1181 Citations
Mitochondrial Carriers MCs are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism The ADP ATP Carriers AACs are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP In human four different AACs isoforms are described which are expressed in tissue-specific manner They are involved in different genetic diseases and play a role in cancerogenesis Up to now only the structures of the bovine isoform and yeast isoforms and AAC have been determined in one particular conformation obtained in complex ... More
Mitochondrial Carriers (MCs) are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism. The ADP/ATP Carriers (AACs) are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP. In human, four different AACs isoforms are described which are expressed in tissue-specific manner. They are involved in different genetic diseases and play a role in cancerogenesis. Up to now only the structures of the bovine (isoform 1) and yeast (isoforms 2 and 3) AAC have been determined in one particular conformation, obtained in complex with the CATR inhibitor. Herein, we report that full-length human ADP/ATP Carriers isoform 1 and 3 were successfully expressed in cell-free system and purified in milligram amounts in detergent-solubilized state. The proteins exhibited the expected secondary structure content. Thermostability profiles showing stabilization by the CATR inhibitor suggest that the carriers are well folded. Less
Development of a highly effective vaccine or antibodies for the prevention and ultimately elimination of malaria is urgently needed Here we report the isolation of a number of human monoclonal antibodies directed against the Plasmodium falciparum Pf circumsporozoite protein PfCSP from several subjects immunized with an attenuated Pf whole-sporozoite SPZ vaccine Sanaria PfSPZ Vaccine Passive transfer of one of these antibodies monoclonal antibody CIS conferred high-level sterile protection in two different mouse models of malaria infection The affinity and stoichiometry of CIS binding to PfCSP indicate that there are two sequential multivalent binding events encompassing the repeat domain The first ... More
Development of a highly effective vaccine or antibodies for the prevention and ultimately elimination of malaria is urgently needed. Here we report the isolation of a number of human monoclonal antibodies directed against the Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) from several subjects immunized with an attenuated Pf whole-sporozoite (SPZ) vaccine (Sanaria PfSPZ Vaccine). Passive transfer of one of these antibodies, monoclonal antibody CIS43, conferred high-level, sterile protection in two different mouse models of malaria infection. The affinity and stoichiometry of CIS43 binding to PfCSP indicate that there are two sequential multivalent binding events encompassing the repeat domain. The first binding event is to a unique 'junctional' epitope positioned between the N terminus and the central repeat domain of PfCSP. Moreover, CIS43 prevented proteolytic cleavage of PfCSP on PfSPZ. Analysis of crystal structures of the CIS43 antigen-binding fragment in complex with the junctional epitope determined the molecular interactions of binding, revealed the epitope's conformational flexibility and defined Asn-Pro-Asn (NPN) as the structural repeat motif. The demonstration that CIS43 is highly effective for passive prevention of malaria has potential application for use in travelers, military personnel and elimination campaigns and identifies a new and conserved site of vulnerability on PfCSP for next-generation rational vaccine design. Less
As a protective envelope surrounding the bacterial cell the peptidoglycan sacculus is a site of vulnerability and an antibiotic target Peptidoglycan components assembled in the cytoplasm are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate A product of peptidoglycan synthesis undecaprenyl-pyrophosphate is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase BacA To understand how BacA functions we determine its crystal structure at resolution The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane Conserved residues map to the pocket where pyrophosphorolysis occurs BacA ... More
As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase. Less
Investigation of the biochemical basis of life is a long standing challenge for scientists The field of molecular biology was established based on the pioneering studies of macromolecular structures including the structural characterization of the DNA double helix and the description of the first protein structures These early achievements along with the technical development e g automatic data collection and computer-assisted data analysis enables extremely efficient and fast determination of structure and dynamic properties of proteins
High-throughput single-cell RNA-seq methods assign limited unique molecular identifier UMI counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts We thus developed a high-throughput single-cell RNA-seq method Quartz-Seq to overcome these issues Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts at a rate of and detect more genes To demonstrate the power of Quartz-Seq we analyzed approximately transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads
The low limits of detection afforded by second harmonic generation SHG microscopy coupled with image analysis algorithms enabled quantitative modeling of the temperature-dependent crystallization of active pharmaceutical ingredients APIs within amorphous solid dispersions ASDs ASDs in which an API is maintained in an amorphous state within a polymer matrix are finding increasing use to address solubility limitations of small-molecule APIs Extensive stability testing is typically performed for ASD characterization the time frame for which is often dictated by the earliest detectable onset of crystal formation Here a study of accelerated stability testing on ritonavir a human immunodeficiency virus HIV protease ... More
The low limits of detection afforded by second harmonic generation (SHG) microscopy coupled with image analysis algorithms enabled quantitative modeling of the temperature-dependent crystallization of active pharmaceutical ingredients (APIs) within amorphous solid dispersions (ASDs). ASDs, in which an API is maintained in an amorphous state within a polymer matrix, are finding increasing use to address solubility limitations of small-molecule APIs. Extensive stability testing is typically performed for ASD characterization, the time frame for which is often dictated by the earliest detectable onset of crystal formation. Here a study of accelerated stability testing on ritonavir, a human immunodeficiency virus (HIV) protease inhibitor, has been conducted. Under the condition for accelerated stability testing at 50 �C/75%RH and 40 �C/75%RH, ritonavir crystallization kinetics from amorphous solid dispersions were monitored by SHG microscopy. SHG microscopy coupled by image analysis yielded limits of detection for ritonavir crystals as low as 10 ppm, which is about 2 orders of magnitude lower than other methods currently available for crystallinity detection in ASDs. The four decade dynamic range of SHG microscopy enabled quantitative modeling with an established (JMAK) kinetic model. From the SHG images, nucleation and crystal growth rates were independently determined. Less
Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the -oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA RNA production This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring To provide validation for this approach as a drug target we have RNAi silenced the three -oxopurine phosphoribosyltransferase PRTase isoforms in the infectious stage of Trypanosoma brucei demonstrating that ... More
Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites’ viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery. Less
Plastidial thioredoxin TRX -like proteins are atypical thioredoxins possessing a WCRKC active site signature and using glutathione for recycling To obtain structural information supporting the peculiar catalytic mechanisms and target proteins of these TRXs we solved the crystal structures of poplar TRX-like in oxidized and reduced states and of mutated variants These structures share similar folding with TRXs exhibiting the canonical WCGPC signature Moreover the overall conformation is not altered by reduction of the catalytic disulfide bond or in a C S C S variant that formed a disulfide-bridged dimer possibly mimicking reaction intermediates with target proteins Modeling of the ... More
Plastidial thioredoxin (TRX)-like2.1 proteins are atypical thioredoxins possessing a WCRKC active site signature and using glutathione for recycling. To obtain structural information supporting the peculiar catalytic mechanisms and target proteins of these TRXs, we solved the crystal structures of poplar TRX-like2.1 in oxidized and reduced states and of mutated variants. These structures share similar folding with TRXs exhibiting the canonical WCGPC signature. Moreover, the overall conformation is not altered by reduction of the catalytic disulfide bond or in a C45S/C67S variant that formed a disulfide-bridged dimer possibly mimicking reaction intermediates with target proteins. Modeling of the interaction of TRX-like2.1 with both NADPH- and ferredoxin-thioredoxin reductases (FTR) indicates that the presence of Arg43 and Lys44 residues likely precludes reduction by the plastidial FTR. Less
Methods are provided for analyzing one or more genetic samples comprising procuring one or more genetic samples comprising genetic material from one or more individuals and sequencing the genetic material using non-targeted ultra-low coverage sequencing to obtain genetic information for individual associated with the one or more genetic samples Personal and genetic information associated with the individuals is stored in a database for retrieval and manipulation
In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains ECDs comparable to the water channels nm of the mesophase Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid Ia d double-diamond Pn m and double-primitive Im m space groups with water channels five times larger than traditional lipidic mesophases and showing re-entrant behavior upon increasing hydration of sequences Ia d ... More
In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains (ECDs), comparable to the water channels (3�5 nm) of the mesophase. Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs. We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid (Ia3d), double-diamond (Pn3m), and double-primitive (Im3m) space groups, with water channels five times larger than traditional lipidic mesophases, and showing re-entrant behavior upon increasing hydration, of sequences Ia3d?Pn3m?Ia3d and Pn3m?Im3m?Pn3m, unknown in lipid self-assembly. We use these mesophases to crystallize membrane proteins with ECDs inaccessible to conventional in meso crystallization, demonstrating the methodology on the Gloeobacter ligand-gated ion channel (GLIC) protein, and show substantial modulation of packing, molecular contacts and activation state of the ensued proteins crystals, illuminating a general strategy in protein structural biology. Less
Drugs frequently require interactions with multiple targets via a process known as polypharmacology to achieve their therapeutic actions Currently drugs targeting several serotonin receptors including the -HT C receptor are useful for treating obesity drug abuse and schizophrenia The competing challenges of developing selective -HT C receptor ligands or creating drugs with a defined polypharmacological profile especially aimed at G protein-coupled receptors GPCRs remain extremely difficult Here we solved two structures of the -HT C receptor in complex with the highly promiscuous agonist ergotamine and the -HT A-C receptor-selective inverse agonist ritanserin at resolutions of and respectively We analyzed their ... More
Drugs frequently require interactions with multiple targets — via a process known as polypharmacology — to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin, at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs. Less
Cells require some metals such as zinc and manganese but excess levels of these metals can be toxic As a result cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication Here we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity together with transcriptomics data for wild-type or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation Altogether we identified genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment and putative zinc-responsive genes Analysis of the omics data points to ... More
Cells require some metals, such as zinc and manganese, but excess levels of these metals can be toxic. As a result, cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication. Here, we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity, together with transcriptomics data for wild-type or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation. Altogether, we identified 47 genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment, and >1800 putative zinc-responsive genes. Analysis of the ‘omics data points to the relevance of ion transporters, glutathione (GSH)-related factors, and conserved disease-associated genes in zinc detoxification. Specific genes identified in the zinc screen include orthologs of human disease-associated genes CTNS, PTPRN (also known as IA-2), and ATP13A2 (also known as PARK9). We show that knockdown of red dog mine (rdog; CG11897), a candidate zinc detoxification gene encoding an ABCC-type transporter family protein related to yeast cadmium factor (YCF1), confers sensitivity to zinc intoxication in cultured cells, and that rdog is transcriptionally upregulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the ‘omics data sets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes. Less
The ageing suppressor -klotho binds to the fibroblast growth factor receptor FGFR This commits FGFR to respond to FGF a key hormone in the regulation of mineral ion and vitamin D homeostasis The role and mechanism of this co-receptor are unknown Here we present the atomic structure of a ternary complex that consists of the shed extracellular domain of -klotho the FGFR c ligand-binding domain and FGF In this complex -klotho simultaneously tethers FGFR c by its D domain and FGF by its C-terminal tail thus implementing FGF FGFR c proximity and conferring stability Dimerization of the stabilized ternary complexes ... More
The ageing suppressor α-klotho binds to the fibroblast growth factor receptor (FGFR). This commits FGFR to respond to FGF23, a key hormone in the regulation of mineral ion and vitamin D homeostasis. The role and mechanism of this co-receptor are unknown. Here we present the atomic structure of a 1:1:1 ternary complex that consists of the shed extracellular domain of α-klotho, the FGFR1c ligand-binding domain, and FGF23. In this complex, α-klotho simultaneously tethers FGFR1c by its D3 domain and FGF23 by its C-terminal tail, thus implementing FGF23–FGFR1c proximity and conferring stability. Dimerization of the stabilized ternary complexes and receptor activation remain dependent on the binding of heparan sulfate, a mandatory cofactor of paracrine FGF signalling. The structure of α-klotho is incompatible with its purported glycosidase activity. Thus, shed α-klotho functions as an on-demand non-enzymatic scaffold protein that promotes FGF23 signalling. Less
The maturation of Ras GTPases and other cellular CaaX proteins involves three enzymatic steps addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine C in the C-terminal CaaX motif proteolytic cleavage of the aaX residues and methylation of the exposed prenylcysteine residue at its terminal carboxylate This final step is catalyzed by isoprenylcysteine carboxyl methyltransferase ICMT a eukaryotic-specific integral membrane enzyme of the endoplasmic reticulum ER ICMT is the only cellular enzyme known to methylate prenylcysteine substrates methylation is important for their biological functions including the membrane localisations and subsequent activities of Ras prelamin A and Rab ICMT ... More
The maturation of Ras GTPases, and ~200 other cellular CaaX proteins, involves three enzymatic steps: addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine (C) in the C-terminal CaaX motif, proteolytic cleavage of the aaX residues, and methylation of the exposed prenylcysteine residue at its terminal carboxylate1. This final step is catalyzed by isoprenylcysteine carboxyl methyltransferase (ICMT), a eukaryotic-specific integral membrane enzyme of the endoplasmic reticulum (ER)2. ICMT is the only cellular enzyme known to methylate prenylcysteine substrates; methylation is important for their biological functions, including the membrane localisations and subsequent activities of Ras1, prelamin A3, and Rab4. ICMT inhibition has potential for combating progeria3 and cancer5–8. Here we present an X-ray structure of ICMT, at 2.3 Å resolution, in complex with its cofactor, an ordered lipid molecule and a monobody inhibitor. The active site spans cytosolic and membrane-exposed regions, indicating distinct entry routes for its cytosolic methyl donor, S-adenosyl-L-methionine (AdoMet), and for prenylcysteine substrates, which are associated with the ER membrane. The structure suggests how ICMT overcomes the topographical challenge and unfavourable energetics of bringing two reactants that have different cellular localisations together in a membrane environment – a relatively uncharacterized, but defining feature of many integral membrane enzymes. Less
Canonical FGFs activate FGF receptors FGFR via paracrine or autocrine mechanisms in a process requiring cooperation with heparan sulfate proteoglycans that function as co-receptors for FGFR activation By contrast endocrine FGFs FGF and are circulating hormones that regulate critical metabolic processes in a variety of tissues FGF regulates bile acid synthesis and lipogenesis while FGF stimulate insulin sensitivity energy expenditure and weight loss Endocrine FGFs signal through FGFRs in a manner that requires Klothos which are cell surface proteins with tandem glycosidase domains Here we describe the crystal structures of free and ligand-bound -Klotho extracellular regions revealing the molecular mechanism ... More
Canonical FGFs activate FGF receptors (FGFR) via paracrine or autocrine mechanisms, in a process requiring cooperation with heparan sulfate proteoglycans that function as co-receptors for FGFR activation1,2. By contrast, endocrine FGFs (FGF 19, 21, and 23) are circulating hormones that regulate critical metabolic processes in a variety of tissues3,4. FGF19 regulates bile acid synthesis and lipogenesis, while FGF21 stimulate insulin sensitivity, energy expenditure and weight loss5. Endocrine FGFs signal through FGFRs in a manner that requires Klothos, which are cell surface proteins with tandem glycosidase domains3,4. Here we describe the crystal structures of free and ligand-bound β-Klotho extracellular regions, revealing the molecular mechanism underlying the specificity of FGF21 towards β-Klotho and demonstrating how FGFR is activated in a Klotho-dependent manner. β-Klotho serves as a primary “Zip code”-like receptor for FGF21 with an FGFR functioning as a catalytic subunit that mediates intracellular signaling. Our structures also show how a sugar cutting enzyme (glycosidase) has evolved to become a specific receptor for hormones that regulate metabolic processes including lowering of blood sugar. Finally, we describe a superior agonistic variant of FGF21 and present structural insights offering development of novel therapeutics for diseases linked to endocrine FGFs. Less
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
In recent years highly detailed characterization of adult bone marrow BM myeloid progenitors has been achieved and as a result the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined Fetal liver FL hematopoietic progenitor cells HPCs are poorly characterized in comparison potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis Numerous disorders for example infant acute leukemias have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets We previously demonstrated that a Runx distal promoter P -GFP proximal promoter P -hCD ... More
In recent years, highly detailed characterization of adult bone marrow (BM) myeloid progenitors has been achieved and, as a result, the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined. Fetal liver (FL) hematopoietic progenitor cells (HPCs) are poorly characterized in comparison, potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis. Numerous disorders, for example infant acute leukemias, have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets. We previously demonstrated that a Runx1 distal promoter (P1)-GFP::proximal promoter (P2)-hCD4 dual-reporter mouse (Mus musculus) model can be used to identify adult BM progenitor subsets with distinct lineage preferences. In this study, we undertook the characterization of the expression of Runx1-P1-GFP and P2-hCD4 in FL. Expression of P2-hCD4 in the FL immunophenotypic Megakaryocyte-Erythroid Progenitor (MEP) and Common Myeloid Progenitor (CMP) compartments corresponded to increased granulocytic/monocytic/megakaryocytic and decreased erythroid specification. Moreover, Runx1-P2-hCD4 expression correlated with several endogenous cell surface markers’ expression, including CD31 and CD45, providing a new strategy for prospective identification of highly purified fetal myeloid progenitors in transgenic mouse models. We utilized this methodology to compare the impact of the deletion of either total RUNX1 or RUNX1C alone and to determine the fetal HPCs lineages most substantially affected. This new prospective identification of FL progenitors therefore raises the prospect of identifying the underlying gene networks responsible with greater precision than previously possible. 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
There is a pressing need to improve approaches for drug discovery related to neuropsychiatric disorders NSDs Therapeutic discovery in neuropsychiatric disorders would benefit from screening assays that can measure changes in complex phenotypes linked to disease mechanisms However traditional assays that track complex neuronal phenotypes such as neuronal connectivity exhibit poor scalability and are not compatible with high-throughput screening HTS procedures Therefore we created a neuronal phenotypic assay platform that focused on improving the scalability and affordability of neuron-based assays capable of tracking disease-relevant phenotypes First using inexpensive laboratory-level automation we industrialized primary neuronal culture production which enabled the creation ... More
There is a pressing need to improve approaches for drug discovery related to neuropsychiatric disorders (NSDs). Therapeutic discovery in neuropsychiatric disorders would benefit from screening assays that can measure changes in complex phenotypes linked to disease mechanisms. However, traditional assays that track complex neuronal phenotypes, such as neuronal connectivity, exhibit poor scalability and are not compatible with high-throughput screening (HTS) procedures. Therefore, we created a neuronal phenotypic assay platform that focused on improving the scalability and affordability of neuron-based assays capable of tracking disease-relevant phenotypes. First, using inexpensive laboratory-level automation, we industrialized primary neuronal culture production, which enabled the creation of scalable assays within functioning neural networks. We then developed a panel of phenotypic assays based on culturing of primary neurons from genetically modified mice expressing HTS-compatible reporters that capture disease-relevant phenotypes. We demonstrated that a library of 1,280 compounds was quickly screened against both assays using only a few litters of mice in a typical academic laboratory setting. Finally, we implemented one assay in a fully automated high-throughput academic screening facility, illustrating the scalability of assays designed using this platform. These methodological improvements simplify the creation of highly scalable neuron-based phenotypic assays designed to improve drug discovery in CNS disorders. 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
This review provides an overview of the science of high-throughput screening HTS within the pharmaceutical industry and the greater drug discovery community From its origins in the early s to the current state of the art key aspects of the process are introduced and described in increasing levels of detail Examples of technologies employed in the automation of HTS are provided together with an evaluation of their applicability and limitations The various detection modalities typically encountered and their suitability to high-density screening formats or well are described The increasing importance of informatics in screen design data interpretation quality control and ... More
This review provides an overview of the science of high-throughput screening (HTS) within the pharmaceutical industry and the greater drug discovery community. From its origins in the early 1990s to the current state of the art, key aspects of the process are introduced and described in increasing levels of detail. Examples of technologies employed in the automation of HTS are provided, together with an evaluation of their applicability and limitations. The various detection modalities typically encountered, and their suitability to high-density screening formats (96, 386, 1536, or 3456 well) are described. The increasing importance of informatics in screen design, data interpretation, quality control, and its contribution to the development of future HTS strategies is introduced. Current and future trends of how HTS is employed to meet the changing needs for new drug discovery are explored, including the parallel use of complementary screening modalities to sample diverse chemical matter and identify the best starting points for drug discovery programs. 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
The growing demand for fast highly sensitive and low cost diagnostic devices has stimulated efforts to generate simplified analytical systems capable of being applied in the field This scientific work involved the study of different approaches to enable the qualitative and quantitative detection of biomarkers for clinical diagnosis in paper-based platforms The first strategy consisted of understanding the operating principles of the lateral flow immunoassay device allowing the assessment of critical steps and consequently providing the most appropriate experimental conditions for the early diagnosis of malaria caused by Plasmodium falciparum by identifying the HRP biomarker The best device performance was ... More
The growing demand for fast, highly sensitive and low cost diagnostic devices has stimulated efforts to generate simplified analytical systems capable of being applied in the field. This scientific work involved the study of different approaches to enable the qualitative and quantitative detection of biomarkers for clinical diagnosis in paper-based platforms. The first strategy consisted of understanding the operating principles of the lateral flow immunoassay device, allowing the assessment of critical steps and, consequently, providing the most appropriate experimental conditions for the early diagnosis of malaria caused by Plasmodium falciparum, by identifying the HRP2 biomarker. The best device performance was achieved by using 0,05 µg (1 µL / 50 µg mL-1) of the capture antibody and incubation for 5 minutes for its adsorption; blocking of nitrocellulose with 1.5% BSA (m/v) containing 0.1% of the surfactant Tween-20 (v/v) by immersion and incubation for 10 minutes; 0.04 µg (20 µL/2 µg mL-1) detection antibody conjugated to the enzyme peroxidase; washing with 0.01 mol L-1 Tris-HCl buffer solution pH 7.4 containing 0.15 mol L-1 NaCl and 0.1% Tween 20 (v/v); addition of 5 µL of TMB chromogenic substrate and reading within 5-20 minutes after this addition. The colorimetric detection system had a visual detection limit of 5 ng mL-1 (135 pmol L-1), which value should be sufficient to identify malaria contamination on the first day of symptom onset.The developed platform was then applied to blood samples from patients infected by the disease, demonstrating efficiency in the qualitative discrimination of a positive and negative result and the generation of reliable results. In another study, with the aim of providing a second generation of the device, a paper-based 3D detection system capable of being coupled to a lateral flow immunoassay platform was built. This system enables quantitative, automated diagnosis with signal amplification through the incorporation of a new polymeric material of the class of poly(benzyl ethers) that selectively responds to hydrogen peroxide, in addition to requiring only color and color visualization. a timer for obtaining data and later analyzing the results.The delimitation of hydrophobic barriers on the paper was carried out by means of wax printing and the orientation processes and type of paper to be used in the layer containing the polymer were evaluated. The material deposition process on the paper surface demonstrated greater repeatability when performed automatically by means of a liquid handling robot. The highest sensitivity condition for the 3D detection system was achieved using a polymer concentration of 4.0 mg mL-1 with the achievement of a detection limit of 0.02 mmol L-1 of hydrogen peroxide. This system was then coupled to a lateral flow immunoassay platform and used in initial studies to detect the creatine kinase MB isoenzyme, one of the biomarkers indicated for the diagnosis of acute myocardial infarction (AU)one of the biomarkers indicated for the diagnosis of acute myocardial infarction (AU) 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 androgen receptor AR NR C is a nuclear receptor whose main function is acting as a transcription factor regulating gene expression for male sexual development and maintaining accessory sexual organ function It is also a necessary component of female fertility by affecting the functionality of ovarian follicles and ovulation Pathological processes involving AR include Kennedy s disease and Klinefelter s syndrome as well as prostate ovarian and testicular cancer Strict regulation of sex hormone signaling is required for normal reproductive organ development and function Therefore testing small molecules for their ability to modulate AR is a first step in ... More
The androgen receptor (AR, NR3C4) is a nuclear receptor whose main function is acting as a transcription factor regulating gene expression for male sexual development and maintaining accessory sexual organ function. It is also a necessary component of female fertility by affecting the functionality of ovarian follicles and ovulation. Pathological processes involving AR include Kennedy’s disease and Klinefelter’s syndrome, as well as prostate, ovarian, and testicular cancer. Strict regulation of sex hormone signaling is required for normal reproductive organ development and function. Therefore, testing small molecules for their ability to modulate AR is a first step in identifying potential endocrine disruptors. We screened the Tox21 10K compound library in a quantitative high-throughput format to identify activators of AR using two reporter gene cell lines, AR β-lactamase (AR-bla) and AR-luciferase (AR-luc). Seventy-five compounds identified through the primary assay were characterized as potential agonists or inactives through confirmation screens and secondary assays. Biochemical binding and AR nuclear translocation assays were performed to confirm direct binding and activation of AR from these compounds. The top seventeen compounds identified were found to bind to AR, and sixteen of them translocated AR from the cytoplasm into the nucleus. Five potentially novel or not well-characterized AR agonists were discovered through primary and follow-up studies. We have identified multiple AR activators, including known AR agonists such as testosterone, as well as novel/not well-known compounds such as prulifloxacin. The information gained from the current study can be directly used to prioritize compounds for further in-depth toxicological evaluations, as well as their potential to disrupt the endocrine system via AR activation. 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