888 Citations
Class B G-protein-coupled receptors GPCRs which consist of an extracellular domain ECD and a transmembrane domain TMD respond to secretin peptides to play a key part in hormonal homeostasis and are important therapeutic targets for a variety of diseases Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket Recently three structures of class B GPCRs in complex with peptide ligands have been solved These structures provide essential ... More
Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases1,2,3,4,5,6,7,8. Previous work9,10,11 has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved12,13,14. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR–NNC0640–mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation—which requires conformational changes of the stalk, first extracellular loop and TMD—that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs. Less
Protein crystallography has significantly advanced in recent years with in situ data collection in which crystals are placed in the X-ray beam within their growth medium being a major point of focus In situ methods eliminate the need to harvest crystals a previously unavoidable drawback particularly for often small membrane-protein crystals Here we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature C and under cryogenic conditions The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive easy to make and compatible with automated ... More
Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20–25°C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in ≤90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min. Less
Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle and are thus drug targets for the treatment of insomnia Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source we determined structures of human orexin receptor in complex with the subtype-selective antagonist EMPA N-ethyl- - -methoxy-pyridin- -yl - toluene- -sulfonyl -amino -N-pyridin- -ylmethyl-acetamide at - and - resolution In comparison with the non-subtype-selective antagonist suvorexant EMPA contacted fewer residues through hydrogen bonds at the orthosteric site explaining the faster dissociation rate Comparisons among these OX R structures in complex with selective antagonists and ... More
Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX2R structures in complex with selective antagonists and previously determined OX1R/OX2R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX2R. The importance of these residues for binding selectivity to OX2R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors. Less
A licensed vaccine for respiratory syncytial virus RSV is unavailable and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants Recently isolated antibodies C and D are substantially more potent than palivizumab and a derivative of D is in clinical trials Here we show that unlike D C preferentially neutralizes subtype A viruses The crystal structure of C bound to the RSV fusion F protein reveals that the overall binding mode of C is similar to that of D but their angles of approach are substantially different Mutagenesis and virological studies demonstrate that RSV F residue is largely ... More
A licensed vaccine for respiratory syncytial virus (RSV) is unavailable, and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants. Recently isolated antibodies 5C4 and D25 are substantially more potent than palivizumab, and a derivative of D25 is in clinical trials. Here we show that unlike D25, 5C4 preferentially neutralizes subtype A viruses. The crystal structure of 5C4 bound to the RSV fusion (F) protein reveals that the overall binding mode of 5C4 is similar to that of D25, but their angles of approach are substantially different. Mutagenesis and virological studies demonstrate that RSV F residue 201 is largely responsible for the subtype specificity of 5C4. These results improve our understanding of subtype-specific immunity and the neutralization breadth requirements of next-generation antibodies, and thereby contribute to the design of broadly protective RSV vaccines. Less
A major hurdle in membrane protein crystallography is generating crystals diffracting sufficiently for structure determination This is often attributed not only to the difficulty of obtaining functionally active protein in mg amounts but also to the intrinsic flexibility of its multiple conformations The cocrystallization of membrane proteins with antibody fragments has been reported as an effective approach to improve the diffraction quality of membrane protein crystals by limiting the intrinsic flexibility Isolating suitable antibody fragments recognizing a single conformation of a native membrane protein is not a straightforward task However by a systematic screening approach the time to obtain suitable ... More
A major hurdle in membrane protein crystallography is generating crystals diffracting sufficiently for structure determination. This is often attributed not only to the difficulty of obtaining functionally active protein in mg amounts but also to the intrinsic flexibility of its multiple conformations. The cocrystallization of membrane proteins with antibody fragments has been reported as an effective approach to improve the diffraction quality of membrane protein crystals by limiting the intrinsic flexibility. Isolating suitable antibody fragments recognizing a single conformation of a native membrane protein is not a straightforward task. However, by a systematic screening approach, the time to obtain suitable antibody fragments and consequently the chance of obtaining diffracting crystals can be reduced. In this chapter, we describe a protocol for the generation of Fab fragments recognizing the native conformation of a major facilitator superfamily (MFS)-type MDR transporter MdfA from Escherichia coli. We confirmed that the use of Fab fragments was efficient for stabilization of MdfA and improvement of its crystallization properties. Less
Membrane proteins constitute an integral part of biomembrane and play key roles in fundamental biological and physiological processes such as metabolism signaling and ion homeostasis About half of all drug targets are membrane proteins Elucidation of three-dimensional structures of membrane proteins by X-ray crystallography can provide mechanistic insights for their cellular activity and reveal atomic resolution of architectural details for rational design of novel therapeutics However the pace of IMP crystallography has been relatively slow due to great challenges in crystallization Lipid cubic phase LCP has proven to be promising in tackling the crystallization problem by providing a membrane-alike environment ... More
Membrane proteins constitute an integral part of biomembrane and play key roles in fundamental biological and physiological processes such as metabolism, signaling, and ion homeostasis. About half of all drug targets are membrane proteins. Elucidation of three-dimensional structures of membrane proteins by X-ray crystallography can provide mechanistic insights for their cellular activity and reveal atomic resolution of architectural details for rational design of novel therapeutics. However, the pace of IMP crystallography has been relatively slow due to great challenges in crystallization. Lipid cubic phase (LCP) has proven to be promising in tackling the crystallization problem by providing a membrane-alike environment. Its bilayer is composed of neutral lipids, such as monoacylglycerols, and can accommodate a substantial amount of native lipids such as phospholipids and cholesterol. Thus, the structure and composition of LCP mimic biomembranes and therefore offer a native-like environment for membrane proteins, which is favorable for functionality and crystallization. Here, the principles for LCP formation, membrane protein reconstitution, and crystallization process are described. The successful application of LCP crystallization for a wide range of membrane proteins including receptors, complexes, transporters, channels, enzymes, membrane protein insertion chaperons, and outer membrane �-barrels is summarized. General methods and protocols for this method are also described. Less
Ferredoxin-dependent bilin reductases FDBRs are a class of enzymes reducing the heme metabolite biliverdin IX BV to form open-chain tetrapyrroles used for light-perception and light-harvesting in photosynthetic organisms Thus far seven FDBR families have been identified each catalysing a distinct reaction and either transferring two or four electrons from ferredoxin onto the substrate The newest addition to the family is PcyX originally identified from metagenomics data derived from phage Phylogenetically PcyA is the closest relative catalysing the reduction of BV to phycocyanobilin PcyX however converts the same substrate to phycoerythrobilin resembling the reaction catalysed by cyanophage PebS Within this study ... More
Ferredoxin-dependent bilin reductases (FDBRs) are a class of enzymes reducing the heme metabolite biliverdin IXα (BV) to form open-chain tetrapyrroles used for light-perception and light-harvesting in photosynthetic organisms. Thus far, seven FDBR families have been identified, each catalysing a distinct reaction and either transferring two or four electrons from ferredoxin onto the substrate. The newest addition to the family is PcyX, originally identified from metagenomics data derived from phage. Phylogenetically, PcyA is the closest relative catalysing the reduction of BV to phycocyanobilin. PcyX, however, converts the same substrate to phycoerythrobilin, resembling the reaction catalysed by cyanophage PebS. Within this study, we aimed at understanding the evolution of catalytic activities within FDBRs using PcyX as an example. Additional members of the PcyX clade and a remote member of the PcyA family were investigated to gain insights into catalysis. Biochemical data in combination with the PcyX crystal structure revealed that a conserved aspartate-histidine pair is critical for activity. Interestingly, the same residues are part of a catalytic Asp-His-Glu triad in PcyA, including an additional Glu. While this Glu residue is replaced by Asp in PcyX, it is not involved in catalysis. Substitution back to a Glu failed to convert PcyX to a PcyA. Therefore, the change in regiospecificity is not only caused by individual catalytic amino acid residues. Rather the combination of the architecture of the active site with the positioning of the substrate triggers specific proton transfer yielding the individual phycobilin products. Less
Peroxide sensing is essential for bacterial survival during aerobic metabolism and host infection Peroxide stress regulators PerRs are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites PerR binding to gene promoters is controlled by the presence of iron in the regulatory site and iron-catalyzed oxidation of PerR by H O leads to the dissociation of PerR from DNA In addition to a regulatory metal most PerRs require a structural metal for proper dimeric assembly We present here a structural and functional characterization of the PerR from the pathogenic spirochete Leptospira interrogans a rare example ... More
Peroxide sensing is essential for bacterial survival during aerobic metabolism and host infection. Peroxide stress regulators (PerRs) are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites. PerR binding to gene promoters is controlled by the presence of iron in the regulatory site, and iron-catalyzed oxidation of PerR by H2O2 leads to the dissociation of PerR from DNA. In addition to a regulatory metal, most PerRs require a structural metal for proper dimeric assembly. We present here a structural and functional characterization of the PerR from the pathogenic spirochete Leptospira interrogans, a rare example of PerR lacking a structural metal-binding site. In vivo studies showed that the leptospiral PerR belongs to the peroxide stimulon in pathogenic species and is involved in controlling resistance to peroxide. Moreover, a perR mutant had decreased fitness in other host-related stress conditions, including at 37 °C or in the presence of superoxide anion. In vitro, leptospiral PerR could bind to the perR promoter region in a metal-dependent manner. The crystal structure of the leptospiral PerR revealed an asymmetric homodimer, with one monomer displaying complete regulatory metal coordination in the characteristic caliper-like DNA-binding conformation and the second monomer exhibiting disrupted regulatory metal coordination in an open non-DNA–binding conformation. This structure showed that leptospiral PerR assembles into a dimer in which a metal-induced conformational switch can occur independently in the two monomers. Our study demonstrates that structural metal binding is not compulsory for PerR dimeric assembly and for regulating peroxide stress. Less
Antimicrobial peptides as part of the mammalian innate immune system target and remove major bacterial pathogens often through irreversible damage of their cellular membranes To explore the mechanism by which the important cathelicidin peptide LL- of the human innate immune system interacts with membranes we performed biochemical biophysical and structural studies The crystal structure of LL- displays dimers of anti-parallel helices and the formation of amphipathic surfaces Peptide-detergent interactions introduce remodeling of this structure after occupation of defined hydrophobic sites at the dimer interface Furthermore hydrophobic nests are shaped between dimer structures providing another scaffold enclosing detergents Both scaffolds underline ... More
Antimicrobial peptides as part of the mammalian innate immune system target and remove major bacterial pathogens, often through irreversible damage of their cellular membranes. To explore the mechanism by which the important cathelicidin peptide LL-37 of the human innate immune system interacts with membranes, we performed biochemical, biophysical and structural studies. The crystal structure of LL-37 displays dimers of anti-parallel helices and the formation of amphipathic surfaces. Peptide-detergent interactions introduce remodeling of this structure after occupation of defined hydrophobic sites at the dimer interface. Furthermore, hydrophobic nests are shaped between dimer structures providing another scaffold enclosing detergents. Both scaffolds underline the potential of LL-37 to form defined peptide-lipid complexes in vivo. After adopting the activated peptide conformation LL-37 can polymerize and selectively extract bacterial lipids whereby the membrane is destabilized. The supramolecular fibril-like architectures formed in crystals can be reproduced in a peptide-lipid system after nanogold-labelled LL-37 interacted with lipid vesicles as followed by electron microscopy. We suggest that these supramolecular structures represent the LL-37-membrane active state. Collectively, our study provides new insights into the fascinating plasticity of LL-37 demonstrated at atomic resolution and opens the venue for LL-37-based molecules as novel antibiotics. Less
The present work contributes to the eld of process development and optimization for the manufacturing of recombinant proteins Recombinant proteins are biological macromolecules which are produced using genetically modi ed organisms GMOs In addition to the food industry and for the synthesis of organic compounds industrially produced proteins are mainly used in the eld of medicine There they make a decisive contribution to the diagnosis prevention and treatment of various human diseases The production of protein-based drugs biopharmaceuticals and their provision in a stable and bioavailable dosage form formulation are a challenge due to the size and complexity of these ... More
The present work contributes to the eld of process development and optimization for the manufacturing of recombinant proteins. Recombinant proteins are biological macromolecules, which are produced using genetically modi ed organisms (GMOs). In addition to the food industry and for the synthesis of organic compounds, industrially produced proteins are mainly used in the eld of medicine. There, they make a decisive contribution to the diagnosis, prevention and treatment of various human diseases. The production of protein-based drugs (biopharmaceuticals) and their provision in a stable and bioavailable dosage form (formulation) are a challenge due to the size and complexity of these molecules. On the way to the nal drug product, the target protein undergoes a long and complex processing chain consisting of GMO cultivation (upstream processing), puri - cation operations (downstream processing) and formulation steps. The reaction medium for the entire manufacturing process is mainly based on aqueous solutions. Some of the process steps used require extreme conditions such as unphysiological salt concentrations (e.g. in chromatographic puri cation) or acidic pH values (e.g. virus inactivation); all of which are potential stress factors for the protein integrity. Resulting irreversible changes in protein structure and physical instabilities, e.g. aggregation can a ect both drug safety and ecacy. In order to ensure the product quality during the entire manufacturing process, the development of strategies for the stabilization of proteins in aqueous solutions is of paramount importance. From an economic perspective, short development times are desirable in order to reduce the `time to market' of a drug product. Standardized procedures are of great interest for shortening the development times of stabilized protein products and are therefore the subject of this dissertation. Less
CLYBL encodes a ubiquitously expressed mitochondrial enzyme conserved across all vertebrates whose cellular activity and pathway assignment are unknown Its homozygous loss is tolerated in seemingly healthy individuals with reduced circulating B levels being the only and consistent phenotype reported to date Here by combining enzymology structural biology and activity-based metabolomics we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells Cells lacking CLYBL accumulate citramalyl-CoA an intermediate in the C -dicarboxylate metabolic pathway that includes itaconate a recently identified human antimicrobial metabolite and immunomodulator We report that CLYBL loss leads to a cell autonomous defect in the ... More
CLYBL encodes a ubiquitously expressed mitochondrial enzyme, conserved across all vertebrates, whose cellular activity and pathway assignment are unknown. Its homozygous loss is tolerated in seemingly healthy individuals, with reduced circulating B12 levels being the only and consistent phenotype reported to date. Here, by combining enzymology, structural biology and activity-based metabolomics we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells. Cells lacking CLYBL accumulate citramalyl-CoA, an intermediate in the C5-dicarboxylate metabolic pathway that includes itaconate, a recently identified human antimicrobial metabolite and immunomodulator. We report that CLYBL loss leads to a cell autonomous defect in the mitochondrial B12 metabolism and that itaconyl-CoA is a cofactor-inactivating, substrate-analogue inhibitor of the mitochondrial B12-dependent methylmalonyl-CoA mutase (MUT). Our work de-orphans the function of human CLYBL and reveals that a consequence of exposure to the immunomodulatory metabolite itaconate is B12 inactivation. Less
Deubiquitinating enzymes DUBs have garnered significant attention as drug targets in the last years The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins especially proteins that are challenging to directly target but which are stabilized by DUB family members Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools Most reported DUB inhibitors however are polypharmacological agents possessing weak micromolar potency toward their primary target limiting their utility in target validation and mechanism studies Due to a lack of high-resolution DUB small-molecule ligand complex structures no structure-guided ... More
Deubiquitinating enzymes (DUBs) have garnered significant attention as drug targets in the last 5–10 years. The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins, especially proteins that are challenging to directly target but which are stabilized by DUB family members. Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools. Most reported DUB inhibitors, however, are polypharmacological agents possessing weak (micromolar) potency toward their primary target, limiting their utility in target validation and mechanism studies. Due to a lack of high-resolution DUB⋅small-molecule ligand complex structures, no structure-guided optimization efforts have been reported for a mammalian DUB. Here, we report a small-molecule⋅ubiquitin-specific protease (USP) family DUB co-structure and rapid design of potent and selective inhibitors of USP7 guided by the structure. Interestingly, the compounds are non-covalent active-site inhibitors. Less
Multiprotein assemblies play major roles in most pathways involved in cell regulation and signaling Weak binary interactions are transformed co-operatively into very specific systems which achieve sensitivity specificity and temporal control Due to the complexity and transience of these regulatory and signaling systems a combination of in vivo cell biochemical biophysical and structural approaches is needed to investigate their structures and dynamics Here we describe the architecture and spatial organisation of the complexes mediating Non-Homologous End Joining NHEJ one of the two major pathways involved in DNA double-strand break repair Our example illustrates the experimental challenges and conceptual questions that ... More
Multiprotein assemblies play major roles in most pathways involved in cell regulation and signaling. Weak binary interactions are transformed co-operatively into very specific systems, which achieve sensitivity, specificity and temporal control. Due to the complexity and transience of these regulatory and signaling systems, a combination of in vivo, cell, biochemical, biophysical, and structural approaches is needed to investigate their structures and dynamics. Here we describe the architecture and spatial organisation of the complexes mediating Non-Homologous End Joining (NHEJ), one of the two major pathways involved in DNA double-strand break repair. Our example illustrates the experimental challenges and conceptual questions that are raised by studying such complex systems. We discuss the potential of using knowledge of the spatial and temporal organization of multiprotein systems not only to give insights into the mechanisms of pathway regulation but also to help in the design of chemical tools and ultimately new therapeutic agents. Less
Solution stability attributes are one of the key parameters within the production and launching phase of new biopharmaceuticals Instabilities of active biological compounds can reduce the yield of biopharmaceutical productions and may induce undesired reactions in patients such as immunogenic rejections Protein solution stability thus needs to be engineered and monitored throughout production and storage In contrast to the gold standard of long-term storage experiments applied in industry novel experimental and in silico molecular dynamics tools for predicting protein solution stability can be applied within several minutes or hours Here a rheological approach in combination with molecular dynamics simulations are ... More
Solution stability attributes are one of the key parameters within the production and launching phase of new biopharmaceuticals. Instabilities of active biological compounds can reduce the yield of biopharmaceutical productions, and may induce undesired reactions in patients, such as immunogenic rejections. Protein solution stability thus needs to be engineered and monitored throughout production and storage. In contrast to the gold standard of long-term storage experiments applied in industry, novel experimental and in silico molecular dynamics tools for predicting protein solution stability can be applied within several minutes or hours. Here, a rheological approach in combination with molecular dynamics simulations are presented, for determining and predicting long-term phase behavior of highly concentrated protein solutions. A diversity of liquid phase conditions, including salt type, ionic strength, pH and protein concentration are tested in a Glutathione-S-Transferase (GST) case study, in combination with the enzyme with and without solubility-enhancing Cherry-Tag™. The rheological characterization of GST and Cherry-GST solutions enabled a fast and efficient prediction of protein instabilities without the need of long-term protein phase diagrams. Finally, the strong solubility enhancing properties of the Cherry-Tag™ were revealed by investigating protein surface properties in MD simulations. The tag highly altered the overall surface charge and hydrophobicity of GST, making it less accessible to alteration by the chemical surrounding. Less
The purpose of this work was to evaluate the impact of polymer s on the dissolution rate supersaturation and precipitation of indomethacin amorphous solid dispersions ASD and to understand the link between precipitate characteristics and redissolution kinetics The crystalline and amorphous solubilities of indomethacin were determined in the absence and presence of hydroxypropylmethyl cellulose HPMC and or Eudragit EPO to establish relevant phase boundaries At acidic pH HPMC could maintain supersaturation of the drug by effectively inhibiting solution crystallization while EPO increased both the crystalline and amorphous solubility of the drug but did not inhibit crystallization The HPMC dispersion dissolved ... More
The purpose of this work was to evaluate the impact of polymer(s) on the dissolution rate, supersaturation and
precipitation of indomethacin amorphous solid dispersions (ASD), and to understand the link between precipitate characteristics and redissolution kinetics. The crystalline and amorphous solubilities of indomethacin
were determined in the absence and presence of hydroxypropylmethyl cellulose (HPMC) and/or Eudragit � EPO
to establish relevant phase boundaries. At acidic pH, HPMC could maintain supersaturation of the drug by
effectively inhibiting solution crystallization while EPO increased both the crystalline and amorphous solubility
of the drug, but did not inhibit crystallization. The HPMC dispersion dissolved relatively slowly without undergoing crystallization while the supersaturation generated by rapid dissolution of the EPO ASD was short-lived
due to crystallization. The crystals thus generated underwent rapid redissolution upon pH increase, dissolving
faster than the reference crystalline material, and at a comparable rate to the amorphous HPMC dispersion. A
ternary dispersion containing both EPO and HPMC dissolved rapidly, generating an apparent drug concentration
that exceeded the amorphous solubility of indomethacin, leading to the formation of a new nanosized droplet
phase. These nanodroplets dissolved virtually immediately when the pH was increased. In conclusion, the
concentration-time profiles achieved from indomethacin ASD dissolution are a complex interplay of drug release
rate, precipitation kinetics and outcome, and precipitate redissolution rate, whereby each of these processes is
highly dependent on the polymer(s) employed in the formulation. Less
precipitation of indomethacin amorphous solid dispersions (ASD), and to understand the link between precipitate characteristics and redissolution kinetics. The crystalline and amorphous solubilities of indomethacin
were determined in the absence and presence of hydroxypropylmethyl cellulose (HPMC) and/or Eudragit � EPO
to establish relevant phase boundaries. At acidic pH, HPMC could maintain supersaturation of the drug by
effectively inhibiting solution crystallization while EPO increased both the crystalline and amorphous solubility
of the drug, but did not inhibit crystallization. The HPMC dispersion dissolved relatively slowly without undergoing crystallization while the supersaturation generated by rapid dissolution of the EPO ASD was short-lived
due to crystallization. The crystals thus generated underwent rapid redissolution upon pH increase, dissolving
faster than the reference crystalline material, and at a comparable rate to the amorphous HPMC dispersion. A
ternary dispersion containing both EPO and HPMC dissolved rapidly, generating an apparent drug concentration
that exceeded the amorphous solubility of indomethacin, leading to the formation of a new nanosized droplet
phase. These nanodroplets dissolved virtually immediately when the pH was increased. In conclusion, the
concentration-time profiles achieved from indomethacin ASD dissolution are a complex interplay of drug release
rate, precipitation kinetics and outcome, and precipitate redissolution rate, whereby each of these processes is
highly dependent on the polymer(s) employed in the formulation. Less
Automated sample changers are now standard equipment for modern macromolecular crystallography synchrotron beamlines Nevertheless most are only compatible with a single type of sample holder and puck Recent work aimed at reducing sample-handling efforts and crystal-alignment times at beamlines has resulted in a new generation of compact and precise sample holders for cryocrystallography miniSPINE and NewPin see the companion paper by Papp et al Acta Cryst D With full data collection now possible within seconds at most advanced beamlines and future fourth-generation synchrotron sources promising to extract data in a few tens of milliseconds the time taken to mount and ... More
Automated sample changers are now standard equipment for modern macromolecular crystallography synchrotron beamlines. Nevertheless, most are only compatible with a single type of sample holder and puck. Recent work aimed at reducing sample-handling efforts and crystal-alignment times at beamlines has resulted in a new generation of compact and precise sample holders for cryocrystallography: miniSPINE and NewPin [see the companion paper by Papp et al. (2017, Acta Cryst., D73, 829�840)]. With full data collection now possible within seconds at most advanced beamlines, and future fourth-generation synchrotron sources promising to extract data in a few tens of milliseconds, the time taken to mount and centre a sample is rate-limiting. In this context, a versatile and fast sample changer, FlexED8, has been developed that is compatible with the highly successful SPINE sample holder and with the miniSPINE and NewPin sample holders. Based on a six-axis industrial robot, FlexED8 is equipped with a tool changer and includes a novel open sample-storage dewar with a built-in ice-filtering system. With seven versatile puck slots, it can hold up to 112 SPINE sample holders in uni-pucks, or 252 miniSPINE or NewPin sample holders, with 36 samples per puck. Additionally, a double gripper, compatible with the SPINE sample holders and uni-pucks, allows a reduction in the sample-exchange time from 40 s, the typical time with a standard single gripper, to less than 5 s. Computer vision-based sample-transfer monitoring, sophisticated error handling and automatic error-recovery procedures ensure high reliability. The FlexED8 sample changer has been successfully tested under real conditions on a beamline. Less
Generation of an electrochemical proton gradient is the first step of cell bioenergetics In prokaryotes the gradient is created by outward membrane protein proton pumps Inward plasma membrane native proton pumps are yet unknown We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins They are inward proton pumps as we demonstrate in model membrane systems Escherichia coli cells human embryonic kidney cells neuroblastoma cells and rat hippocampal neuronal cells We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina NsXeR and suggest a mechanism of inward proton pumping ... More
Generation of an electrochemical proton gradient is the first step of cell bioenergetics. In prokaryotes, the gradient is created by outward membrane protein proton pumps. Inward plasma membrane native proton pumps are yet unknown. We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins. They are inward proton pumps as we demonstrate in model membrane systems, Escherichia coli cells, human embryonic kidney cells, neuroblastoma cells, and rat hippocampal neuronal cells. We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina (NsXeR) and suggest a mechanism of inward proton pumping. We demonstrate that the NsXeR is a powerful pump, which is able to elicit action potentials in rat hippocampal neuronal cells up to their maximal intrinsic firing frequency. Hence, inwardly directed proton pumps are suitable for light-induced remote control of neurons, and they are an alternative to the well-known cation-selective channelrhodopsins. Less
Tepsin is currently the only accessory trafficking protein identified in adaptor-related protein AP coated vesicles originating at the trans-Golgi network TGN The molecular basis for interactions between AP subunits and motifs in the tepsin C-terminus have been characterized but the biological role of tepsin remains unknown We determined X-ray crystal structures of the tepsin ENTH and VHS ENTH-like domains Our data reveal unexpected structural features that suggest key functional differences between these and similar domains in other trafficking proteins The tepsin ENTH domain lacks helix helix and a lipid binding pocket found in epsin These results explain why tepsin requires ... More
Tepsin is currently the only accessory trafficking protein identified in adaptor-related protein 4 (AP4) coated vesicles originating at the trans-Golgi network (TGN). The molecular basis for interactions between AP4 subunits and motifs in the tepsin C-terminus have been characterized, but the biological role of tepsin remains unknown. We determined X-ray crystal structures of the tepsin ENTH and VHS/ENTH-like domains. Our data reveal unexpected structural features that suggest key functional differences between these and similar domains in other trafficking proteins. The tepsin ENTH domain lacks helix0, helix8, and a lipid binding pocket found in epsin1/2/3. These results explain why tepsin requires AP4 for its membrane recruitment and further suggest ENTH domains cannot be defined solely as lipid binding modules. The VHS domain lacks helix8 and thus contains fewer helices than other VHS domains. Structural data explain biochemical and biophysical evidence that tepsin VHS does not mediate known VHS functions, including recognition of dileucine-based cargo motifs or ubiquitin. Structural comparisons indicate the domains are very similar to each other, and phylogenetic analysis reveals their evolutionary pattern within the domain superfamily. Phylogenetics and comparative genomics further show tepsin within a monophyletic clade that diverged away from epsins early in evolutionary history (~1,500 million years ago). Together, these data provide the first detailed molecular view of tepsin and suggest tepsin structure and function diverged away from other epsins. More broadly, these data highlight the challenges inherent in classifying and understanding protein function based only on sequence and structure. Less
American trypanosomiasis or Chagas Disease is caused by the protozoan Trypanosoma cruzi The treatment is based on drugs that present serious side effects and are inefficient against the chronic phase of the disease The research in new metabolic targets may lead to the development of safer and more efficient drugs Malic enzyme ME is considered a promising target due to its ability to produce NADPH reducing agent that participates in a number of biosynthetic pathways and in detoxication of reactive oxygen species produced from endogenous metabolic reactions or from exogenous immune insults generated by mammalian host cells T cruzi expresses ... More
American trypanosomiasis, or Chagas Disease, is caused by the protozoan
Trypanosoma cruzi. The treatment is based on drugs that present serious side effects
and are inefficient against the chronic phase of the disease. The research in new
metabolic targets may lead to the development of safer and more efficient drugs. Malic
enzyme (ME) is considered a promising target due to its ability to produce NADPH,
reducing agent that participates in a number of biosynthetic pathways and in
detoxication of reactive oxygen species produced from endogenous metabolic
reactions or from exogenous immune insults generated by mammalian host cells. T.
cruzi expresses two isoforms of the enzyme, one mitochondrial (TcMEm) and other
cytosolic (TcMEc). In this thesis, the first characterized inhibitors for these enzymes
are reported, identified by a biochemical high-throughput screening (HTS) assay
against a library of 30 thousand compounds. IC50 values of 262 molecules were
determined for both TcMEs as well as for three human ME isoforms, with the inhibitors
clustered into six groups according to their chemical similarity. The most potent hits
belonged to group ATR3, comprised of 218 aryl-sulfonamides that specifically target
TcMEc. Moreover, several selected inhibitors of both TcMEs showed trypanocidal
effect against the replicative forms of T. cruzi. Also, in this work the first crystallographic
structures of both TcMEs are shown, revealing different features from the human
homologues. In addition, the complex of TcMEc with 6 different ATR3 molecules were
determined, unravelling the inhibition site at the dimer interface. In conclusion, the HTS
results demonstrate that TcMEs are druggable, being modulated by small hydrophobic
molecules, which is an essential requirement for a drug target. Moreover, the identified
compounds can be used as chemical probes in the validation of the enzymes. The
enzyme structures are important tools which may be employed to design new inhibitors
or analogues. Less
Trypanosoma cruzi. The treatment is based on drugs that present serious side effects
and are inefficient against the chronic phase of the disease. The research in new
metabolic targets may lead to the development of safer and more efficient drugs. Malic
enzyme (ME) is considered a promising target due to its ability to produce NADPH,
reducing agent that participates in a number of biosynthetic pathways and in
detoxication of reactive oxygen species produced from endogenous metabolic
reactions or from exogenous immune insults generated by mammalian host cells. T.
cruzi expresses two isoforms of the enzyme, one mitochondrial (TcMEm) and other
cytosolic (TcMEc). In this thesis, the first characterized inhibitors for these enzymes
are reported, identified by a biochemical high-throughput screening (HTS) assay
against a library of 30 thousand compounds. IC50 values of 262 molecules were
determined for both TcMEs as well as for three human ME isoforms, with the inhibitors
clustered into six groups according to their chemical similarity. The most potent hits
belonged to group ATR3, comprised of 218 aryl-sulfonamides that specifically target
TcMEc. Moreover, several selected inhibitors of both TcMEs showed trypanocidal
effect against the replicative forms of T. cruzi. Also, in this work the first crystallographic
structures of both TcMEs are shown, revealing different features from the human
homologues. In addition, the complex of TcMEc with 6 different ATR3 molecules were
determined, unravelling the inhibition site at the dimer interface. In conclusion, the HTS
results demonstrate that TcMEs are druggable, being modulated by small hydrophobic
molecules, which is an essential requirement for a drug target. Moreover, the identified
compounds can be used as chemical probes in the validation of the enzymes. The
enzyme structures are important tools which may be employed to design new inhibitors
or analogues. Less
Various techniques have been used to detect crystallization in amorphous solid dispersions ASD However most of these techniques do not enable the detection of very low levels of crystallinity The aim ofthe current study was to compare the sensitivity of second harmonic generation SHG microscopy with powder X-ray diffraction XRPD in detecting the presence of crystals in low drug loading amorphous solid dispersions Amorphous solid dispersions of the poorly water soluble compounds flutamide FTM wt drug loading and ezetimibe EZT wt drug loading with hydroxypropyl methylcellulose acetate succinate HPMCAS were prepared by spray drying To induce crystallization samples were subsequently ... More
Various techniques have been used to detect crystallization in amorphous solid dispersions (ASD). However, most of these techniques do not enable the detection of very low levels of crystallinity (<1%). The
aim ofthe current study was to compare the sensitivity of second harmonic generation (SHG) microscopy
with powder X-ray diffraction (XRPD) in detecting the presence of crystals in low drug loading amorphous solid dispersions. Amorphous solid dispersions of the poorly water soluble compounds, flutamide
(FTM, 15 wt.% drug loading) and ezetimibe (EZT, 30 wt.% drug loading) with hydroxypropyl methylcellulose acetate succinate (HPMCAS) were prepared by spray drying. To induce crystallization, samples
were subsequently stored at 75% or 82% relative humidity (RH) and 40 ?C. Crystallization was monitored
by XRPD and by SHG microscopy. Solid state nuclear magnetic resonance spectroscopy (ssNMR) was
used to further investigate crystallinity in selected samples. For flutamide, crystals were detected by
SHG microscopy after 8 days of storage at 40 ?C/82% RH, whereas no evidence of crystallinity could be
observed by XRPD until 26 days. Correspondingly, for FTM samples stored at 40 ?C/75% RH, crystals were
detected after 11 days by SHG microscopy and after 53 days by XRPD. The evolution of crystals, that is
an increase in the number and size of crystalline regions, with time could be readily monitored from the
SHG images, and revealed the formation of needle-shaped crystals. Further investigation with scanning
electron microscopy indicated an unexpected mechanism of crystallization, whereby flutamide crystals
grew as needle-shaped projections from the surface of the spray dried particles. Similarly, EZT crystals
could be detected at earlier time points (15 days) with SHG microscopy relative to with XRPD (60 days).
Thus, SHG microscopy was found to be a highly sensitive method for detecting and monitoring the evolution of crystals formed from spray dried particles, providing much earlier detection of crystallinity than
XRPD under comparable run times. Less
aim ofthe current study was to compare the sensitivity of second harmonic generation (SHG) microscopy
with powder X-ray diffraction (XRPD) in detecting the presence of crystals in low drug loading amorphous solid dispersions. Amorphous solid dispersions of the poorly water soluble compounds, flutamide
(FTM, 15 wt.% drug loading) and ezetimibe (EZT, 30 wt.% drug loading) with hydroxypropyl methylcellulose acetate succinate (HPMCAS) were prepared by spray drying. To induce crystallization, samples
were subsequently stored at 75% or 82% relative humidity (RH) and 40 ?C. Crystallization was monitored
by XRPD and by SHG microscopy. Solid state nuclear magnetic resonance spectroscopy (ssNMR) was
used to further investigate crystallinity in selected samples. For flutamide, crystals were detected by
SHG microscopy after 8 days of storage at 40 ?C/82% RH, whereas no evidence of crystallinity could be
observed by XRPD until 26 days. Correspondingly, for FTM samples stored at 40 ?C/75% RH, crystals were
detected after 11 days by SHG microscopy and after 53 days by XRPD. The evolution of crystals, that is
an increase in the number and size of crystalline regions, with time could be readily monitored from the
SHG images, and revealed the formation of needle-shaped crystals. Further investigation with scanning
electron microscopy indicated an unexpected mechanism of crystallization, whereby flutamide crystals
grew as needle-shaped projections from the surface of the spray dried particles. Similarly, EZT crystals
could be detected at earlier time points (15 days) with SHG microscopy relative to with XRPD (60 days).
Thus, SHG microscopy was found to be a highly sensitive method for detecting and monitoring the evolution of crystals formed from spray dried particles, providing much earlier detection of crystallinity than
XRPD under comparable run times. Less
Pseudomonas aeruginosa a prevalent pathogen in nosocomial infections and a major burden in cystic fibrosis uses three interconnected quorum-sensing systems to coordinate virulence processes At variance with other Gram-negative bacteria one of these systems relies on -alkyl- H -quinolones Pseudomonas quinolone signal PQS and might hence be an attractive target for new anti-infective agents Here we report crystal structures of the N-terminal domain of anthranilate-CoA ligase PqsA the first enzyme of PQS biosynthesis in complex with anthraniloyl-AMP and with -fluoroanthraniloyl-AMP FABA-AMP at and resolution We find that PqsA belongs to an unrecognized subfamily of anthranilate-CoA ligases that recognize the amino ... More
Pseudomonas aeruginosa, a prevalent pathogen in nosocomial infections and a major burden in cystic fibrosis, uses three interconnected quorum-sensing systems to coordinate virulence processes. At variance with other Gram-negative bacteria, one of these systems relies on 2-alkyl-4(1H)-quinolones (Pseudomonas quinolone signal, PQS) and might hence be an attractive target for new anti-infective agents. Here we report crystal structures of the N-terminal domain of anthranilate-CoA ligase PqsA, the first enzyme of PQS biosynthesis, in complex with anthraniloyl-AMP and with 6-fluoroanthraniloyl-AMP (6FABA-AMP) at 1.4 and 1.7 Å resolution. We find that PqsA belongs to an unrecognized subfamily of anthranilate-CoA ligases that recognize the amino group of anthranilate through a water-mediated hydrogen bond. The complex with 6FABA-AMP explains why 6FABA, an inhibitor of PQS biosynthesis, is a good substrate of PqsA. Together, our data might pave a way to new pathoblockers in P. aeruginosa infections. Less
NADH and NADPH are redox coenzymes broadly required for energy metabolism biosynthesis and detoxification Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes a holistic understanding of the regulation and compartmentalization of NADH and NADPH-dependent pathways is lacking in part because of a lack of tools with which to investigate them in living cells We previously reported the use of the naturally occurring Lactobacillus brevis H O-forming NADH oxidase LbNOX as a genetic tool for manipulation of the NAD NADH ratio in human cells Here we present TPNOX triphosphopyridine nucleotide oxidase a rationally designed and engineered mutant ... More
NADH and NADPH are redox coenzymes broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH and NADPH-dependent pathways is lacking, in part because of a lack of tools with which to investigate them in living cells. We previously reported the use of the naturally occurring Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD+/NADH ratio in human cells. Here we present TPNOX (triphosphopyridine nucleotide oxidase), a rationally designed and engineered mutant of LbNOX that is strictly specific towards NADPH. We characterize the effects of TPNOX expression on cellular metabolism and use it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected. Less
DHHC enzymes catalyze palmitoylation a major post-translational modification that regulates a number of key cellular processes There are up to DHHCs in mammals and hundreds of substrate proteins that get palmitoylated However how DHHC enzymes engage with their substrates is still poorly understood There is currently no structural information about the interaction between any DHHC enzyme and protein substrates In this study we have investigated the structural and thermodynamic bases of interaction between the ankyrin repeat domain of Human DHHC ANK and Snap b We solved a high-resolution crystal structure of the complex between ANK and a peptide fragment of ... More
DHHC enzymes catalyze palmitoylation, a major post-translational modification that regulates a number of key cellular processes. There are up to 24 DHHCs in mammals and hundreds of substrate proteins that get palmitoylated. However, how DHHC enzymes engage with their substrates is still poorly understood. There is currently no structural information about the interaction between any DHHC enzyme and protein substrates. In this study we have investigated the structural and thermodynamic bases of interaction between the ankyrin repeat domain of Human DHHC17 (ANK17) and Snap25b. We solved a high-resolution crystal structure of the complex between ANK17 and a peptide fragment of Snap25b. Through structure-guided mutagenesis, we discovered key residues in DHHC17 that are critically important for interaction with Snap25b. We further extended our finding by showing that the same residues are also crucial for the interaction of DHHC17 with Huntingtin, one of its most relevant substrates. Less
Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases Given the importance of G protein-coupled receptors GPCRs as pharmaceutical targets there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs Here we present the - resolution structure of a complex between the human -hydroxytryptamine B -HT B receptor and an antibody Fab fragment bound to the extracellular side of the receptor determined by serial femtosecond crystallography with an X-ray free-electron laser The antibody binds to a D epitope of the receptor that includes all three extracellular loops The -HT ... More
Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å resolution structure of a complex between the human 5-hydroxytryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femtosecond crystallography with an X-ray free-electron laser. The antibody binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT2B receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies. Less
Vancomycin is known to bind to Zn II and can induce a zinc starvation response in bacteria Here we identify a novel polymerization of vancomycin dimers by structural analysis of vancomycin-Zn II crystals and fibre X-ray diffraction Bioassays indicate that this structure is associated with an increased antibiotic activity against bacterial strains possessing high level vancomycin resistance mediated by the reprogramming of peptidoglycan biosynthesis to use precursors terminating in D-Ala-D-Lac in place of D-Ala-D-Ala Polymerization occurs via interaction of Zn II with the N-terminal methylleucine group of vancomycin and we show that the activity of other glycopeptide antibiotics with this ... More
Vancomycin is known to bind to Zn(II) and can induce a zinc starvation response in bacteria. Here we identify a novel polymerization of vancomycin dimers by structural analysis of vancomycin-Zn(II) crystals and fibre X-ray diffraction. Bioassays indicate that this structure is associated with an increased antibiotic activity against bacterial strains possessing high level vancomycin resistance mediated by the reprogramming of peptidoglycan biosynthesis to use precursors terminating in D-Ala-D-Lac in place of D-Ala-D-Ala. Polymerization occurs via interaction of Zn(II) with the N-terminal methylleucine group of vancomycin, and we show that the activity of other glycopeptide antibiotics with this feature can also be similarly augmented by Zn(II). Construction and analysis of a model strain predominantly using D-Ala-D-Lac precursors for peptidoglycan biosynthesis during normal growth supports the hypothesis that Zn(II) mediated vancomycin polymerization enhances the binding affinity towards these precursors. Less
The cannabinoid receptor CB is the principal target of the psychoactive constituent of marijuana the partial agonist -tetrahydrocannabinol -THC Here we report two agonist-bound crystal structures of human CB in complex with a tetrahydrocannabinol AM and a hexahydrocannabinol AM at and resolution respectively The two CB agonist complexes reveal important conformational changes in the overall structure relative to the antagonist-bound state including a reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region In addition a twin toggle switch of Phe and Trp superscripts denote Ballesteros Weinstein numbering is experimentally observed ... More
The cannabinoid receptor 1 (CB1) is the principal target of the psychoactive constituent of marijuana, the partial agonist Δ9-tetrahydrocannabinol (Δ9-THC)1. Here we report two agonist-bound crystal structures of human CB1 in complex with a tetrahydrocannabinol (AM11542) and a hexahydrocannabinol (AM841) at 2.80 Å and 2.95 Å resolution, respectively. The two CB1–agonist complexes reveal important conformational changes in the overall structure, relative to the antagonist-bound state2, including a 53% reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region. In addition, a ‘twin toggle switch’ of Phe2003.36 and Trp3566.48 (superscripts denote Ballesteros–Weinstein numbering3) is experimentally observed and appears to be essential for receptor activation. The structures reveal important insights into the activation mechanism of CB1 and provide a molecular basis for predicting the binding modes of Δ9-THC, and endogenous and synthetic cannabinoids. The plasticity of the binding pocket of CB1 seems to be a common feature among certain class A G-protein-coupled receptors. These findings should inspire the design of chemically diverse ligands with distinct pharmacological properties. Less
Lipoproteins serve essential roles in the bacterial cell envelope The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes All are potential targets for the development of new antibiotics Here we report the crystal structure of the last enzyme in the pathway apolipoprotein N-acyltransferase Lnt responsible for adding a third acyl chain to the lipoprotein s invariant diacylated N-terminal cysteine Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved they are remarkably similar Both consist of a membrane domain on which sits a globular periplasmic domain The active site resides above the membrane interface where the ... More
Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein�s invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects. Less
High protein titers are gaining importance in biopharmaceutical industry A major challenge in the development of highly concentrated mAb solutions is their long-term stability and often incalculable viscosity The complexity of the molecule itself as well as the various molecular interactions make it difficult to describe their solution behavior To study the formulation stability long- and short-range interactions and the formation of complex network structures have to be taken into account For a better understanding of highly concentrated solutions we combined established and novel analytical tools to characterize the effect of solution properties on the stability of highly concentrated mAb ... More
High protein titers are gaining importance in biopharmaceutical industry. A major challenge in the development of highly concentrated mAb solutions is their long-term stability and often incalculable viscosity. The complexity of the molecule itself, as well as the various molecular interactions, make it difficult to describe their solution behavior. To study the formulation stability, long- and short-range interactions and the formation of complex network structures have to be taken into account. For a better understanding of highly concentrated solutions, we combined established and novel analytical tools to characterize the effect of solution properties on the stability of highly concentrated mAb formulations. In this study, monoclonal antibody solutions in a concentration range of 50–200 mg/ml at pH 5–9 with and without glycine, PEG4000, and Na2SO4 were analyzed. To determine the monomer content, analytical size-exclusion chromatography runs were performed. ζ-potential measurements were conducted to analyze the electrophoretic properties in different solutions. The melting and aggregation temperatures were determined with the help of fluorescence and static light scattering measurements. Additionally, rheological measurements were conducted to study the solution viscosity and viscoelastic behavior of the mAb solutions. The so-determined analytical parameters were scored and merged in an analytical toolbox. The resulting scoring was then successfully correlated with long-term storage (40 d of incubation) experiments. Our results indicate that the sensitivity of complex rheological measurements, in combination with the applied techniques, allows reliable statements to be made with respect to the effect of solution properties, such as protein concentration, ionic strength, and pH shift, on the strength of protein-protein interaction and solution colloidal stability. Less
The human glucagon receptor GCGR belongs to the class B G protein-coupled receptor GPCR family and plays a key role in glucose homeostasis and the pathophysiology of type diabetes Here we report the crystal structure of full-length GCGR containing both extracellular domain ECD and transmembrane domain TMD in an inactive conformation The two domains are connected by a -residue segment termed the stalk which adopts a -strand conformation instead of forming an -helix as observed in the previously solved structure of GCGR-TMD The first extracellular loop ECL exhibits a -hairpin conformation and interacts with the stalk to form a compact ... More
The human glucagon receptor (GCGR) belongs to the class B G protein-coupled receptor (GPCR) family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both extracellular domain (ECD) and transmembrane domain (TMD) in an inactive conformation. The two domains are connected by a 12-residue segment termed the ‘stalk’, which adopts a β-strand conformation, instead of forming an α-helix as observed in the previously solved structure of GCGR-TMD. The first extracellular loop (ECL1) exhibits a β-hairpin conformation and interacts with the stalk to form a compact β-sheet structure. Hydrogen/deuterium exchange, disulfide cross-linking and molecular dynamics studies suggest that the stalk and ECL1 play critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding about the signaling mechanisms of class B GPCRs. Less
Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis the most common chronic inflammatory disease in humans worldwide Its comorbidity with systemic diseases such as type diabetes oral cancers and cardiovascular diseases continues to generate considerable interest Surprisingly these two microorganisms do not ferment carbohydrates rather they use proteinaceous substrates as carbon and energy sources However the underlying biochemical mechanisms of their energy metabolism remain unknown Here we show that dipeptidyl peptidase DPP a central metabolic enzyme in these bacteria undergoes a conformational change upon peptide binding to distinguish substrates from end products It binds substrates through an ... More
Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme (“entropy reservoirs”). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors. Less
Bacteria and archaea use the CRISPR Cas system as an adaptive response against infection by foreign nucleic acids Owing to its remarkable flexibility this mechanism has been harnessed and adopted as a powerful tool for genome editing The CRISPR Cas system includes two classes that are subdivided into six types and subtypes according to conservation of the cas gene and loci organization Recently a new protein with endonuclease activity belonging to class type V has been identified This endonuclease termed Cpf in complex with a single CRISPR RNA crRNA is able to recognize and cleave a target DNA preceded by ... More
Bacteria and archaea use the CRISPR�Cas system as an adaptive response
against infection by foreign nucleic acids. Owing to its remarkable flexibility, this
mechanism has been harnessed and adopted as a powerful tool for genome
editing. The CRISPR�Cas system includes two classes that are subdivided into
six types and 19 subtypes according to conservation of the cas gene and loci
organization. Recently, a new protein with endonuclease activity belonging to
class 2 type V has been identified. This endonuclease, termed Cpf1, in complex
with a single CRISPR RNA (crRNA) is able to recognize and cleave a target
DNA preceded by a 50
-TTN-30 protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of
Cpf1, the crystallization of an active complex containing the full extent of the
crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding
Cpf1 from Francisella novicida was cloned, overexpressed and purified.
The crRNA was transcribed and purified in vitro. Finally, the ternary
FnCpf1�crRNA�DNA complex was assembled and purified by preparative
electrophoresis before crystallization. Crystals belonging to space group C2221,
with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 A� , were obtained and
subjected to preliminary diffraction experiments. Less
against infection by foreign nucleic acids. Owing to its remarkable flexibility, this
mechanism has been harnessed and adopted as a powerful tool for genome
editing. The CRISPR�Cas system includes two classes that are subdivided into
six types and 19 subtypes according to conservation of the cas gene and loci
organization. Recently, a new protein with endonuclease activity belonging to
class 2 type V has been identified. This endonuclease, termed Cpf1, in complex
with a single CRISPR RNA (crRNA) is able to recognize and cleave a target
DNA preceded by a 50
-TTN-30 protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of
Cpf1, the crystallization of an active complex containing the full extent of the
crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding
Cpf1 from Francisella novicida was cloned, overexpressed and purified.
The crRNA was transcribed and purified in vitro. Finally, the ternary
FnCpf1�crRNA�DNA complex was assembled and purified by preparative
electrophoresis before crystallization. Crystals belonging to space group C2221,
with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 A� , were obtained and
subjected to preliminary diffraction experiments. Less
Apelin receptor APJR is a key regulator of human cardiovascular function and is activated by two different endogenous peptide ligands apelin and Elabela each with different isoforms diversified by length and amino acid sequence Here we report the - resolution crystal structure of human APJR in complex with a designed -amino-acid apelin mimetic peptide agonist The structure reveals that the peptide agonist adopts a lactam constrained curved two-site ligand binding mode Combined with mutation analysis and molecular dynamics simulations with apelin- binding to the wild-type APJR this structure provides a mechanistic understanding of apelin recognition and binding specificity Comparison of ... More
Apelin receptor (APJR) is a key regulator of human cardiovascular function and is activated by two different endogenous peptide ligands, apelin and Elabela, each with different isoforms diversified by length and amino acid sequence. Here we report the 2.6-� resolution crystal structure of human APJR in complex with a designed 17-amino-acid apelin mimetic peptide agonist. The structure reveals that the peptide agonist adopts a lactam constrained curved two-site ligand binding mode. Combined with mutation analysis and molecular dynamics simulations with apelin-13 binding to the wild-type APJR, this structure provides a mechanistic understanding of apelin recognition and binding specificity. Comparison of this structure with that of other peptide receptors suggests that endogenous peptide ligands with a high degree of conformational flexibility may bind and modulate the receptors via a similar two-site binding mechanism. Less
Clinical studies indicate that partial agonists of the G-protein-coupled free fatty acid receptor GPR enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type diabetes mellitus Full allosteric agonists AgoPAMs of GPR bind to a site distinct from partial agonists and can provide additional efficacy We report the - crystal structure of human GPR hGPR in complex with both the partial agonist MK- and an AgoPAM which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle Comparison with an additional - structure of the hGPR MK- binary complex reveals an induced-fit conformational coupling between ... More
Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands. Less
Schistosoma mansoni is the parasite responsible for schistosomiasis a disease that affects about million people worldwide Currently both direct treatment and disease control initiatives rely on chemotherapy using a single drug praziquantel Concerns over the possibility of resistance developing to praziquantel have stimulated efforts to develop new drugs for the treatment of schistosomiasis Schistosomes do not have the de novo purine biosynthetic pathway and instead depend entirely on the purine salvage pathway to supply its need for purines The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis Adenylosuccinate lyase SmADSL is an ... More
Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 218 million people worldwide. Currently, both direct treatment and disease control initiatives rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, have stimulated efforts to develop new drugs for the treatment of schistosomiasis. Schistosomes do not have the de novo purine biosynthetic pathway, and instead depend entirely on the purine salvage pathway to supply its need for purines. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenylosuccinate lyase (SmADSL) is an enzyme in this pathway, which cleaves adenylosuccinate (ADS) into adenosine 5′-monophosphate (AMP) and fumarate. SmADSL kinetic characterization was performed by isothermal titration calorimetry (ITC) using both ADS and SAICAR as substrates. Structures of SmADSL in Apo form and in complex with AMP were elucidated by x-ray crystallography revealing a highly conserved tetrameric structure required for their function since the active sites are formed from residues of three different subunits. The active sites are also highly conserved between species and it is difficult to identify a potent species-specific inhibitor for the development of new therapeutic agents. In contrast, several mutagenesis studies have demonstrated the importance of dimeric interface residues in the stability of the quaternary structure of the enzyme. The lower conservation of these residues between SmADSL and human ADSL could be used to lead the development of anti-schistosomiasis drugs based on disruption of subunit interfaces. These structures and kinetics data add another layer of information to Schistosoma mansoni purine salvage pathway. Less
PP C phosphatases control biological processes including stress responses development and cell division in all kingdoms of life Diverse regulatory domains adapt PP C phosphatases to specific functions but how these domains control phosphatase activity was unknown We present structures representing active and inactive states of the PP C phosphatase SpoIIE from Bacillus subtilis Based on structural analyses and genetic and biochemical experiments we identify an a-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor Our analysis indicates that this switch is widely conserved among PP C family members serving as a platform ... More
PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an a-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activity in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site. Less
The Smoothened receptor SMO belongs to the Class Frizzled of the G protein-coupled receptor GPCR superfamily constituting a key component of the Hedgehog signalling pathway Here we report the crystal structure of the multi-domain human SMO bound and stabilized by a designed tool ligand TC using an X-ray free-electron laser source at The structure reveals a precise arrangement of three distinct domains a seven-transmembrane helices domain TMD a hinge domain HD and an intact extracellular cysteine-rich domain CRD This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation By combining the structural data ... More
The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs. Less
The glucagon-like peptide- receptor GLP- R and the glucagon receptor GCGR are members of the secretin-like class B family of G-protein-coupled receptors GPCRs and have opposing physiological roles in insulin release and glucose homeostasis The treatment of type diabetes requires positive modulation of GLP- R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner Here we report crystal structures of the human GLP- R transmembrane domain in complex with two different negative allosteric modulators PF- and NNC at and resolution respectively The structures reveal a common binding pocket for negative allosteric modulators present in both GLP- R ... More
The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are members of the secretin-like class B family of G-protein-coupled receptors (GPCRs) and have opposing physiological roles in insulin release and glucose homeostasis1. The treatment of type 2 diabetes requires positive modulation of GLP-1R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner2. Here we report crystal structures of the human GLP-1R transmembrane domain in complex with two different negative allosteric modulators, PF-06372222 and NNC0640, at 2.7 and 3.0 Å resolution, respectively. The structures reveal a common binding pocket for negative allosteric modulators, present in both GLP-1R and GCGR3 and located outside helices V–VII near the intracellular half of the receptor. The receptor is in an inactive conformation with compounds that restrict movement of the intracellular tip of helix VI, a movement that is generally associated with activation mechanisms in class A GPCRs4,5,6. Molecular modelling and mutagenesis studies indicate that agonist positive allosteric modulators target the same general region, but in a distinct sub-pocket at the interface between helices V and VI, which may facilitate the formation of an intracellular binding site that enhances G-protein coupling. Less
We describe a fast easy and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide single-wavelength anomalous diffraction I-SAD The potential universality of the method is based on a common feature of membrane proteins the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins including a human G protein coupled receptor GPCR and we show that I-SAD can be applied using data collection strategies based on either standard or ... More
We describe a fast, easy, and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide–single-wavelength anomalous diffraction (I-SAD). The potential universality of the method is based on a common feature of membrane proteins—the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts. We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins, including a human G protein–coupled receptor (GPCR), and we show that I-SAD can be applied using data collection strategies based on either standard or serial x-ray crystallography techniques. Less
Lipidic cubic phase LCP has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment Application of this material to a wide variety of membrane proteins however is hindered due to a limited number of available host lipids mostly monoacylglycerols MAGs Here we designed synthesized and characterized a series of chemically stable lipids resistant to hydrolysis with properties complementary to the widely used MAGs In order to assess their potential to serve as host lipids for crystallization we characterized the phase properties and lattice parameters of mesophases made of ... More
Lipidic cubic phase (LCP) has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment. Application of this material to a wide variety of membrane proteins, however, is hindered due to a limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed, synthesized and characterized a series of chemically stable lipids resistant to hydrolysis, with properties complementary to the widely used MAGs. In order to assess their potential to serve as host lipids for crystallization, we characterized the phase properties and lattice parameters of mesophases made of two most promising lipids at a variety of different conditions by polarized light microscopy and small-angle X-ray scattering. Both lipids showed remarkable chemical stability and an extended LCP region in the phase diagram covering a wide range of temperatures down to 4 °C. One of these lipids has been used for crystallization and structure determination of a prototypical membrane protein bacteriorhodopsin at 4 °C and 20 °C. Less
Neisserial heparin-binding antigen NHBA is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero the crystal structures of two fragment antigen-binding domains Fabs isolated from human monoclonal antibodies targeting NHBA were determined Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs these structures provide broad insights into the NHBA epitopes recognized by the human immune system As expected these Fabs also show remarkable structural conservation ... More
Neisserial heparin-binding antigen (NHBA) is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero. As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero, the crystal structures of two fragment antigen-binding domains (Fabs) isolated from human monoclonal antibodies targeting NHBA were determined. Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs, these structures provide broad insights into the NHBA epitopes recognized by the human immune system. As expected, these Fabs also show remarkable structural conservation, as shown by a structural comparison of 15 structures of apo Fab 10C3 which were obtained from crystals grown in different crystallization conditions and were solved while searching for a complex with a bound NHBA fragment or epitope peptide. This study also provides indirect evidence for the intrinsically disordered nature of two N-terminal regions of NHBA. Less
Second harmonic generation SHG was integrated with Raman spectroscopy for the analysis of pharmaceutical materials Particulate formulations of clopidogrel bisulphate were prepared in two crystal forms Form I and Form II Image analysis approaches enable automated identification of particles by bright field imaging followed by classification by SHG Quantitative SHG microscopy enabled discrimination of crystal form on a per particle basis with confidence in a total measurement time of ms per particle Complementary measurements by Raman and synchrotron XRD are in excellent agreement with the classifications made by SHG with measurement times of minute and several seconds per particle respectively ... More
Second harmonic generation (SHG) was integrated with Raman spectroscopy for the
analysis of pharmaceutical materials. Particulate formulations of clopidogrel bisulphate were
prepared in two crystal forms (Form I and Form II). Image analysis approaches enable
automated identification of particles by bright field imaging, followed by classification by SHG.
Quantitative SHG microscopy enabled discrimination of crystal form on a per particle basis with
99.95% confidence in a total measurement time of ~10 ms per particle. Complementary
measurements by Raman and synchrotron XRD are in excellent agreement with the
classifications made by SHG, with measurement times of ~1 minute and several seconds per
particle, respectively. Coupling these capabilities with at-line monitoring may enable real-time
feedback for reaction monitoring during pharmaceutical production to favor the more
bioavailable but metastable Form I with limits of detection in the ppm regime. Less
analysis of pharmaceutical materials. Particulate formulations of clopidogrel bisulphate were
prepared in two crystal forms (Form I and Form II). Image analysis approaches enable
automated identification of particles by bright field imaging, followed by classification by SHG.
Quantitative SHG microscopy enabled discrimination of crystal form on a per particle basis with
99.95% confidence in a total measurement time of ~10 ms per particle. Complementary
measurements by Raman and synchrotron XRD are in excellent agreement with the
classifications made by SHG, with measurement times of ~1 minute and several seconds per
particle, respectively. Coupling these capabilities with at-line monitoring may enable real-time
feedback for reaction monitoring during pharmaceutical production to favor the more
bioavailable but metastable Form I with limits of detection in the ppm regime. Less
Biological molecules especially the proteins have a special and important function We study their structure to understand their functions and further make application like the medical research The routine method is diffraction but not work for molecules which cannot grow into crystal and molecules which their crystal are too small Cryo-EM technique provides another way to solve their structures through their images it does not need crystals Meanwhile electron diffraction can work small crystals micro- and nano-crystals after the sample preparation was improved Hence we try to build a method that can restore the structure from the crystal s image ... More
Biological molecules, especially the proteins, have a special and important function. We study their structure to understand their functions, and further make application, like the medical research. The routine method is diffraction, but not work for molecules which cannot grow into crystal and molecules which their crystal are too small. Cryo-EM technique provides another way to solve their structures through their images, it does not need crystals. Meanwhile, electron diffraction can work small crystals (micro- and nano-crystals) after the sample preparation was improved. Hence, we try to build a method that can restore the structure from the crystal�s image. We collect images of protein nano-crystal, and these images were processed to enhance their contrast. The key step is to find the orientations of these images in the procedure of reconstruction, therefore, we create a method that calculates these orientations. Nano-crystals, which cannot be used in diffraction method, then can be used in this method. Less
Structures of enzyme-substrate product complexes have been studied for over four decades but have been limited to either before or after a chemical reaction Recently using in crystallo catalysis combined with X-ray diffraction we have discovered that many enzymatic reactions in nucleic-acid metabolism require additional metal-ion cofactors that are not present in the substrate or product state By controlling metal ions essential for catalysis the in crystallo approach has revealed unprecedented details of reaction intermediates Here we present protocols used for successful studies of Mg -dependent DNA polymerases and ribonucleases that are applicable to analyses of a variety of metal ... More
Structures of enzyme-substrate/product complexes have been studied for over four decades but have been limited to either before or after a chemical reaction. Recently using in crystallo catalysis combined with X-ray diffraction, we have discovered that many enzymatic reactions in nucleic-acid metabolism require additional metal-ion cofactors that are not present in the substrate or product state. By controlling metal ions essential for catalysis, the in crystallo approach has revealed unprecedented details of reaction intermediates. Here we present protocols used for successful studies of Mg2+-dependent DNA polymerases and ribonucleases that are applicable to analyses of a variety of metal ion-dependent reactions. Less
Bacteria sense and adapt to environmental changes using two-component systems These signaling pathways are formed by a histidine kinase that phosphorylates a response regulator RR which finally modulates the transcription of target genes The bacterium Brucella abortus codes for a two-component system formed by the histidine kinase NtrY and the RR NtrX that participates in sensing low oxygen tension and generating an adaptive response NtrX is a modular protein with REC AAA and DNA-binding domains an architecture that classifies it among the NtrC subfamily of RRs However it lacks the signature GAFTGA motif that is essential for activating transcription by ... More
Bacteria sense and adapt to environmental changes using two-component systems. These signaling pathways are formed by a histidine kinase that phosphorylates a response regulator (RR), which finally modulates the transcription of target genes. The bacterium Brucella abortus codes for a two-component system formed by the histidine kinase NtrY and the RR NtrX that participates in sensing low oxygen tension and generating an adaptive response. NtrX is a modular protein with REC, AAA +, and DNA-binding domains, an architecture that classifies it among the NtrC subfamily of RRs. However, it lacks the signature GAFTGA motif that is essential for activating transcription by the mechanism proposed for canonical members of this subfamily. In this article, we present the first crystal structure of full-length NtrX, which is also the first structure of a full-length NtrC-like RR with all the domains solved, showing that the protein is structurally similar to other members of the subfamily. We also report that NtrX binds nucleotides and the structures of the protein bound to ATP and ADP. Despite binding ATP, NtrX does not have ATPase activity and does not form oligomers in response to phosphorylation or nucleotide binding. We also identify a nucleotide sequence recognized by NtrX that allows it to bind to a promoter region that regulates its own transcription and to establish a negative feedback mechanism to modulate its expression. Overall, this article provides a detailed description of the NtrX RR and supports that it functions by a mechanism different to classical NtrC-like RRs. Less
Autophagy is a conserved cellular process involved in the elimination of proteins and organelles It is also used to combat infection with pathogenic microbes The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg LC proteins coupled to phosphatidylethanolamine PE on autophagosomal membranes To understand how RavZ recognizes and deconjugates LC -PE we prepared semisynthetic LC proteins and elucidated the structures of the RavZ LC interaction Semisynthetic LC proteins allowed the analysis of structure-function relationships RavZ extracts LC -PE from the membrane before deconjugation RavZ initially recognizes the LC molecule on membranes via its ... More
Autophagy is a conserved cellular process involved in the elimination of proteins and organelles. It is also used to combat infection with pathogenic microbes. The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3 proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes. To understand how RavZ recognizes and deconjugates LC3-PE, we prepared semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3 interaction. Semisynthetic LC3 proteins allowed the analysis of structure-function relationships. RavZ extracts LC3-PE from the membrane before deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and docking of the acyl chains into the lipid-binding site of RavZ that is related in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has evolved a novel mechanism to specifically evade host autophagy. Less
Angiotensin II receptors AT R and AT R serve as key components of the renin-angiotensin-aldosterone system While AT R plays a central role in the regulation of blood pressure the function of AT R is enigmatic with a variety of reported effects To elucidate the mechanisms for the functional diversity and ligand selectivity between these receptors we report crystal structures of the human AT R bound to an AT R-selective and an AT R AT R-dual ligand respectively capturing the receptor in an active-like conformation Unexpectedly helix VIII was found in a non-canonical position stabilizing the active-like state but at ... More
Angiotensin II receptors, AT1R and AT2R, serve as key components of the renin-angiotensin-aldosterone system. While AT1R plays a central role in the regulation of blood pressure, the function of AT2R is enigmatic with a variety of reported effects. To elucidate the mechanisms for the functional diversity and ligand selectivity between these receptors, we report crystal structures of the human AT2R bound to an AT2R-selective and an AT1R/AT2R-dual ligand, respectively, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins/β-arrestins, in agreement with the lack of signaling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the interactions critical for ligand binding and selectivity. Our results thus provide insights into the structural basis for distinct functions of the angiotensin receptors, and may guide the design of novel selective ligands. Less
Dehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids Given the insolubility of the natural C C fatty acyl substrate of the PpsC dehydratase DH domain we investigated its structure function relationships in the presence of shorter surrogate substrates Since most enzymes belonging to the R -specific enoyl hydratase hydroxyacyl dehydratase family conduct the reverse hydration reaction ... More
Dehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine. The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids. Given the insolubility of the natural C28?C30 fatty acyl substrate of the PpsC dehydratase (DH) domain, we investigated its structure?function relationships in the presence of shorter surrogate substrates. Since most enzymes belonging to the (R)-specific enoyl hydratase/hydroxyacyl dehydratase family conduct the reverse hydration reaction in vitro, we have determined the X-ray structures of the PpsC DH domain, both unliganded (apo) and in complex with trans-but-2-enoyl-CoA or trans-dodec-2-enoyl-CoA derivatives. This study provides for the first time a snapshot of dehydratase?ligand interactions following a hydration reaction. Our structural analysis allowed us to identify residues essential for substrate binding and activity. The structural comparison of the two complexes also sheds light on the need for long acyl chains for this dehydratase to carry out its function, consistent with both its in vitro catalytic behavior and the physiological role of the PpsC enzyme. Less
Insulin-regulated aminopeptidase IRAP is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates although the mechanism behind this wide specificity is not clearly understood We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at resolution In the presence of this inhibitor the enzyme adopts a novel conformation in which domains II and IV are juxtaposed forming a hollow structure that excludes external solvent access to the catalytic center A loop adjacent to the enzyme s GAMEN motif undergoes structural reconfiguration allowing the ... More
Insulin-regulated aminopeptidase (IRAP) is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates, although the mechanism behind this wide specificity is not clearly understood. We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at 2.53 � resolution. In the presence of this inhibitor, the enzyme adopts a novel conformation in which domains II and IV are juxtaposed, forming a hollow structure that excludes external solvent access to the catalytic center. A loop adjacent to the enzyme�s GAMEN motif undergoes structural reconfiguration, allowing the accommodation of bulky inhibitor side chains. Atomic interactions between the inhibitor and IRAP that are unique to this conformation can explain the strong selectivity compared to homologous aminopeptidases ERAP1 and ERAP2. This conformation provides insight on IRAP�s catalytic cycle and reveals significant active-site plasticity that may underlie its substrate permissiveness. Less
This multidisciplinary project begins with one overarching aim to elucidate the role of the rigidity of the lipid tail on the phase transitions of lipidic mesophases Previous studies have demonstrated that the position and the number of cis double bonds in monoacylglycerols determine the chain splay of the molecule establishing how this parameter was essential in influencing the phase behavior Following on from this novel lipids which are inspired by naturally occuring cyclopropanated lipids have been synthesized and their phase behavior elucidated The chain rigidity has been systematically varied by locking the cis configuration of the double bond on the ... More
This multidisciplinary project begins with one overarching aim: to elucidate the role of the
rigidity of the lipid tail on the phase transitions of lipidic mesophases. Previous studies have
demonstrated that the position and the number of cis double bonds in monoacylglycerols
determine the chain splay of the molecule, establishing how this parameter was essential in
influencing the phase behavior. Following on from this, novel lipids which are inspired by
naturally occuring cyclopropanated lipids have been synthesized, and their phase behavior
elucidated. The chain rigidity has been systematically varied by locking the cis configuration
of the double bond on the alkyl chain in a confined geometry. To understand the relationship
between chain rigidity and phase behavior a library of new lipids has been synthesized replacing
the cis double bond by a geometrically confined cyclopropyl ring. The replacement of the
double bond with a chemically analogous cyclopropyl group was designed in order to maintain
a similar chain splay and CPP parameter. The insertion of an additional carbon into the lipidic
chain doesn�t significantly change the length or the curvature of the chain but varies
substantially the packing frustration and the lateral stress of the lipid.
The phase behavior of these novel lipids with identical head group and different alkyl chains
has been investigated with utmost care. Small angle X-ray scattering (SAXS) measurements at
different hydration level and at different temperatures have been used to study the thermal
behavior of these lipid and the effect of this novel motif on the lipidic packing, with particular
attention to low temperature effects.
Since cyclopropanated lipids are present in several dairy products, and since lipidic
nanoparticles have been proved to be excellent drug delivery systems, digestion studies of
cubosomes and hexosomes formed by the novel synthesized cyclopropanated lipids have been
performed. Time resolved synchrotron SAXS has been used to monitor the phase changes
during the enzymatic reaction.
In order to test the utility of the cyclopropanated lipidic systems for membrane protein
crystallization the novel lipidic cubic phase (LCP) matrices have been employed in
crystallization studies with the membrane protein model system bacteriorhodopsin (bR).
IV
Finally, the successful crystallization attempts for membrane protein structural studies of the
chloride channels EcClC and Rm1ClC, as well as the lipopolysaccharide transporter LptD-LptE
show the broad applicability of the LCP crystallization method and the utility of tuning
crystallization conditions, including a screening of different lipids, to optimize crystal growt. Less
rigidity of the lipid tail on the phase transitions of lipidic mesophases. Previous studies have
demonstrated that the position and the number of cis double bonds in monoacylglycerols
determine the chain splay of the molecule, establishing how this parameter was essential in
influencing the phase behavior. Following on from this, novel lipids which are inspired by
naturally occuring cyclopropanated lipids have been synthesized, and their phase behavior
elucidated. The chain rigidity has been systematically varied by locking the cis configuration
of the double bond on the alkyl chain in a confined geometry. To understand the relationship
between chain rigidity and phase behavior a library of new lipids has been synthesized replacing
the cis double bond by a geometrically confined cyclopropyl ring. The replacement of the
double bond with a chemically analogous cyclopropyl group was designed in order to maintain
a similar chain splay and CPP parameter. The insertion of an additional carbon into the lipidic
chain doesn�t significantly change the length or the curvature of the chain but varies
substantially the packing frustration and the lateral stress of the lipid.
The phase behavior of these novel lipids with identical head group and different alkyl chains
has been investigated with utmost care. Small angle X-ray scattering (SAXS) measurements at
different hydration level and at different temperatures have been used to study the thermal
behavior of these lipid and the effect of this novel motif on the lipidic packing, with particular
attention to low temperature effects.
Since cyclopropanated lipids are present in several dairy products, and since lipidic
nanoparticles have been proved to be excellent drug delivery systems, digestion studies of
cubosomes and hexosomes formed by the novel synthesized cyclopropanated lipids have been
performed. Time resolved synchrotron SAXS has been used to monitor the phase changes
during the enzymatic reaction.
In order to test the utility of the cyclopropanated lipidic systems for membrane protein
crystallization the novel lipidic cubic phase (LCP) matrices have been employed in
crystallization studies with the membrane protein model system bacteriorhodopsin (bR).
IV
Finally, the successful crystallization attempts for membrane protein structural studies of the
chloride channels EcClC and Rm1ClC, as well as the lipopolysaccharide transporter LptD-LptE
show the broad applicability of the LCP crystallization method and the utility of tuning
crystallization conditions, including a screening of different lipids, to optimize crystal growt. Less
Effect of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme
During production purification formulation and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability Such harmful conditions include extreme pH changes high ionic strengths or elevated temperatures The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development In this context PEGylation i e the covalent attachment of polyethylene glycol PEG to proteins represents a valuable strategy to improve the physico-chemical properties of proteins In this work the influence ... More
During production, purification, formulation, and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability. Such harmful conditions include extreme pH changes, high ionic strengths or elevated temperatures. The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation, as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development. In this context PEGylation, i.e. the covalent attachment of polyethylene glycol (PEG) to proteins, represents a valuable strategy to improve the physico-chemical properties of proteins. In this work, the influence of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme is investigated. Specifically, conformational and colloidal properties were studied by means of high-throughput melting point determination and automated generation of protein phase diagrams, respectively. Lysozyme from chicken egg-white as a model protein was randomly conjugated to 2 kDa, 5 kDa and 10 kDa mPEG-aldehyde and resulting PEGamer species were purified by chromatographic separation. Besides protein stability assessment, residual enzyme activities were evaluated employing a Micrococcus lysodeikticus based activity assay. PEG molecules with lower molecular weights and lower PEGylation degrees resulted in higher residual activities. Changes in enzyme activities upon PEGylation have shown to result from a combination of steric hindrance and molecular flexibility. In contrast, higher PEG molecular weights and PEGylation degrees enhanced conformational and colloidal stability. By PEGylating lysozyme an increase of the protein solubility by more than 11-fold was achieved. Less