orders of magnitude ... More
Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å. 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
Nisin is a -amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics Nisin displays a high bactericidal activity against various Gram-positive bacteria including some human-pathogenic strains However there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome The encoded protein NSR cleaves off the last six amino acids of nisin thereby reducing its bactericidal efficacy An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops resulting ... More
Nisin is a 34-amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics. Nisin displays a high bactericidal activity against various Gram-positive bacteria, including some human-pathogenic strains. However, there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome. The encoded protein, NSR, cleaves off the last six amino acids of nisin, thereby reducing its bactericidal efficacy. An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops, resulting in crystals that diffracted X-rays to 2.8 and 2.2 Å, respectively. 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
We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems However there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners Here we present a screen that comprehensively explores the parameters affecting the stability of interactions in affinity-captured complexes enabling the discovery of physiological binding partners and the elucidation of their functional interactions in unparalleled detail We have implemented this screen on several macromolecular complexes from a variety of organisms revealing novel profiles even for well-studied proteins Our approach is robust economical ... More
We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems. However, there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners. Here, we present a screen that comprehensively explores the parameters affecting the stability of interactions in affinity-captured complexes, enabling the discovery of physiological binding partners and the elucidation of their functional interactions in unparalleled detail. We have implemented this screen on several macromolecular complexes from a variety of organisms, revealing novel profiles even for well-studied proteins. Our approach is robust, economical and automatable, providing an inroad to the rigorous, systematic dissection of cellular interactomes. 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
In this study we take advantage of a recently described chimera of the a nicotinic acetylcholine receptor nAChR and acetylcholine binding protein AChBP termed a -AChBP To date more than crystal structures have been determined for AChBP in complex with ligands that occupy the orthosteric binding site Here we use an innovative screening strategy to discover molecular fragments that occupy allosteric binding sites In combination with X-ray crystallography we determine a molecular blueprint of three different allosteric sites in a -AChBP Using electrophysiological recordings on the human a nAChR we demonstrate that each of the three sites is involved in ... More
In this study we take advantage of a recently described chimera of the a7 nicotinic acetylcholine receptor (nAChR) and acetylcholine binding protein (AChBP), termed a7-AChBP. To date, more than 70 crystal structures have been determined for AChBP in complex with ligands that occupy the orthosteric binding site. Here, we use an innovative screening strategy to discover molecular fragments that occupy allosteric binding sites. In combination with X-ray crystallography we determine a molecular blueprint of three different allosteric sites in a7-AChBP. Using electrophysiological recordings on the human a7 nAChR we demonstrate that each of the three sites is involved in allosteric modulation of the receptor. Our study contributes to understanding the sites of allosteric binding in ion channels. 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
Angiotensin II type receptor AT R is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance Although several anti-hypertensive drugs have been developed as AT R blockers ARBs the structural basis for AT R ligand-binding and regulation has remained elusive mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser we successfully determined the room-temperature crystal structure of the human AT R in complex with its selective antagonist ZD at resolution The AT R-ZD ... More
Angiotensin II type 1 receptor (AT1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT1R blockers (ARBs), the structural basis for AT1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT1R in complex with its selective antagonist ZD7155 at 2.9 � resolution. The AT1R-ZD7155 complex structure revealed key structural features of AT1R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT1R structure-function relationship and structure-based drug design. 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