1052 Citations
Glucagon-like peptide- receptor GLP- R is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type diabetes Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides however little is known about their extracellular domain ECD conformations in the absence of orthosteric ligands which has limited our understanding of their activation mechanism Here we report the resolution peptide-free crystal structure of the full-length human GLP- R in an inactive state which reveals a unique closed conformation of the ECD Disulfide cross-linking validates the physiological relevance of the ... More
Glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type 2 diabetes. Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides, however, little is known about their extracellular domain (ECD) conformations in the absence of orthosteric ligands, which has limited our understanding of their activation mechanism. Here, we report the 3.2 Å resolution, peptide-free crystal structure of the full-length human GLP-1R in an inactive state, which reveals a unique closed conformation of the ECD. Disulfide cross-linking validates the physiological relevance of the closed conformation, while electron microscopy (EM) and molecular dynamic (MD) simulations suggest a large degree of conformational dynamics of ECD that is necessary for binding GLP-1. Our inactive structure represents a snapshot of the peptide-free GLP-1R and provides insights into the activation pathway of this receptor family. Less
Small-molecule drug discovery can be hindered by the formation of aggregates that act as non-selective inhibitors of drug targets Such aggregates appear as false positives in high-throughput screening campaigns and can bedevil structure-activity relationships during compound optimization Protocols are described for resonant waveguide grating RWG and dynamic light scattering DLS as microplate-based high-throughput approaches to identify compound aggregation Resonant waveguide grating and dynamic light scattering give equivalent results for the compound test set as assessed with Bland-Altman analysis
The human formyl peptide receptor FPR plays a crucial role in host defense and inflammation and has been considered as a drug target for chronic inflammatory diseases A variety of peptides with different structures and origins have been characterized as FPR ligands However the ligand-binding modes of FPR remain elusive thereby limiting the development of potential drugs Here we report the crystal structure of FPR bound to the potent peptide agonist WKYMVm at resolution The structure adopts an active conformation and exhibits a deep ligand-binding pocket Combined with mutagenesis ligand binding and signaling studies key interactions between the agonist and ... More
The human formyl peptide receptor 2 (FPR2) plays a crucial role in host defense and inflammation, and has been considered as a drug target for chronic inflammatory diseases. A variety of peptides with different structures and origins have been characterized as FPR2 ligands. However, the ligand-binding modes of FPR2 remain elusive, thereby limiting the development of potential drugs. Here we report the crystal structure of FPR2 bound to the potent peptide agonist WKYMVm at 2.8 Å resolution. The structure adopts an active conformation and exhibits a deep ligand-binding pocket. Combined with mutagenesis, ligand binding and signaling studies, key interactions between the agonist and FPR2 that govern ligand recognition and receptor activation are identified. Furthermore, molecular docking and functional assays reveal key factors that may define binding affinity and agonist potency of formyl peptides. These findings deepen our understanding about ligand recognition and selectivity mechanisms of the formyl peptide receptor family. Less
One central question surrounding the biosynthesis of fatty acids and polyketide-derived natural products is how the -phosphopantetheinyl transferase PPTase interrogates the essential acyl carrier protein ACP domain to fulfill the initial activation step The triggering factor of this study was the lack of structural information on PPTases at physiological pH which could bias our comprehension of the mechanism of action of these important enzymes Structural and functional studies on the family II PPTase PptAb of Mycobacterium abscessus show that pH has a profound effect on the coordination of metal ions and on the conformation of endogenously bound coenzyme A CoA ... More
One central question surrounding the biosynthesis of fatty acids and polyketide-derived natural products is how the 4′-phosphopantetheinyl transferase (PPTase) interrogates the essential acyl carrier protein (ACP) domain to fulfill the initial activation step. The triggering factor of this study was the lack of structural information on PPTases at physiological pH, which could bias our comprehension of the mechanism of action of these important enzymes. Structural and functional studies on the family II PPTase PptAb of Mycobacterium abscessus show that pH has a profound effect on the coordination of metal ions and on the conformation of endogenously bound coenzyme A (CoA). The observed conformational flexibility of CoA at physiological pH is accompanied by a disordered 4′-phosphopantetheine (Ppant) moiety. Finally, structural and dynamical information on an isolated mycobacterial ACP domain, in its apo form and in complex with the activator PptAb, suggests an alternate mechanism for the post-translational modification of modular megasynthases. Less
The lipid cubic phases LCP have enabled the determination of many important high-resolution structures of membrane proteins such as G-protein-coupled receptors photosensitive proteins enzymes channels and transporters However harvesting the crystals from the glass or plastic plates in which crystals grow is challenging The in meso in situ serial X-ray crystallography IMISX method uses thin plastic windowed plates that minimize LCP crystal manipulation The method which is compatible with high-throughput in situ measurements allows systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without direct crystal harvesting In this chapter we describe an ... More
The lipid cubic phases (LCP) have enabled the determination of many important high-resolution structures of membrane proteins such as G-protein-coupled receptors, photosensitive proteins, enzymes, channels, and transporters. However, harvesting the crystals from the glass or plastic plates in which crystals grow is challenging. The in meso in situ serial X-ray crystallography (IMISX) method uses thin plastic windowed plates that minimize LCP crystal manipulation. The method, which is compatible with high-throughput in situ measurements, allows systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without direct crystal harvesting. In this chapter, we describe an IMISX protocol for in situ serial X-ray data collection of LCP-grown crystals at both cryogenic and room temperatures which includes the crystallization setup, sample delivery, automated serial diffraction data collection, and experimental phasing. We also detail how the IMISX method was applied successfully for the structure determination of two novel targets—the undecaprenyl-pyrophosphate phosphatase BacA and the chemokine G-protein-coupled receptor CCR2A. Less
Membrane proteins are highly interesting targets due to their pivotal role in cell function and disease They are inserted in cell membranes are often intrinsically flexible and can adopt several conformational states to carry out their function Although most overall folds of membrane proteins are known many questions remain about specific functionally relevant intramolecular rearrangements that require experimental structure determination Here using the example of rhodopsin we describe how to prepare and analyze membrane protein crystals for serial crystallography at room temperature a new technique allowing to merge diffraction data from thousands of injector-delivered crystals that are too tiny for ... More
Membrane proteins are highly interesting targets due to their pivotal role in cell function and disease. They are inserted in cell membranes, are often intrinsically flexible, and can adopt several conformational states to carry out their function. Although most overall folds of membrane proteins are known, many questions remain about specific functionally relevant intramolecular rearrangements that require experimental structure determination. Here, using the example of rhodopsin, we describe how to prepare and analyze membrane protein crystals for serial crystallography at room temperature, a new technique allowing to merge diffraction data from thousands of injector-delivered crystals that are too tiny for classical single-crystal analysis even in cryogenic conditions. The application of serial crystallography for studying protein dynamics is mentioned. Less
Two-component signaling systems TCSs are a large and important class of sensory systems in bacteria archaea and some eukaryotes yet their mechanism of action is still not fully understood from the structural point of view Many TCS receptors are elongated flexible proteins with transmembrane TM regions and are difficult to work with Consequently truncated fragments of the receptors are often used in structural studies However it is not fully clear whether the structures of the fragments correspond well to their native structures in the context of full-length proteins Recently we crystallized a fragment of Escherichia coli nitrate nitrite sensor histidine ... More
Two-component signaling systems (TCSs) are a large and important class of sensory systems in bacteria, archaea, and some eukaryotes, yet their mechanism of action is still not fully understood from the structural point of view. Many TCS receptors are elongated flexible proteins with transmembrane (TM) regions, and are difficult to work with. Consequently, truncated fragments of the receptors are often used in structural studies. However, it is not fully clear whether the structures of the fragments correspond well to their native structures in the context of full-length proteins. Recently, we crystallized a fragment of Escherichia coli nitrate/nitrite sensor histidine kinase, NarQ, encompassing the sensor, TM, and HAMP domains. Here we report that a smaller proteolytic fragment consisting of the sensor and TM domains can also be crystallized using the in meso approach. The structure of the fragment is similar to the previously determined one, with minor differences in the vicinity of the truncation site. The results show that the crystallization of such sensor–TM fragments can be accomplished and can provide information on the packing of transmembrane helices, albeit limited, and that the proteolysis may or may not be a problem during crystallization. Less
Rhodopsins are the most abundant light-harvesting proteins A new family of rhodopsins heliorhodopsins HeRs has recently been discovered Unlike in the known rhodopsins in HeRs the N termini face the cytoplasm The function of HeRs remains unknown We present the structures of the bacterial HeR- C in two states at the resolution of which highlight its remarkable difference from all known rhodopsins The interior of HeR s extracellular part is completely hydrophobic while the cytoplasmic part comprises a cavity Schiff base cavity SBC surrounded by charged amino acids and containing a cluster of water molecules presumably being a primary proton ... More
Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR’s extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes. Less
Histone lysine specific demethylase LSD has become a potential therapeutic target for the treatment of cancer Discovery and develop novel and potent LSD inhibitors is a challenge although several of them have already entered into clinical trials Herein for the first time we reported the discovery of a series of -cyano- -phenylpyrimidine derivatives as LSD inhibitors using flavin adenine dinucleotide FAD similarity-based designing strategy of which compound q was finally identified to repress LSD with IC nmol L Docking analysis suggested that compound q fitted well into the FAD-binding pocket Further mechanism studies showed that compound q may inhibit LSD ... More
Histone lysine specific demethylase 1 (LSD1) has become a potential therapeutic target for the treatment of cancer. Discovery and develop novel and potent LSD1 inhibitors is a challenge, although several of them have already entered into clinical trials. Herein, for the first time, we reported the discovery of a series of 5-cyano-6-phenylpyrimidine derivatives as LSD1 inhibitors using flavin adenine dinucleotide (FAD) similarity-based designing strategy, of which compound 14q was finally identified to repress LSD1 with IC50 = 183 nmol/L. Docking analysis suggested that compound 14q fitted well into the FAD-binding pocket. Further mechanism studies showed that compound 14q may inhibit LSD1 activity competitively by occupying the FAD binding sites of LSD1 and inhibit cell migration and invasion by reversing epithelial to mesenchymal transition (EMT). Overall, these findings showed that compound 14q is a suitable candidate for further development of novel FAD similarity-based LSD1 inhibitors. Less
Human muscarinic receptor M belongs to the class A subfamily of the G-protein-coupled receptors GPCRs M has emerged as an attractive drug target for the treatment of Alzheimer s disease and schizophrenia Recent results showed that M -mediated cholinergic transmission is related to motor symptoms in Parkinson s disease Selective ligand design for the five muscarinic acetylcholine receptor mAchR subtypes currently remains challenging owing to the high sequence and structural similarity of their orthosteric binding pockets In order to obtain M -selective antagonists a new approach was tried to lock M into an inactive form by rationally designing an N ... More
Human muscarinic receptor M4 belongs to the class A subfamily of the G-protein-coupled receptors (GPCRs). M4 has emerged as an attractive drug target for the treatment of Alzheimer’s disease and schizophrenia. Recent results showed that M4-mediated cholinergic transmission is related to motor symptoms in Parkinson’s disease. Selective ligand design for the five muscarinic acetylcholine receptor (mAchR) subtypes currently remains challenging owing to the high sequence and structural similarity of their orthosteric binding pockets. In order to obtain M4-selective antagonists, a new approach was tried to lock M4 into an inactive form by rationally designing an N4497.49R mutation, which mimics the allosteric sodium binding in the conserved sodium site usually found in class A GPCRs. In addition, the crystal structure of the mutation-induced inactive M4 was determined. By comparative analysis with other mAchR structures, followed by functional assays, the N4497.49R mutation was shown to stabilize M4 into an inactive state. Virtual screening of a focused ligand library using the crystal structure showed that the inactive M4 prefers antagonists much more than agonists. This study provides a powerful mutation strategy to stabilize GPCRs in inactive states and facilitate their structure determination. Less
GPR is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington s disease and several psychiatric disorders Pathological malfunction of GPR signalling occurs primarily through the heterotrimeric Gs protein but it is unclear how GPR and Gs couple for signal transduction and whether a native ligand or other activating input is required Here we present the high-resolution structures of human GPR in three states a ligand-free state a Gs-coupled self-activation state and a potential allosteric ligand-bound state Together our structures reveal that extracellular loop occupies the ... More
GPR52 is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington’s disease and several psychiatric disorders1,2. Pathological malfunction of GPR52 signalling occurs primarily through the heterotrimeric Gs protein2, but it is unclear how GPR52 and Gs couple for signal transduction and whether a native ligand or other activating input is required. Here we present the high-resolution structures of human GPR52 in three states: a ligand-free state, a Gs-coupled self-activation state and a potential allosteric ligand-bound state. Together, our structures reveal that extracellular loop 2 occupies the orthosteric binding pocket and operates as a built-in agonist, conferring an intrinsically high level of basal activity to GPR523. A fully active state is achieved when Gs is coupled to GPR52 in the absence of an external agonist. The receptor also features a side pocket for ligand binding. These insights into the structure and function of GPR52 could improve our understanding of other self-activated GPCRs, enable the identification of endogenous and tool ligands, and guide drug discovery efforts that target GPR52. Less
Toxoplasma gondii a protozoan parasite undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts Here we applied single-cell RNA-sequencing scRNA-seq on Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development revealing hidden states and transcriptional factors associated with each developmental stage Analysis of SAG -related sequence SRS antigenic repertoire reveals a highly heterogeneous sporadic expression pattern unexplained by measurement noise cell cycle or asexual development Furthermore we identified AP IX- as a transcription factor that controls ... More
Toxoplasma gondii, a protozoan parasite, undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts. Here, we applied single-cell RNA-sequencing (scRNA-seq) on >5,400 Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development, revealing hidden states and transcriptional factors associated with each developmental stage. Analysis of SAG1-related sequence (SRS) antigenic repertoire reveals a highly heterogeneous, sporadic expression pattern unexplained by measurement noise, cell cycle, or asexual development. Furthermore, we identified AP2IX-1 as a transcription factor that controls the switching from the ubiquitous SAG1 to rare surface antigens not previously observed in tachyzoites. In addition, comparative analysis between Toxoplasma and Plasmodium scRNA-seq results reveals concerted expression of gene sets, despite fundamental differences in cell division. Lastly, we built an interactive data-browser for visualization of our atlas resource. Less
Phytoplankton is the base of the marine food chain oxygen carbon cycle playing a global role in climate and ecology Nucleocytoplasmic Large DNA Viruses regulating the dynamics of phytoplankton comprise genes of rhodopsins of two distinct families We present a function-structure characterization of two homologous proteins representatives of family of viral rhodopsins OLPVR and VirChR VirChR is a highly selective Ca -dependent Na K - conducting channel and in contrast to known cation channelrhodopsins ChRs is impermeable to Ca ions In human neuroblastoma cells upon illumination VirChR depolarizes the cell membrane to a level sufficient to fire neurons It suggests ... More
Phytoplankton is the base of the marine food chain, oxygen, carbon cycle playing a global role in climate and ecology. Nucleocytoplasmic Large DNA Viruses regulating the dynamics of phytoplankton comprise genes of rhodopsins of two distinct families. We present a function-structure characterization of two homologous proteins representatives of family 1 of viral rhodopsins, OLPVR1 and VirChR1. VirChR1 is a highly selective, Ca2+-dependent, Na+/K+- conducting channel and, in contrast to known cation channelrhodopsins (ChRs), is impermeable to Ca2+ ions. In human neuroblastoma cells, upon illumination, VirChR1 depolarizes the cell membrane to a level sufficient to fire neurons. It suggests its unique optogenetic potential. 1.4 Å resolution structure of OLPVR1 reveals their remarkable difference from the known channelrhodopsins and a unique ion-conducting pathway. The data suggest that viral channelrhodopsins mediate phototaxis of algae enhancing the host anabolic processes to support virus reproduction, and therefore, their key role in global phytoplankton dynamics. Less
The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level The ability to measure distances of nm is particularly important deformations arising from protein binding commonly fall within this range but the reliable measurement of such distances for a conformational ensemble remains a significant challenge Using several techniques we show that electron paramagnetic resonance EPR spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the position offers a robust and minimally perturbing tool for obtaining such measurements For two nitroxides we present results from EPR spectroscopy X-ray crystal structures of B-form ... More
The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2–10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2′ position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2′-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation. Less
Trematode infections such as schistosomiasis and fascioliasis cause significant morbidity in an estimated million people worldwide and the associated agricultural losses are estimated at more than US billion per year Current chemotherapy is limited Triosephosphate isomerase TIM an enzyme of the glycolytic pathway has emerged as a useful drug target in many parasites including Fasciola hepatica TIM FhTIM We identified novel compounds that selectively inhibit this enzyme Using microscale thermophoresis we explored the interaction between target and compounds and identified a potent interaction between the sulfonyl- -thiadiazole compound and FhTIM which showed an IC of M and a Kd of ... More
Trematode infections such as schistosomiasis and fascioliasis cause significant morbidity in an estimated 250 million people worldwide and the associated agricultural losses are estimated at more than US$ 6 billion per year. Current chemotherapy is limited. Triosephosphate isomerase (TIM), an enzyme of the glycolytic pathway, has emerged as a useful drug target in many parasites, including Fasciola hepatica TIM (FhTIM). We identified 21 novel compounds that selectively inhibit this enzyme. Using microscale thermophoresis we explored the interaction between target and compounds and identified a potent interaction between the sulfonyl-1,2,4-thiadiazole (compound 187) and FhTIM, which showed an IC50 of 5 µM and a Kd of 66 nM. In only 4 hours, this compound killed the juvenile form of F. hepatica with an IC50 of 3 µM, better than the reference drug triclabendazole (TCZ). Interestingly, we discovered in vitro inhibition of FhTIM by TCZ, with an IC50 of 7 µM suggesting a previously uncharacterized role of FhTIM in the mechanism of action of this drug. Compound 187 was also active against various developmental stages of Schistosoma mansoni. The low toxicity in vitro in different cell types and lack of acute toxicity in mice was demonstrated for this compound, as was demonstrated the efficacy of 187 in vivo in F. hepatica infected mice. Finally, we obtained the first crystal structure of FhTIM at 1.9 Å resolution which allows us using docking to suggest a mechanism of interaction between compound 187 and TIM. In conclusion, we describe a promising drug candidate to control neglected trematode infections in human and animal health. Less
Phospholipase D enzymes PLDs are ubiquitous phosphodiesterases that produce phosphatidic acid PA a key second messenger and biosynthetic building block Although an orthologous bacterial Streptomyces sp strain PMF PLD structure was solved two decades ago the molecular basis underlying the functions of the human PLD enzymes hPLD remained unclear based on this structure due to the low homology between these sequences Here we describe the first crystal structures of hPLD and hPLD catalytic domains and identify novel structural elements and functional differences between the prokaryotic and eukaryotic enzymes Furthermore structure-based mutation studies and structures of inhibitor hPLD complexes allowed us ... More
Phospholipase D enzymes (PLDs) are ubiquitous phosphodiesterases that produce phosphatidic acid (PA), a key second messenger and biosynthetic building block. Although an orthologous bacterial Streptomyces sp. strain PMF PLD structure was solved two decades ago, the molecular basis underlying the functions of the human PLD enzymes (hPLD) remained unclear based on this structure due to the low homology between these sequences. Here, we describe the first crystal structures of hPLD1 and hPLD2 catalytic domains and identify novel structural elements and functional differences between the prokaryotic and eukaryotic enzymes. Furthermore, structure-based mutation studies and structures of inhibitor–hPLD complexes allowed us to elucidate the binding modes of dual and isoform-selective inhibitors, highlight key determinants of isoenzyme selectivity and provide a basis for further structure-based drug discovery and functional characterization of this therapeutically important superfamily of enzymes. Less
Rhodopsins are the most abundant light-harvesting proteins A new family of rhodopsins heliorhodopsins HeRs was recently discovered In opposite to the known rhodopsins their N-termini face the cytoplasm HeRs structure and function remain unknown We present structures of two HeR- C states at showing its remarkable difference from all known rhodopsins Its internal extracellular part is completely hydrophobic while the cytoplasmic part comprises a cavity active site surrounded by charged amino acids and containing a cluster of water molecules presumably being a primary proton acceptor from the Schiff base At acidic pH a planar triangle molecule acetate is present in ... More
Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), was recently discovered. In opposite to the known rhodopsins their N-termini face the cytoplasm. HeRs structure and function remain unknown. We present structures of two HeR-48C12 states at 1.5 Å showing its remarkable difference from all known rhodopsins. Its internal extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (’active site’), surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH a planar triangle molecule (acetate) is present in the ‘active site’ which demonstrated its ability to maintain such anions as carbonate or nitrate. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement into fundamental redox biological processes. Less
The process of macromolecular crystallisation almost always begins by setting up crystallisation trials using commercial or other premade screens followed by cycles of optimisation where the crystallisation cocktails are focused towards a particular small region of chemical space The screening process is relatively straightforward but still requires an understanding of the plethora of commercially available screens Optimisation is complicated by requiring both the design and preparation of the appropriate secondary screens Software has been developed in the C lab to aid the process of choosing initial screens to analyse the results of the initial trials and to design and describe ... More
The process of macromolecular crystallisation almost always begins by setting up crystallisation trials using commercial or other premade screens, followed by cycles of optimisation where the crystallisation cocktails are focused towards a particular small region of chemical space. The screening process is relatively straightforward, but still requires an understanding of the plethora of commercially available screens. Optimisation is complicated by requiring both the design and preparation of the appropriate secondary screens. Software has been developed in the C3 lab to aid the process of choosing initial screens, to analyse the results of the initial trials, and to design and describe how to prepare optimisation screens. Less
In high-throughput systems the crystallization experiments require the inspection and analysis of a large number of trial images The visualization and analysis tools are needed to view and analyze the experimental results and recommend novel crystalline conditions by analyzing prior results It is essential to integrate all these components into a single system Therefore we developed Visual-X an interactive visualization software developed to aid the user for quick and efficient visualization and analysis of the results of the experiments Visual-X has a number of useful features for visualization and analysis dual plate view thumbnail and symbolic detailed well view with ... More
In high-throughput systems, the crystallization experiments require the inspection and analysis of a large number of trial images. The visualization and analysis tools are needed to view and analyze the experimental results, and recommend novel crystalline conditions by analyzing prior results. It is essential to integrate all these components into a single system. Therefore, we developed Visual-X2, an interactive visualization software developed to aid the user for quick and efficient visualization and analysis of the results of the experiments. Visual-X2 has a number of useful features for visualization and analysis: dual plate view (thumbnail and symbolic), detailed well view with scoring option, multiple-scan and time-course views, support for screening analysis based on multiple screens, three novel screen analysis methods (associative experimental design, GenScreen, and novelty methods), and generating pipetting file with a family of conditions varying concentrations based on stock concentration. Less
Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB and CB Their high sequence similarity low agonist selectivity and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications Importantly missing structural information has significantly held back the development of promising CB -selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB Here we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB and CB in complex with Gi as well as agonist-bound CB crystal structure Of important scientific and therapeutic benefit our results reveal a ... More
Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role. Less
Amorphous solid dispersions ASDs are single-phase amorphous systems where drug molecules are molecularly dispersed dissolved in a polymer matrix The molecular dispersion of the drug molecules is responsible for their improved dissolution properties Unambiguously establishing the phase behavior of the ASDs is of utmost importance In this paper we focused on the complementary nature of modulated differential scanning calorimetry m DSC and X-ray powder diffraction XRPD to elucidate the phase behavior of ASDs as demonstrated by a critical discussion of practical real-life examples observed in our research group The ASDs were manufactured by either applying a solvent-based technique spray drying ... More
Amorphous solid dispersions (ASDs) are single-phase amorphous systems, where drug molecules are molecularly dispersed (dissolved) in a polymer matrix. The molecular dispersion of the drug molecules is responsible for their improved dissolution properties. Unambiguously establishing the phase behavior of the ASDs is of utmost importance. In this paper, we focused on the complementary nature of (modulated) differential scanning calorimetry ((m)DSC) and X-ray powder diffraction (XRPD) to elucidate the phase behavior of ASDs as demonstrated by a critical discussion of practical real-life examples observed in our research group. The ASDs were manufactured by either applying a solvent-based technique (spray drying), a heat-based technique (hot melt extrusion) or mechanochemical activation (cryo-milling). The encountered limiting factors of XRPD were the lack of sensitivity for small traces of crystallinity, the impossibility to differentiate between distinct amorphous phases and its impossibility to detect nanocrystals in a polymer matrix. In addition, the limiting factors of (m)DSC were defined as the well-described heat-induced sample alteration upon heating, the interfering of residual solvent evaporation with other thermal events and the coinciding of enthalpy recovery with melting events. In all of these cases, the application of a single analytical technique would have led to erroneous conclusions, whilst the combination of (m)DSC and XRPD elucidated the true phases of the ASD. Less
There is an urgent need to develop new methods for male contraception however a major barrier to drug discovery has been the lack of validated targets and the absence of an effective high-throughput phenotypic screening system To address this deficit we developed a fully-automated robotic screening platform that provided quantitative evaluation of compound activity against two key attributes of human sperm function motility and acrosome reaction In order to accelerate contraceptive development we screened the comprehensive collection of molecules that make up the ReFRAME repurposing library comprising nearly all the small molecules that have been approved or have undergone clinical ... More
There is an urgent need to develop new methods for male contraception, however a major barrier to drug discovery has been the lack of validated targets and the absence of an effective high-throughput phenotypic screening system. To address this deficit, we developed a fully-automated robotic screening platform that provided quantitative evaluation of compound activity against two key attributes of human sperm function: motility and acrosome reaction. In order to accelerate contraceptive development, we screened the comprehensive collection of 12,000 molecules that make up the ReFRAME repurposing library, comprising nearly all the small molecules that have been approved or have undergone clinical development, or have significant preclinical profiling. We identified several compounds that potently inhibit motility representing either novel drug candidates or routes to target identification. This platform will now allow for major drug discovery programmes that address the critical gap in the contraceptive portfolio as well as uncover novel human sperm biology. Less
Carbapenem-resistant Enterobacteriaceae CRE represent an urgent threat to human health Here we report the application of several complementary whole-genome sequencing WGS technologies to characterise a hospital outbreak of blaIMP- carbapenemase-producing E hormaechei Using Illumina sequencing we determined that all outbreak strains were sequence type ST and near-identical Comparison to publicly available data linked all outbreak isolates to a isolate from the same ward suggesting an environmental source in the hospital Using Pacific Biosciences sequencing we resolved the complete context of the blaIMP- gene on a large IncHI plasmid carried by all IMP- -producing strains across different hospitals Shotgun metagenomic sequencing ... More
Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat to human health. Here we report the application of several complementary whole-genome sequencing (WGS) technologies to characterise a hospital outbreak of blaIMP-4 carbapenemase-producing E. hormaechei. Using Illumina sequencing, we determined that all outbreak strains were sequence type 90 (ST90) and near-identical. Comparison to publicly available data linked all outbreak isolates to a 2013 isolate from the same ward, suggesting an environmental source in the hospital. Using Pacific Biosciences sequencing, we resolved the complete context of the blaIMP-4 gene on a large IncHI2 plasmid carried by all IMP-4-producing strains across different hospitals. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing. Finally, Oxford Nanopore sequencing rapidly resolved the true relationship of subsequent isolates to the initial outbreak. Overall, our strategic application of three WGS technologies provided an in-depth analysis of the outbreak. Less
Developing antibody agonists targeting the human apelin receptor APJ is a promising therapeutic approach for the treatment of chronic heart failure Here we report the structure-guided discovery of a single-domain antibody sdAb agonist JN - based on the cocrystal structure of APJ with an sdAb antagonist JN the first cocrystal structure of a class A G protein coupled receptor GPCR with a functional antibody As revealed by the structure JN binds to the extracellular side of APJ makes critical contacts with the second extracellular loop and inserts the CDR into the ligand-binding pocket We converted JN into a full agonist ... More
Developing antibody agonists targeting the human apelin receptor (APJ) is a promising therapeutic approach for the treatment of chronic heart failure. Here, we report the structure-guided discovery of a single-domain antibody (sdAb) agonist JN241-9, based on the cocrystal structure of APJ with an sdAb antagonist JN241, the first cocrystal structure of a class A G protein–coupled receptor (GPCR) with a functional antibody. As revealed by the structure, JN241 binds to the extracellular side of APJ, makes critical contacts with the second extracellular loop, and inserts the CDR3 into the ligand-binding pocket. We converted JN241 into a full agonist JN241-9 by inserting a tyrosine into the CDR3. Modeling and molecular dynamics simulation shed light on JN241-9–stimulated receptor activation, providing structural insights for finding agonistic antibodies against class A GPCRs. Less
Antimicrobial resistance is a major global threat that calls for new antibiotics Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme LspA thereby compromising the integrity of the bacterial cell envelope As part of a project aimed at understanding their mechanism of action and for drug development we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus MRSA complexed with globomycin and with myxovirescin Our results reveal an instance of convergent evolution The two antibiotics possess different molecular structures Yet they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs Remarkably the ... More
Antimicrobial resistance is a major global threat that calls for new antibiotics. Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme, LspA, thereby compromising the integrity of the bacterial cell envelope. As part of a project aimed at understanding their mechanism of action and for drug development, we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) complexed with globomycin and with myxovirescin. Our results reveal an instance of convergent evolution. The two antibiotics possess different molecular structures. Yet, they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs. Remarkably, the two antibiotics superpose along nineteen contiguous atoms that interact similarly with LspA. This 19-atom motif recapitulates a part of the substrate lipoprotein in its proposed binding mode. Incorporating this motif into a scaffold with suitable pharmacokinetic properties should enable the development of effective antibiotics with built-in resistance hardiness. Less
The most abundant member of the collagen protein family collagen I also known as type I collagen COL is composed of one unique chain B and two similar chain A polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix Given the offset chain B can occupy either the leading BAA middle ABA or trailing AAB position of the triple helix yielding three isomeric biomacromolecules with different protein recognition properties Despite five decades of intensive research there is no consensus on the position of chain B in COL Here three triple-helical heterotrimers that each contain a putative ... More
The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register. Less
This chapter discusses that high-throughput HT macromolecular X-ray crystallography MX plays in the drug discovery process It focuses on the pivotal element in MX namely the diffraction experiment and describes the evolution of the main protagonists viz the crystal and the beam the means of producing each of them and of manipulating them so as to make them interact most efficiently and productively and the ways of deriving structure-based drug discovery-relevant structural information from observations of that interaction The chapter describes the all-human nine-step workflow that prevailed before systematic efforts were made to move from interactivity to automation serving as ... More
This chapter discusses that high-throughput (HT) macromolecular X-ray crystallography (MX) plays in the drug discovery process. It focuses on the pivotal element in MX, namely, the diffraction experiment, and describes the evolution of the main protagonists, viz. the crystal and the beam, the means of producing each of them and of manipulating them so as to make them interact most efficiently and productively, and the ways of deriving structure-based drug discovery-relevant structural information from observations of that interaction. The chapter describes the "all-human" nine-step workflow that prevailed before systematic efforts were made to move from interactivity to automation, serving as a "baseline" against which to motivate and describe those subsequent efforts. The ability of a crystalline specimen to yield Bragg reflections corresponding to atomic-scale detail is called its "diffraction quality" - higher quality meaning higher resolution limits. Less
Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery It provides a robustness check prior to any extensive crystal screening a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported and the different physicochemical requirements of a successful antibiotic are ... More
Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery. It provides a robustness check prior to any extensive crystal screening, a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements. The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported, and the different physicochemical requirements of a successful antibiotic are compared with traditional medicines. Less
Serial crystallography at both synchrotron and X-ray free-electron laser light sources is becoming increasingly popular However the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization These case studies aim to show i how vapour diffusion conditions can be converted into batch by ... More
Serial crystallography, at both synchrotron and X-ray free-electron laser light sources, is becoming increasingly popular. However, the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography. This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization. These case studies aim to show (i) how vapour diffusion conditions can be converted into batch by optimizing the length of time crystals take to appear; (ii) how an understanding of the crystallization phase diagram can act as a guide when designing batch crystallization protocols; and (iii) an accessible methodology when attempting to scale batch conditions to larger volumes. These methods are needed to minimize the sample preparation gap between standard rotation crystallography and dedicated serial laboratories, ultimately making serial crystallography more accessible to all crystallographers. Less
NEMO is a scaffolding protein which plays an essential role in the NF- B pathway by assembling the IKK-complex with the kinases IKK and IKK Upon activation the IKK complex phosphorylates the I B molecules leading to NF- B nuclear translocation and activation of target genes Inhibition of the NEMO IKK interaction is an attractive therapeutic paradigm for the modulation of NF- B pathway activity making NEMO a target for inhibitors design and discovery To facilitate the process of discovery and optimization of NEMO inhibitors we engineered an improved construct of the IKK-binding domain of NEMO that would allow for ... More
NEMO is a scaffolding protein which plays an essential role in the NF-κB pathway by assembling the IKK-complex with the kinases IKKα and IKKβ. Upon activation, the IKK complex phosphorylates the IκB molecules leading to NF-κB nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-κB pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors. Less
The hormone melatonin secreted from the pineal gland mediates multiple physiological effects including modulation of Wnt -catenin signalling The Wnt palmitoleate lipid modification is essential for its signalling activity while the carboxylesterase Notum can remove the lipid from Wnt and inactivate it Notum enzyme inhibition can therefore upregulate Wnt signalling While searching for Notum inhibitors by crystallographic fragment screening a hit compound N- - -fluoro- H-indol- -yl ethyl acetamide that is structurally similar to melatonin came to our attention We then soaked melatonin and its precursor N-acetylserotonin into Notum crystals and obtained high-resolution structures of their complexes In each of ... More
The hormone melatonin, secreted from the pineal gland, mediates multiple physiological effects including modulation of Wnt/β-catenin signalling. The Wnt palmitoleate lipid modification is essential for its signalling activity, while the carboxylesterase Notum can remove the lipid from Wnt and inactivate it. Notum enzyme inhibition can therefore upregulate Wnt signalling. While searching for Notum inhibitors by crystallographic fragment screening, a hit compound N-[2-(5-fluoro-1H-indol-3-yl)ethyl]acetamide that is structurally similar to melatonin came to our attention. We then soaked melatonin and its precursor N-acetylserotonin into Notum crystals and obtained high-resolution structures (≤1.5 Å) of their complexes. In each of the structures, two compound molecules bind with Notum: one at the enzyme's catalytic pocket, overlapping the space occupied by the acyl tail of the Wnt palmitoleate lipid, and the other at the edge of the pocket opposite the substrate entrance. Although the inhibitory activity of melatonin shown by in vitro enzyme assays is low (IC50 75 µmol/L), the structural information reported here provides a basis for the design of potent and brain accessible drugs for neurodegenerative diseases such as Alzheimer's disease, in which upregulation of Wnt signalling may be beneficial. Less
Sex is a key modifier of neurological disease outcomes Microglia are implicated in neurological diseases and modulated by microRNAs but it is unknown whether microglial microRNAs have sex-specific influences on disease We show in mice that microglial microRNA expression differs in males and females and that loss of microRNAs leads to sex-specific changes in the microglial transcriptome and tau pathology These findings suggest that microglial microRNAs influence tau pathogenesis in a sex-specific manner
In tropical iron ore regions biologically mediated reduction of crystalline iron oxides drives ongoing iron cycling that contributes to the stability of surface duricrusts This represents a biotechnological opportunity with respect to post-mining rehabilitation attempts requiring re-formation of these duricrusts However cultivated dissimilatory iron reducing bacteria typically reduce crystalline iron oxides quite poorly A glucose-fermenting microbial consortium capable of reducing at least mmol L goethite was enriched from an iron duricrust region Metagenome analysis led to the recovery of a metagenome assembled genome MAG of an iron reducer belonging to the alphaproteobacterial genus Telmatospirillum This is the first report of ... More
In tropical iron ore regions, biologically mediated reduction of crystalline iron oxides drives ongoing iron cycling that contributes to the stability of surface duricrusts. This represents a biotechnological opportunity with respect to post-mining rehabilitation attempts, requiring re-formation of these duricrusts. However, cultivated dissimilatory iron reducing bacteria typically reduce crystalline iron oxides quite poorly. A glucose-fermenting microbial consortium capable of reducing at least 27 mmol/L goethite was enriched from an iron duricrust region. Metagenome analysis led to the recovery of a metagenome assembled genome (MAG) of an iron reducer belonging to the alphaproteobacterial genus Telmatospirillum. This is the first report of iron reduction within the Telmatospirillum and the first reported genome of an iron-reducing, neutrophilic member of the Alphaproteobacteria. The Telmatospirillum MAG encodes putative metal transfer reductases (MtrA, MtrB) and a novel, multi-heme outer membrane cytochrome for extracellular electron transfer. In the presence of goethite, short chain fatty acid production shifted significantly in favor of acetate rather than propionate, indicating goethite is a hydrogen sink in the culture. Therefore, the presence of fermentative bacteria likely promotes iron reduction via hydrogen production. Stimulating microbial fermentation has potential to drive reduction of crystalline iron oxides, the rate limiting step for iron duricrust re-formation. Less
Shiga toxin Stx is the major virulence factor of Shiga toxin-producing Escherichia coli STEC Stx evolves rapidly and as such new subtypes continue to emerge that challenge the efficacy of existing disease management and surveillance strategies A new subtype Stx k was recently identified in E coli isolated from a wide range of sources including diarrheal patients animals and raw meats and was poorly detected by existing immunoassays In this study the structure of Stx kE Q was determined at resolution and the conservation of structure with Stx a was revealed A novel polyclonal antibody capable of neutralizing Stx k ... More
Shiga toxin (Stx) is the major virulence factor of Shiga toxin-producing Escherichia coli (STEC). Stx evolves rapidly and, as such, new subtypes continue to emerge that challenge the efficacy of existing disease management and surveillance strategies. A new subtype, Stx2k, was recently identified in E. coli isolated from a wide range of sources including diarrheal patients, animals, and raw meats, and was poorly detected by existing immunoassays. In this study, the structure of Stx2kE167Q was determined at 2.29 Å resolution and the conservation of structure with Stx2a was revealed. A novel polyclonal antibody capable of neutralizing Stx2k and an immunoassay, with a 10-fold increase in sensitivity compared to assays using extant antibodies, were developed. Stx2k is less toxic than Stx2a in Vero cell assays but is similar to Stx2a in receptor-binding preference, thermostability, and acid tolerance. Although Stx2k does not appear to be as potent as Stx2a to Vero cells, the wide distribution and blended virulence profiles of the Stx2k-producing strains suggest that horizontal gene transfer through Stx2k-converting phages could result in the emergence of new and highly virulent pathogens. This study provides useful information and tools for early detection and control of Stx2k-producing E. coli, which could reduce public risk of infection by less-known STECs. Less
Membrane integral ATP synthases produce adenosine triphosphate the universal energy currency of most organisms However important details of proton driven energy conversion are still unknown We present the first high-resolution structure of the in meso crystallized c-ring of subunits from spinach chloroplasts The structure reveals molecular mechanisms of intersubunit contacts in the c -ring and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane Similar densities were found in all known high-resolution structures of c-rings of F FO ATP synthases from archaea and bacteria to eukaryotes The densities might originate from isoprenoid quinones ... More
Membrane integral ATP synthases produce adenosine triphosphate, the universal “energy currency” of most organisms. However, important details of proton driven energy conversion are still unknown. We present the first high-resolution structure (2.3 Å) of the in meso crystallized c-ring of 14 subunits from spinach chloroplasts. The structure reveals molecular mechanisms of intersubunit contacts in the c14-ring, and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane. Similar densities were found in all known high-resolution structures of c-rings of F1FO ATP synthases from archaea and bacteria to eukaryotes. The densities might originate from isoprenoid quinones (such as coenzyme Q in mitochondria and plastoquinone in chloroplasts) that is consistent with differential UV-Vis spectroscopy of the c-ring samples, unusually large distance between polar/apolar interfaces inside the c-ring and universality among different species. Although additional experiments are required to verify this hypothesis, coenzyme Q and its analogues known as electron carriers of bioenergetic chains may be universal cofactors of ATP synthases, stabilizing c-ring and prevent ion leakage through it. Less
Cysteinyl leukotriene G protein-coupled receptors CysLT and CysLT regulate pro-inflammatory responses associated with allergic disorders While selective inhibition of CysLT R has been used for treating asthma and associated diseases for over two decades CysLT R has recently started to emerge as a potential drug target against atopic asthma brain injury and central nervous system disorders as well as several types of cancer Here we describe four crystal structures of CysLT R in complex with three dual CysLT R CysLT R antagonists The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular ... More
Cysteinyl leukotriene G protein-coupled receptors CysLT1 and CysLT2 regulate pro-inflammatory responses associated with allergic disorders. While selective inhibition of CysLT1R has been used for treating asthma and associated diseases for over two decades, CysLT2R has recently started to emerge as a potential drug target against atopic asthma, brain injury and central nervous system disorders, as well as several types of cancer. Here, we describe four crystal structures of CysLT2R in complex with three dual CysLT1R/CysLT2R antagonists. The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular determinants of CysLTR ligand selectivity and specific effects of disease-related single nucleotide variants. Less
The availability of whole-genome sequence data made possible by significant advances in DNA sequencing technology led to the emergence of structural genomics projects in the late s These projects not only significantly increased the number of D structures deposited in the Protein Data Bank in the last two decades but also influenced present crystallographic strategies by introducing automation and high-throughput approaches in the structure-determination pipeline Today dedicated crystallization facilities many of which are open to the general user community routinely set up and track thousands of crystallization screening trials per day Here we review the current methods for high-throughput crystallization ... More
The availability of whole-genome sequence data, made possible by significant advances in DNA sequencing technology, led to the emergence of structural genomics projects in the late 1990s. These projects not only significantly increased the number of 3D structures deposited in the Protein Data Bank in the last two decades, but also influenced present crystallographic strategies by introducing automation and high-throughput approaches in the structure-determination pipeline. Today, dedicated crystallization facilities, many of which are open to the general user community, routinely set up and track thousands of crystallization screening trials per day. Here, we review the current methods for high-throughput crystallization and procedures to obtain crystals suitable for X-ray diffraction studies, and we describe the crystallization pipeline implemented in the medium-scale crystallography platform at the Institut Pasteur (Paris) as an example. Less
Adrenergic G-protein-coupled receptors GPCRs mediate different cellular signaling pathways in the presence of endogenous catecholamines and play important roles in both physiological and pathological conditions Extensive studies have been carried out to investigate the structure and function of adrenergic receptors ARs However the structure of a adrenergic receptors aARs remains to be determined Here we report the structure of the human a C adrenergic receptor a CAR with the non-selective antagonist RS at Our structure mutations modeling and functional experiments indicate that a a CAR-specific D ECL -R ECL -Y network plays a role in determining a adrenergic subtype selectivity ... More
Adrenergic G-protein-coupled receptors (GPCRs) mediate different cellular signaling pathways in the presence of endogenous catecholamines and play important roles in both physiological and pathological conditions. Extensive studies have been carried out to investigate the structure and function of � adrenergic receptors (�ARs). However, the structure of a adrenergic receptors (aARs) remains to be determined. Here, we report the structure of the human a2C adrenergic receptor (a2CAR) with the non-selective antagonist, RS79948, at 2.8 �. Our structure, mutations, modeling, and functional experiments indicate that a a2CAR-specific D206ECL2-R409ECL3-Y4056.58 network plays a role in determining a2 adrenergic subtype selectivity. Furthermore, our results show that a specific loosened helix at the top of TM4 in a2CAR is involved in receptor activation. Together, our structure of human a2CAR-RS79948 provides key insight into the mechanism underlying the a2 adrenergic receptor activation and subtype selectivity. Less
Broadly HIV- neutralizing VRC class antibodies target the CD -binding site of Env They are derived from VH - antibody heavy chains paired with rare light chains expressing -amino acid-long CDRL s They have been isolated from infected subjects but have not yet been elicited by immunization Env-derived immunogens capable of binding the germline forms of VRC B cell receptors on naive B cells have been designed and evaluated in knockin mice However the elicited antibodies cannot bypass glycans present on the conserved position N of Env which restricts access to the CD -binding site Efforts to guide the appropriate ... More
Broadly HIV-1 neutralizing VRC01 class antibodies target the CD4-binding site of Env. They are derived from VH1-2*02 antibody heavy chains paired with rare light chains expressing 5-amino acid-long CDRL3s. They have been isolated from infected subjects but have not yet been elicited by immunization. Env-derived immunogens capable of binding the germline forms of VRC01 B cell receptors on naive B cells have been designed and evaluated in knockin mice. However, the elicited antibodies cannot bypass glycans present on the conserved position N276 of Env, which restricts access to the CD4-binding site. Efforts to guide the appropriate maturation of these antibodies by sequential immunization have not yet been successful. Here, we report on a two-step immunization scheme that leads to the maturation of VRC01-like antibodies capable of accommodating the N276 glycan and displaying autologous tier 2 neutralizing activities. Our results are relevant to clinical trials aiming to elicit VRC01 antibodies. Less
Adrenergic receptors are highly homologous while at the same time display a wide diversity of ligand and G-protein binding and understanding this diversity is key for designing selective or biased drugs for them Here we determine two crystal structures of the a A adrenergic receptor a AAR in complex with a partial agonist and an antagonist Key non-conserved residues from the ligand-binding pocket Phe and Tyr to G-protein coupling region Ile and Lys are discovered to play a key role in the interplay between partial agonism and biased signaling of a AAR which provides insights into the diversity of ligand ... More
Adrenergic receptors are highly homologous while at the same time display a wide diversity of ligand and G-protein binding, and understanding this diversity is key for designing selective or biased drugs for them. Here, we determine two crystal structures of the a2A adrenergic receptor (a2AAR) in complex with a partial agonist and an antagonist. Key non-conserved residues from the ligand-binding pocket (Phe7.39 and Tyr6.55) to G-protein coupling region (Ile34.51 and Lys34.56) are discovered to play a key role in the interplay between partial agonism and biased signaling of a2AAR, which provides insights into the diversity of ligand binding and G-protein coupling preference of adrenergic receptors and lays the foundation for the discovery of next-generation drugs targeting these receptors. Less
Crystallization processes have been widely used in the pharmaceutical industry for the manufacture storage and delivery of small-molecule and small protein therapeutics However the identification of crystallization processes for biologics particularly monoclonal antibodies has been prohibitive due to the size and the flexibility of their overall structure There remains a challenge and an opportunity to utilize the benefits of crystallization of biologics The research laboratories of Merck Sharp Dome Corp MSD in collaboration with the International Space Station ISS National Laboratory performed crystallization experiments with pembrolizumab Keytruda on the SpaceX-Commercial Resupply Services- mission to the ISS By leveraging microgravity effects ... More
Crystallization processes have been widely used in the pharmaceutical industry for the manufacture, storage, and delivery of small-molecule and small protein therapeutics. However, the identification of crystallization processes for biologics, particularly monoclonal antibodies, has been prohibitive due to the size and the flexibility of their overall structure. There remains a challenge and an opportunity to utilize the benefits of crystallization of biologics. The research laboratories of Merck Sharp & Dome Corp. (MSD) in collaboration with the International Space Station (ISS) National Laboratory performed crystallization experiments with pembrolizumab (Keytruda�) on the SpaceX-Commercial Resupply Services-10 mission to the ISS. By leveraging microgravity effects such as reduced sedimentation and minimal convection currents, conditions producing crystalline suspensions of homogeneous monomodal particle size distribution (39 �m) in high yield were identified. In contrast, the control ground experiments produced crystalline suspensions with a heterogeneous bimodal distribution of 13 and 102 �m particles. In addition, the flight crystalline suspensions were less viscous and sedimented more uniformly than the comparable ground-based crystalline suspensions. These results have been applied to the production of crystalline suspensions on earth, using rotational mixers to reduce sedimentation and temperature gradients to induce and control crystallization. Using these techniques, we have been able to produce uniform crystalline suspensions (1�5 �m) with acceptable viscosity (<12 cP), rheological, and syringeability properties suitable for the preparation of an injectable formulation. The results of these studies may help widen the drug delivery options to improve the safety, adherence, and quality of life for patients and caregivers. Less
Reaching the full potential of optoelectronic materials is often hindered by the years of necessary trial-and-error Perovskites are an example of materials having exceptional optoelectronic properties but require improvement with respect to stability and toxicity as they approach commercialization Exploring new types of perovskites is key to achieving these goals In this thesis I develop an accelerated materials discovery pipeline aimed at discovering new perovskite materials This pipeline incorporates image recognition that detects crystals via convolutional neural networks with accuracy and uses parameter exploration to predict an optimal material with experimental data With this framework I discovered a new type ... More
Reaching the full potential of optoelectronic materials is often hindered by the years of necessary trial-and-error. Perovskites are an example of materials having exceptional optoelectronic properties, but require improvement with respect to stability and toxicity as they approach commercialization. Exploring new types of perovskites is key to achieving these goals. In this thesis I develop an accelerated materials discovery pipeline aimed at discovering new perovskite materials. This pipeline incorporates image recognition that detects crystals via convolutional neural networks with 95% accuracy and uses parameter exploration to predict an optimal material with experimental data. With this framework, I discovered a new type of perovskite single crystal, (3-PLA)2PbCl4, that employs a new ligand, 3-PLA, offering avenues to higher efficiency and more stable devices. This work develops a framework for discovering and optimizing materials in a wide chemical space and provides the groundwork for identifying new materials that lie beyond known chemical spaces. Less
Selective activation of the -opioid receptor DOP has great potential for the treatment of chronic pain benefitting from ancillary anxiolytic and antidepressant-like effects Moreover DOP agonists show reduced adverse effects as compared to -opioid receptor MOP agonists that are in the spotlight of the current opioid crisis Here we report the first crystal structures of the DOP in an activated state in complex with two relevant and structurally diverse agonists the potent opioid agonist peptide KGCHM and the small-molecule agonist DPI- at and resolution respectively Our study identifies key determinants for agonist recognition receptor activation and DOP selectivity revealing crucial ... More
Selective activation of the δ-opioid receptor (DOP) has great potential for the treatment of chronic pain, benefitting from ancillary anxiolytic and antidepressant-like effects. Moreover, DOP agonists show reduced adverse effects as compared to μ-opioid receptor (MOP) agonists that are in the spotlight of the current “opioid crisis.” Here, we report the first crystal structures of the DOP in an activated state, in complex with two relevant and structurally diverse agonists: the potent opioid agonist peptide KGCHM07 and the small-molecule agonist DPI-287 at 2.8 and 3.3 Å resolution, respectively. Our study identifies key determinants for agonist recognition, receptor activation, and DOP selectivity, revealing crucial differences between both agonist scaffolds. Our findings provide the first investigation into atomic-scale agonist binding at the DOP, supported by site-directed mutagenesis and pharmacological characterization. These structures will underpin the future structure-based development of DOP agonists for an improved pain treatment with fewer adverse effects. Less
The release of synthetic chemical pollutants in the environment is posing serious health risks Enzymes including oxygenases play a crucial role in xenobiotic degradation In the present study we employed a functional metagenomics approach to overcome the limitation of cultivability of microbes under standard laboratory conditions in order to isolate novel dioxygenases capable of degrading recalcitrant pollutants Fosmid clones possessing dioxygenase activity were further sequenced and their genes were identified using bioinformatics tools Two positive fosmid clones SD and RW suggested the presence of -dihydroxybiphenyl -dioxygenase BphC-SD and catechol -dioxygenase C O-RW respectively Recombinant versions of these enzymes were purified ... More
The release of synthetic chemical pollutants in the environment is posing serious health risks. Enzymes, including oxygenases, play a crucial role in xenobiotic degradation. In the present study, we employed a functional metagenomics approach to overcome the limitation of cultivability of microbes under standard laboratory conditions in order to isolate novel dioxygenases capable of degrading recalcitrant pollutants. Fosmid clones possessing dioxygenase activity were further sequenced, and their genes were identified using bioinformatics tools. Two positive fosmid clones, SD3 and RW1, suggested the presence of 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC-SD3) and catechol 2,3-dioxygenase (C23O-RW1), respectively. Recombinant versions of these enzymes were purified to examine their pollutant-degrading abilities. The crystal structure of BphC-SD3 was determined at 2.6-Å resolution, revealing a two-domain architecture, i.e., N-terminal and C-terminal domains, with the sequential arrangement of βαβββ in each domain, characteristic of Fe-dependent class II type I extradiol dioxygenases. The structure also reveals the presence of conserved amino acids lining the catalytic pocket and Fe3+ metal ion in the large funnel-shaped active site in the C-terminal domain. Further studies suggest that Fe3+ bound in the BphC-SD3 active site probably imparts aerobic stability. We further demonstrate the potential application of BphC-SD3 in biosensing of catecholic compounds. The halotolerant and oxygen-resistant properties of these enzymes reported in this study make them potential candidates for bioremediation and biosensing applications. Less
Staphylococcus aureus strains produce a unique family of immunostimulatory exotoxins termed as bacterial superantigens SAgs which cross-link major histocompatibility complex class II MHC II molecule and T-cell receptor TCR to stimulate large numbers of T cells at extremely low concentrations SAgs are associated with food poisoning and toxic shock syndrome To date genetically distinct staphylococcal SAgs have been reported This study reports the first X-ray structure of newly characterized staphylococcal enterotoxin N SEN SEN possesses the classical two domain architecture that includes an N-terminal oligonucleotide-binding fold and a C-terminal -grasp domain Amino acid and structure alignments revealed that several critical ... More
Staphylococcus aureus strains produce a unique family of immunostimulatory exotoxins termed as bacterial superantigens (SAgs), which cross-link major histocompatibility complex class II (MHC II) molecule and T-cell receptor (TCR) to stimulate large numbers of T cells at extremely low concentrations. SAgs are associated with food poisoning and toxic shock syndrome. To date, 26 genetically distinct staphylococcal SAgs have been reported. This study reports the first X-ray structure of newly characterized staphylococcal enterotoxin N (SEN). SEN possesses the classical two domain architecture that includes an N-terminal oligonucleotide-binding fold and a C-terminal β-grasp domain. Amino acid and structure alignments revealed that several critical amino acids that are proposed to be responsible for MHC II and TCR molecule engagements are variable in SEN, suggesting that SEN may adopt a different binding mode to its cellular receptors. This work helps better understand the mechanisms of action of SAgs. Less
Short-term parameters correlating to long-term protein stability such as the protein cloud point temperature Tcloud are of interest to improve efficiency during protein product development Such efficiency is reached if short-term parameters are obtained in a low volume and high-throughput HT manner This study presents a low volume HT detection method for sub-zero Tcloud determination of lysozyme as such an experimental method is not available yet The setup consists of a cryogenic device with an automated imaging system Measurement reproducibility median absolute deviation of C and literature-based parameter validation Pearson correlation coefficient of were shown by a robustness and validation ... More
Short-term parameters correlating to long-term protein stability, such as the protein cloud point temperature (Tcloud), are of interest to improve efficiency during protein product development. Such efficiency is reached if short-term parameters are obtained in a low volume and high-throughput (HT) manner. This study presents a low volume HT detection method for (sub-zero) Tcloud determination of lysozyme, as such an experimental method is not available yet. The setup consists of a cryogenic device with an automated imaging system. Measurement reproducibility (median absolute deviation of 0.2 °C) and literature-based parameter validation (Pearson correlation coefficient of 0.996) were shown by a robustness and validation study. The subsequent case study demonstrated a partial correlation between the obtained apparent Tcloud parameter and long-term protein stability as a function of lysozyme concentration, ion type, ionic strength, and freeze/thaw stress. The presented experimental setup demonstrates its ability to advance short-term strategies for efficient protein formulation development. Less
The selective downregulation of activated intracellular proteins is a key challenge in cell biology RHO small GTPases switch between a guanosine diphosphate GDP -bound and a guanosine triphosphate GTP -bound state that drives downstream signaling At present no tool is available to study endogenous RHO-GTPinduced conformational changes in live cells Here we established a cell-based screen to selectively degrade RHOB-GTP using F-box-intracellular single-domain antibody fusion We identified one intracellular antibody intrabody that shows selective targeting of endogenous RHOB-GTP mediated by interactions between the CDR loop of the domain antibody and the GTP-binding pocket of RHOB Our results suggest that while ... More
The selective downregulation of activated intracellular proteins is a key challenge in cell biology. RHO small GTPases switch between a guanosine diphosphate (GDP)-bound and a guanosine triphosphate (GTP)-bound state that drives downstream signaling. At present, no tool is available to study endogenous RHO-GTPinduced conformational changes in live cells. Here, we established a cell-based screen to selectively degrade RHOB-GTP using F-box-intracellular single-domain antibody fusion. We identified one intracellular antibody (intrabody) that shows selective targeting of endogenous RHOB-GTP mediated by interactions between the CDR3 loop of the domain antibody and the GTP-binding pocket of RHOB. Our results suggest that, while RHOB is highly regulated at the expression level, only the GTP-bound pool, but not its global expression, mediates RHOB functions in genomic instability and in cell invasion. The F-box/intrabody-targeted protein degradation represents a unique approach to knock down the active form of small GTPases or other proteins with multiple cellular activities. Less
The over-expression and aggregation of -synuclein Syn are linked to the onset and pathology of Parkinson s disease Native monomeric Syn exists in an intrinsically disordered ensemble of interconverting conformations which has made its therapeutic targeting by small molecules highly challenging Nonetheless here we successfully target the monomeric structural ensemble of Syn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes Using a surface plasmon resonance high-throughput screen in which monomeric Syn is incubated with microchips arrayed with tethered compounds we identified novel Syn interacting drug-like compounds Because these small molecules could impact a variety of Syn ... More
The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson’s disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson’s disease. Less
In vitro models cell culture models microtissue organ-on-a-chip microfabrication micropumps membrane manufacturing filtration vascularization perfusion microfluidics channels cleanroom silicon glass polydimethylsiloxane additive manufacturing bioprinting bioprinter three-dimensional printing bioreactor stimulation biointerfaces scaffold barrier junction sterilization biodegradable polymers functionalization hydrogels coatings etching photolithography microinjection fluid control automation disposable autoclavable media exchange sampling pipetting viscous drag pump peristaltic pump capillary pump valves bubble traps flow sensor pressure sensor noninvasive monitoring oxygen sensor pH sensor mechanotransduction shear stress compression tensile stress gauge sensor TEER electrochemical sensor biosensor calibration immunosensor ion-sensitive field-effect transistor electrical impedance spectroscopy label-free sensor lactate sensor glucose sensor temperature sensor
The elicitation of broadly neutralizing antibodies bNAbs against the HIV- envelope glycoprotein Env trimer remains a major vaccine challenge Most cross-conserved protein determinants are occluded by self-N-glycan shielding limiting B cell recognition of the underlying polypeptide surface The exceptions to the contiguous glycan shield include the conserved receptor CD binding site CD bs and glycoprotein gp elements proximal to the furin cleavage site Accordingly we performed heterologous trimer-liposome prime boosting in rabbits to drive B cells specific for cross-conserved sites To preferentially expose the CD bs to B cells we eliminated proximal N-glycans while maintaining the native-like state of the ... More
The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination Less