1209 Citations
There is an increasing demand for rapid effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray free-electron lasers or high-intensity micro-focus synchrotron radiation sources Here we demonstrate a compact multimodal multiphoton microscope driven by a fiber-based ultrafast laser enabling excitation wavelengths at nm and nm for nonlinear optical imaging which simultaneously records second-harmonic generation third-harmonic generation and three-photon excited ultraviolet fluorescence to identify and detect protein crystals with high sensitivity The instrument serves as a valuable and important tool supporting sample scoring and sample optimization in biomolecular crystallography which we hope ... More
There is an increasing demand for rapid, effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray free-electron lasers or high-intensity micro-focus synchrotron radiation sources. Here, we demonstrate a compact multimodal, multiphoton microscope, driven by a fiber-based ultrafast laser, enabling excitation wavelengths at 775 nm and 1300 nm for nonlinear optical imaging, which simultaneously records second-harmonic generation, third-harmonic generation and three-photon excited ultraviolet fluorescence to identify and detect protein crystals with high sensitivity. The instrument serves as a valuable and important tool supporting sample scoring and sample optimization in biomolecular crystallography, which we hope will increase the capabilities and productivity of serial diffraction data collection in the future. Less
Mutations in the calcium-binding protein calsequestrin cause a highly lethal familial arrhythmia catecholaminergic polymorphic ventricular tachycardia CPVT In vivo calsequestrin multimerizes into filaments but a compelling atomic-resolution structure of a calsequestrin filament is lacking We report a crystal structure of a cardiac calsequestrin filament with supporting mutation analysis provided by an in vitro fomentation assay We also report and characterize a novel disease-associated calsequestrin mutation S I which localizes to the filament-forming interface In addition we show that a previously reported dominant disease mutation K R maps to the same multimerization surface Both mutations disrupt filamentation suggesting that dominant disease ... More
Mutations in the calcium-binding protein calsequestrin cause a highly lethal familial arrhythmia, catecholaminergic polymorphic ventricular tachycardia (CPVT). In vivo, calsequestrin multimerizes into filaments, but a compelling atomic-resolution structure of a calsequestrin filament is lacking. We report a crystal structure of a cardiac calsequestrin filament with supporting mutation analysis provided by an in vitro fomentation assay. We also report and characterize a novel disease-associated calsequestrin mutation, S173I, which localizes to the filament-forming interface. In addition, we show that a previously reported dominant disease mutation, K180R, maps to the same multimerization surface. Both mutations disrupt filamentation, suggesting that dominant disease arises from defects in multimer formation. A ytterbium-derivatized structure pinpoints multiple credible calcium sites at filament-forming interfaces, explaining the atomic basis of calsequestrin filamentation in the presence of calcium. This work advances our understanding of calsequestrin biochemistry and provides a unifying structure-function molecular mechanism by which dominant-acting calsequestrin mutations provoke lethal arrhythmias. Less
Bright yellow BY- tobacco cells combined with the XVE chemically inducible system are one of the most promising plant-based platforms for recombinant protein production This offers a range of benefits including the separation of the cell growth and heterologous gene expression lack of risk of infecting the end product with prions and human viruses or appropriate protein glycosylation and folding However low protein productivity remains a major obstacle that limits the extensive commercialization of bioproduction in plants A number of molecular cell culture and down processing approaches have been made to overcome this problem Media development for the specific nutritional ... More
Bright yellow (BY-2) tobacco cells combined with the XVE chemically inducible system are one of the most promising plant-based platforms for recombinant protein production. This offers a range of benefits, including the separation of the cell growth and heterologous gene expression, lack of risk of infecting the end product with prions and human viruses or appropriate protein glycosylation and folding. However, low protein productivity remains a major obstacle that limits the extensive commercialization of bioproduction in plants. A number of molecular, cell culture and down processing approaches have been made to overcome this problem. Media development for the specific nutritional and hormonal requirements of transgenic plant cells is one of the most efficient cell-culture approaches. We optimized the induction medium towards recombinant protein production in BY-2 and demonstrated the usefulness of evolutionary medium optimization for high-yield protein production in liquid plant cultures. A reliable XVE/GFP model, parallel conducting experiments in a microscale on 96-well plates, and dedicated Gene Game evolutionary optimization software allowed for an effective search of 7611 possible solutions of 11-component media. Within the 4608 formulations tested, the Induct X medium was found with a significant 107.14% increase in protein expression in relation to the standard BY-2 medium. Less
Fungal aryl-alcohol oxidases AAOx are extracellular flavoenzymes that belong to glucose-methanol-choline oxidoreductase family and are responsible for the selective conversion of primary aromatic alcohols into aldehydes and aromatic aldehydes to their corresponding acids with concomitant production of hydrogen peroxide H O as by-product The H O can be provided to lignin degradation pathway a biotechnological property explored in biofuel production In the thermophilic fungus Thermothelomyces thermophilus formerly Myceliophthora thermophila just one AAOx was identified in the exo-proteome
Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates one from the main group M and the other from the minor group O of HIV- we identify in the C-terminal domain CTD of integrase a new functional motif constituted by four non-contiguous amino acids N K N K Mutating the lysines abolishes integration through decreased -processing and inefficient nuclear import of reverse transcribed genomes Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration The number of charges in the motif appears ... More
Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four non-contiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3’-processing and inefficient nuclear import of reverse transcribed genomes. Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K could be permutated or additional K could be inserted in the motif, generally without affecting integration per se. Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions integrase exerts. We propose that the existence of several amino acids arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution. Less
Immuno-oncology approaches that utilize T cell receptors TCRs are becoming highly attractive because of their potential to target virtually all cellular proteins including cancer-specific epitopes via the recognition of peptide-human leukocyte antigen pHLA complexes presented at the cell surface However because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities efforts have been made to enhance their binding strength in some cases by several million-fold In this study we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors Additionally we performed molecular dynamics simulations ... More
Immuno-oncology approaches that utilize T cell receptors (TCRs) are becoming highly attractive because of their potential to target virtually all cellular proteins, including cancer-specific epitopes, via the recognition of peptide-human leukocyte antigen (pHLA) complexes presented at the cell surface. However, because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities, efforts have been made to enhance their binding strength, in some cases by several million-fold. In this study, we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors. Additionally, we performed molecular dynamics simulations to better understand the energetic mechanisms driving the affinity enhancements. These data demonstrate that supra-physiological binding affinities can be achieved without altering native TCR-pHLA binding modes via relatively subtle modifications to the interface contacts, often driven through the addition of buried hydrophobic residues. Individual energetic components of the TCR-pHLA interaction governing affinity enhancements were distinct and highly variable for each TCR, often resulting from additive, or knock-on, effects beyond the mutated residues. This comprehensive analysis of affinity-enhanced TCRs has important implications for the future rational design of engineered TCRs as efficacious and safe drugs for cancer treatment. Less
The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases EHs reveals its resourceful microscopic machinery The whole-genome sequence of Streptomyces sp CBMAI an endophytic actinobacterium isolated from Citrus sinensis branches was explored by genome mining and a putative -epoxide hydrolase named B EPH and encoded by amino acids was selected for functional and structural studies The crystal structure of B EPH was obtained at a resolution of and ... More
The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities. The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases (EHs) reveals its resourceful microscopic machinery. The whole-genome sequence of Streptomyces sp. CBMAI 2042, an endophytic actinobacterium isolated from Citrus sinensis branches, was explored by genome mining, and a putative α/β-epoxide hydrolase named B1EPH2 and encoded by 344 amino acids was selected for functional and structural studies. The crystal structure of B1EPH2 was obtained at a resolution of 2.2 Å and it was found to have a similar fold to other EHs, despite its hexameric quaternary structure, which contrasts with previously solved dimeric and monomeric EH structures. While B1EPH2 has a high sequence similarity to EHB from Mycobacterium tuberculosis, its cavity is similar to that of human EH. A group of 12 aromatic and aliphatic racemic epoxides were assayed to determine the activity of B1EPH2; remarkably, this enzyme was able to hydrolyse all the epoxides to the respective 1,2-diols, indicating a wide-range substrate scope acceptance. Moreover, the (R)- and (S)-enantiomers of styrene oxide, epichlorohydrin and 1,2-epoxybutane were used to monitor enantiopreference. Taken together, the functional and structural analyses indicate that this enzyme is an attractive biocatalyst for future biotechnological applications. Less
Cation-chloride cotransporters CCCs regulate the movement of chloride across membranes controlling physiological processes from cell volume maintenance to neuronal signaling Human CCCs are clinical targets for existing diuretics and potentially additional indications Here we report the X-ray crystal structure of the soluble C-terminal regulatory domain of a eukaryotic potassium-chloride cotransporter Caenorhabditis elegans KCC- We observe a core a fold conserved among CCCs Using structure-based sequence alignment we analyze similarities and differences to the C-terminal domains of other CCC family members We find that important regulatory motifs are in less-structured regions and residues important for dimerization are not widely conserved suggesting ... More
Cation-chloride cotransporters (CCCs) regulate the movement of chloride across membranes, controlling physiological processes from cell volume maintenance to neuronal signaling. Human CCCs are clinical targets for existing diuretics and potentially additional indications. Here, we report the X-ray crystal structure of the soluble C-terminal regulatory domain of a eukaryotic potassium-chloride cotransporter, Caenorhabditis elegans KCC-1. We observe a core a/� fold conserved among CCCs. Using structure-based sequence alignment, we analyze similarities and differences to the C-terminal domains of other CCC family members. We find that important regulatory motifs are in less-structured regions and residues important for dimerization are not widely conserved, suggesting that oligomerization and its effects may vary within the larger family. This snapshot of a eukaryotic KCC is a valuable starting point for the rational design of studies of cellular chloride regulation. Less
Ageing is characterised by cellular senescence leading to imbalanced tissue maintenance cell death and compromised organ function This is first observed in the thymus the primary lymphoid organ that generates and selects T cells However the molecular and cellular mechanisms underpinning these ageing processes remain unclear Here we show that mouse ageing leads to less efficient T cell selection decreased self-antigen representation and increased T cell receptor repertoire diversity Using a combination of single-cell RNA-seq and lineage-tracing we find that progenitor cells are the principal targets of ageing whereas the function of individual mature thymic epithelial cells is compromised only ... More
Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of individual mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus. Less
Lung cancer the leading cause of cancer mortality exhibits heterogeneity that enables adaptability limits therapeutic success and remains incompletely understood Single-cell RNA sequencing scRNA-seq of metastatic lung cancer was performed using clinical biopsies obtained from patients before and during targeted therapy Over cancer and tumor microenvironment TME single-cell profiles exposed a rich and dynamic tumor ecosystem scRNA-seq of cancer cells illuminated targetable oncogenes beyond those detected clinically Cancer cells surviving therapy as residual disease RD expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell-state transition whereas those present at on-therapy progressive disease PD upregulated kynurenine plasminogen and gap-junction pathways ... More
Lung cancer, the leading cause of cancer mortality, exhibits heterogeneity that enables adaptability, limits therapeutic success, and remains incompletely understood. Single-cell RNA sequencing (scRNA-seq) of metastatic lung cancer was performed using 49 clinical biopsies obtained from 30 patients before and during targeted therapy. Over 20,000 cancer and tumor microenvironment (TME) single-cell profiles exposed a rich and dynamic tumor ecosystem. scRNA-seq of cancer cells illuminated targetable oncogenes beyond those detected clinically. Cancer cells surviving therapy as residual disease (RD) expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell-state transition, whereas those present at on-therapy progressive disease (PD) upregulated kynurenine, plasminogen, and gap-junction pathways. Active T-lymphocytes and decreased macrophages were present at RD and immunosuppressive cell states characterized PD. Biological features revealed by scRNA-seq were biomarkers of clinical outcomes in independent cohorts. This study highlights how therapy-induced adaptation of the multi-cellular ecosystem of metastatic cancer shapes clinical outcomes. Less
Pathogenic coronaviruses are a major threat to global public health as exemplified by severe acute respiratory syndrome coronavirus SARS-CoV Middle East respiratory syndrome coronavirus MERS-CoV and the newly emerged SARS-CoV- the causative agent of coronavirus disease COVID- We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus C-like protease CLpro an enzyme essential for viral replication The optimized compounds were effective against several human coronaviruses including MERS-CoV SARS-CoV and SARS-CoV- in an enzyme assay and in cell-based assays using Huh- and Vero E cell lines Two selected compounds showed antiviral effects against SARS-CoV- in cultured primary ... More
Pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses. Less
Ghrelin is a gastric peptide hormone with important physiological functions The unique feature of ghrelin is its Serine acyl-modification which is essential for ghrelin s activity However it remains to be elucidated why the acyl-modification of ghrelin is necessary for activity To address these questions we solved the crystal structure of the ghrelin receptor bound to antagonist The ligand-binding pocket of the ghrelin receptor is bifurcated by a salt bridge between E and R A striking feature of the ligand-binding pocket of the ghrelin receptor is a wide gap crevasse between the TM and TM bundles that is rich in ... More
Ghrelin is a gastric peptide hormone with important physiological functions. The unique feature of ghrelin is its Serine 3 acyl-modification, which is essential for ghrelin’s activity. However, it remains to be elucidated why the acyl-modification of ghrelin is necessary for activity. To address these questions, we solved the crystal structure of the ghrelin receptor bound to antagonist. The ligand-binding pocket of the ghrelin receptor is bifurcated by a salt bridge between E124 and R283. A striking feature of the ligand-binding pocket of the ghrelin receptor is a wide gap (crevasse) between the TM6 and TM7 bundles that is rich in hydrophobic amino acids, including a cluster of phenylalanine residues. Mutagenesis analyses suggest that the interaction between the gap structure and the acyl acid moiety of ghrelin may participate in transforming the ghrelin receptor into an active conformation. Less
In biology it is often critical to determine the identity of an organism and phenotypic traits of interest Whole-genome sequencing can be useful for this but has limited power for trait prediction However we can take advantage of the inherent information content of phenotypes to bypass these limitations We demonstrate in clinical and environmental bacterial isolates that growth dynamics in standardized conditions can differentiate between genotypes even among strains from the same species We find that for pairs of isolates there is little correlation between genetic distance according to phylogenetic analysis and phenotypic distance as determined by growth dynamics This ... More
In biology, it is often critical to determine the identity of an organism and phenotypic traits of interest. Whole-genome sequencing can be useful for this but has limited power for trait prediction. However, we can take advantage of the inherent information content of phenotypes to bypass these limitations. We demonstrate, in clinical and environmental bacterial isolates, that growth dynamics in standardized conditions can differentiate between genotypes, even among strains from the same species. We find that for pairs of isolates, there is little correlation between genetic distance, according to phylogenetic analysis, and phenotypic distance, as determined by growth dynamics. This absence of correlation underscores the challenge in using genomics to infer phenotypes and vice versa. Bypassing this complexity, we show that growth dynamics alone can robustly predict antibiotic responses. These findings are a foundation for a method to identify traits not easily traced to a genetic mechanism. Less
T cell-mediated immunity is governed primarily by T cell receptor TCR recognition of peptide-human leukocyte antigen pHLA complexes and is essential for immunosurveillance and disease control This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region CDR loops and whereas peptide selectivity is guided by direct interactions with the TCR CDR loops Here we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A MAGE-A The TCR bound pHLA in a position shifted toward the peptide's N terminus This enabled the ... More
T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide-human leukocyte antigen (pHLA) complexes and is essential for immunosurveillance and disease control. This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region (CDR) loops 1 and 2, whereas peptide selectivity is guided by direct interactions with the TCR CDR3 loops. Here, we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A4 (MAGE-A4). The TCR bound pHLA in a position shifted toward the peptide's N terminus. This enabled the TCR to achieve peptide selectivity via an indirect mechanism, whereby the TCR sensed the first residue of the peptide through HLA residue Trp-167, which acted as a tunable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp-167 side-chain conformation abrogated TCR binding, indicating that this indirect binding mechanism is essential for peptide recognition. These findings extend our understanding of the molecular rules that underpin antigen recognition by TCRs and have important implications for the development of TCR-based therapies. Less
Baculovirus mediated-insect cell expression systems have been widely used for producing heterogeneous proteins However to date there is still the lack of an easy-to-manipulate system that enables the high-throughput protein characterization in insect cells by taking advantage of large existing Gateway clone libraries To resolve this limitation we have constructed a suite of Gateway-compatible pIEx-derived baculovirus expression vectors that allow the rapid and cost-effective construction of expression clones for mass parallel protein expression in insect cells This vector collection also supports the attachment of a variety of fusion tags to target proteins to meet the needs for different research applications ... More
Baculovirus mediated-insect cell expression systems have been widely used for producing heterogeneous proteins. However, to date, there is still the lack of an easy-to-manipulate system that enables the high-throughput protein characterization in insect cells by taking advantage of large existing Gateway clone libraries. To resolve this limitation, we have constructed a suite of Gateway-compatible pIEx-derived baculovirus expression vectors that allow the rapid and cost-effective construction of expression clones for mass parallel protein expression in insect cells. This vector collection also supports the attachment of a variety of fusion tags to target proteins to meet the needs for different research applications. We first demonstrated the utility of these vectors for protein expression and purification using a set of 40 target proteins of various sizes, cellular localizations and host organisms. We then established a scalable pipeline coupled with the SONICC and TEM techniques to screen for microcrystal formation within living insect cells. Using this pipeline, we successfully identified microcrystals for ~ 16% of the tested protein set, which can be potentially used for structure elucidation by X-ray crystallography. In summary, we have established a versatile pipeline enabling parallel gene cloning, protein expression and purification, and in vivo microcrystal screening for structural studies. Less
Knowledge of both apo and holo states of riboswitches aid in elucidating the various mechanisms of ligand-induced conformational switching that underpin their gene-regulating capabilities Previous structural studies on the flavin mononucleotide FMN -binding aptamer of the FMN riboswitch however have revealed minimal conformational changes associated with ligand binding that do not adequately explain the basis for the switching behavior We have determined a - resolution crystal structure of the ligand-free FMN riboswitch aptamer that is distinct from previously reported structures particularly in the conformation and orientation of the P and P helices The nearly symmetrical tertiary structure provides a mechanism ... More
Knowledge of both apo and holo states of riboswitches aid in elucidating the various mechanisms of ligand-induced conformational “switching” that underpin their gene-regulating capabilities. Previous structural studies on the flavin mononucleotide (FMN)-binding aptamer of the FMN riboswitch, however, have revealed minimal conformational changes associated with ligand binding that do not adequately explain the basis for the switching behavior. We have determined a 2.7-Å resolution crystal structure of the ligand-free FMN riboswitch aptamer that is distinct from previously reported structures, particularly in the conformation and orientation of the P1 and P4 helices. The nearly symmetrical tertiary structure provides a mechanism by which one of two pairs of adjacent helices (P3/P4 or P1/P6) undergo collinear stacking in a mutually exclusive manner, in the absence or presence of ligand, respectively. Comparison of these structures suggests the stem-loop that includes P4 and L4 is important for maintaining a global conformational state that, in the absence of ligand, disfavors formation of the P1 regulatory helix. Together, these results provide further insight to the structural basis for conformational switching of the FMN riboswitch. Less
Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi Trypanosoma brucei and Leishmania major Tritryp published in opened up new perspectives for drug development targeting Chagas disease African sleeping sickness and Leishmaniasis neglected diseases affecting millions of most economically disadvantaged people Still half of the Tritryp genes code for proteins of unknown function Moreover almost of conserved eukaryotic protein domains are missing in the Tritryp genomes This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes Furthermore proteins without homologous counterparts in humans may provide ... More
Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major (Tritryp), published in 2005, opened up new perspectives for drug development targeting Chagas disease, African sleeping sickness and Leishmaniasis, neglected diseases affecting millions of most economically disadvantaged people. Still, half of the Tritryp genes code for proteins of unknown function. Moreover, almost 50% of conserved eukaryotic protein domains are missing in the Tritryp genomes. This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes. Furthermore, proteins without homologous counterparts in humans may provide potential targets for therapeutic intervention. Here we describe the crystal structure of the T. cruzi protein Q4D6Q6, a conserved and kinetoplastid-specific protein essential for cell viability. Q4D6Q6 is a representative of a family of 20 orthologs, all annotated as proteins of unknown function. Q4D6Q6 monomers adopt a ββαββαββ topology and form a propeller-like tetramer. Oligomerization was verified in solution using NMR, SAXS, analytical ultra-centrifugation and gel filtration chromatography. A rigorous search for similar structures using the DALI server revealed similarities with propeller-like structures of several different functions. Although a Q4D6Q6 function could not be inferred from such structural comparisons, the presence of an oxidized cysteine at position 69, part of a cluster with phosphorylated serines and hydrophobic residues, identifies a highly reactive site and suggests a role of this cysteine as a nucleophile in a post-translational modification reaction. Less
The Mantis microfluidics liquid handler was acquired as a tool for our core facility to improve our workflows and facilitate research projects for others The robot dispensing system can be employed to set up complex dilution patterns which can be advantageous to development projects The Mantis has been utilized in a proof of concept project with our Bio-Rad Droplet DigitalTM PCR ddPCRTM to determine its effectiveness in optimizing reaction primer and template concentrations Protocols were run testing the effectiveness of chip cleaning protocols in removing residual DNA from chips before reusing them in a ddPCR assay Mantis dispensing and dilution ... More
The Mantis microfluidics liquid handler was acquired as a tool for our core facility to improve our workflows and facilitate research projects for others. The robot dispensing system can be employed to set up complex dilution patterns which can be advantageous to development projects. The Mantis has been utilized in a proof of concept project with our Bio-Rad Droplet DigitalTM PCR (ddPCRTM) to determine its effectiveness in optimizing reaction primer and template concentrations. Protocols were run testing the effectiveness of chip cleaning protocols in removing residual DNA from chips before reusing them in a ddPCR assay. Mantis dispensing and dilution series were compared to manual pipetting. In addition, a variable primer setup for ddPCR was run to determine the best conditions for a ddPCR assay. The Mantis has a much speedier delivery than manual setup or our automated pipetting robot. A normalization test of dispensing one reagent with one tip to a plate in variable amounts to each well was completed in less than 2 minutes by the Mantis, while our pipetting robot took over 12 minutes. The cleaning protocols removed DNA below the sensitivity of our ddPCR. The variability of replicate counts of droplets on plate set up with the Mantis has similar spread and deviation compared to a careful manual pipetting setup of a plate. The number of reagents or samples that can be included in a protocol are limited by the number of chips loaded on the Mantis or timeliness between cleanings. Within some limitations the Mantis can be a useful instrument in a core environment. Less
The vast majority of biomolecular structural information is derived from macromolecular X-ray crystallography methods which serve as a foundation for structural biology and account for nearly of the more than biomolecular structures available in the PDB Crystallography requires high-quality well-diffracting crystals coaxing biomolecules into crystalline form is a rate-limiting step in structure determination Searching for conditions in which a biomolecule will crystallize often entails screening multiple different constructs against thousands of crystallization conditions requiring large sample amounts and many person-hours in a typical laboratory set-up In recent circumstances due to the COVID- pandemic being physically in the laboratory for setting ... More
The vast majority of biomolecular structural information is derived from macromolecular X-ray crystallography
methods, which serve as a foundation for structural biology and account for nearly 90% of the more than 165,000
biomolecular structures available in the PDB. Crystallography requires high-quality, well-diffracting crystals;
coaxing biomolecules into crystalline form is a rate-limiting step in structure determination. Searching for
conditions in which a biomolecule will crystallize often entails screening multiple different constructs against
thousands of crystallization conditions, requiring large sample amounts and many person-hours in a typical
laboratory set-up. In recent circumstances due to the COVID-19 pandemic, being physically in the laboratory for
setting up crystallization screening has become even more difficult. The Crystallization Center at HWI has been in
continuous operation as a crystallization resource for 20 years providing mail-in crystallization and remote access
to crystal growth monitoring. These services have become even more critical in the face of restrictions due to
COVID-19. The Crystallization Center is a high-throughput facility that provides expertise and access to state-ofthe-
art instrumentation to facilitate efficient and cost-effective crystallization. We have extensive robotics for
automated sample handling with very small sample volumes integrated with advanced imaging and a Formulatrix
Rock Imager with SONICC for rapid detection of crystal growth. The current pipeline in the Crystallization Center
screens for 1,536 conditions in one experimental plate and employs a robust imaging schedule, all of which is then
accessible remotely. Here, we will present details about the current capacity for high-throughput crystal growth
screening. We will also discuss innovations we are developing and opportunities for enhanced crystallization
services that will further facilitate crystallization for biomolecular structure determination, including scale up and
optimization, in situ diffraction experiments and enhanced imaging for crystal detection. Less
methods, which serve as a foundation for structural biology and account for nearly 90% of the more than 165,000
biomolecular structures available in the PDB. Crystallography requires high-quality, well-diffracting crystals;
coaxing biomolecules into crystalline form is a rate-limiting step in structure determination. Searching for
conditions in which a biomolecule will crystallize often entails screening multiple different constructs against
thousands of crystallization conditions, requiring large sample amounts and many person-hours in a typical
laboratory set-up. In recent circumstances due to the COVID-19 pandemic, being physically in the laboratory for
setting up crystallization screening has become even more difficult. The Crystallization Center at HWI has been in
continuous operation as a crystallization resource for 20 years providing mail-in crystallization and remote access
to crystal growth monitoring. These services have become even more critical in the face of restrictions due to
COVID-19. The Crystallization Center is a high-throughput facility that provides expertise and access to state-ofthe-
art instrumentation to facilitate efficient and cost-effective crystallization. We have extensive robotics for
automated sample handling with very small sample volumes integrated with advanced imaging and a Formulatrix
Rock Imager with SONICC for rapid detection of crystal growth. The current pipeline in the Crystallization Center
screens for 1,536 conditions in one experimental plate and employs a robust imaging schedule, all of which is then
accessible remotely. Here, we will present details about the current capacity for high-throughput crystal growth
screening. We will also discuss innovations we are developing and opportunities for enhanced crystallization
services that will further facilitate crystallization for biomolecular structure determination, including scale up and
optimization, in situ diffraction experiments and enhanced imaging for crystal detection. Less
Tankyrases catalyse poly-ADP-ribosylation of their binding partners and the modification serves as a signal for the subsequent proteasomal degradation of these proteins Tankyrases thereby regulate the turnover of many proteins involved in multiple and diverse cellular processes such as mitotic spindle formation telomere homeostasis and Wnt -catenin signalling In recent years tankyrases have become attractive targets for the development of inhibitors as potential therapeutics against cancer and fibrosis Further it has become clear that tankyrases are not only enzymes but also act as scaffolding proteins forming large cellular signalling complexes While many potent and selective tankyrase inhibitors of the poly-ADP-ribosylation ... More
Tankyrases catalyse poly-ADP-ribosylation of their binding partners and the modification serves as a signal for the subsequent proteasomal degradation of these proteins. Tankyrases thereby regulate the turnover of many proteins involved in multiple and diverse cellular processes, such as mitotic spindle formation, telomere homeostasis and Wnt/β-catenin signalling. In recent years, tankyrases have become attractive targets for the development of inhibitors as potential therapeutics against cancer and fibrosis. Further, it has become clear that tankyrases are not only enzymes, but also act as scaffolding proteins forming large cellular signalling complexes. While many potent and selective tankyrase inhibitors of the poly-ADP-ribosylation function exist, the inhibition of tankyrase scaffolding functions remains scarcely explored. In this work we present a robust, simple and cost-effective high-throughput screening platform based on FRET for the discovery of small molecule probes targeting the protein–protein interactions of tankyrases. Validatory screening with the platform led to the identification of two compounds with modest binding affinity to the tankyrase 2 ARC4 domain, demonstrating the applicability of this approach. The platform will facilitate identification of small molecules binding to tankyrase ARC or SAM domains and help to advance a structure-guided development of improved chemical probes targeting tankyrase oligomerization and substrate protein interactions. Less
NKp is one of the main human natural killer NK cell activating receptors used in directed immunotherapy The oligomerization of the NKp ligand binding domain depends on the length of the C-terminal stalk region but our structural knowledge of NKp oligomerization and its role in signal transduction remains limited Moreover ligand binding of NKp is affected by the presence and type of N-glycosylation In this study we assessed whether NKp oligomerization depends on its N-glycosylation Our results show that NKp forms oligomers when expressed in HEK S GnTI cell lines with simple N-glycans However NKp was detected only as monomers ... More
NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI− cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics. Less
Background Immune checkpoint inhibition ICI alone is not efficacious for a large number of patients with melanoma brain metastases We previously established an in situ vaccination ISV regimen combining radiation and immunocytokine to enhance response to ICIs Here we tested whether ISV inhibits the development of brain metastases in a murine melanoma model Methods B GD melanoma primary tumors were engrafted on the right flank of C BL mice After weeks primary tumors were treated with ISV radiation Gy day -GD immunocytokine hu -IL days and ICI -CTLA- days Complete response CR was defined as no residual tumor observed at ... More
Background Immune checkpoint inhibition (ICI) alone is not efficacious for a large number of patients with melanoma brain metastases. We previously established an in situ vaccination (ISV) regimen combining radiation and immunocytokine to enhance response to ICIs. Here, we tested whether ISV inhibits the development of brain metastases in a murine melanoma model. Methods B78 (GD2+) melanoma ‘primary’ tumors were engrafted on the right flank of C57BL/6 mice. After 3–4 weeks, primary tumors were treated with ISV (radiation (12 Gy, day 1), α-GD2 immunocytokine (hu14.18-IL2, days 6–10)) and ICI (α-CTLA-4, days 3, 6, 9). Complete response (CR) was defined as no residual tumor observed at treatment day 90. Mice with CR were tested for immune memory by re-engraftment with B78 in the left flank and then the brain. To test ISV efficacy against metastases, tumors were also engrafted in the left flank and brain of previously untreated mice. Tumors were analyzed by quantitative reverse transcription-PCR, immunohistochemistry, flow cytometry and multiplex cytokine assay. Results ISV+α-CTLA-4 resulted in immune memory and rejection of B78 engraftment in the brain in 11 of 12 mice. When B78 was engrafted in brain prior to treatment, ISV+α-CTLA-4 increased survival compared with ICI alone. ISV+α-CTLA-4 eradicated left flank tumors but did not elicit CR at brain sites when tumor cells were engrafted in brain prior to ISV. ISV+α-CTLA-4 increased CD8+ and CD4+ T cells in flank and brain tumors compared with untreated mice. Among ISV + α-CTLA-4 treated mice, left flank tumors showed increased CD8+ infiltration and CD8+:FOXP3+ ratio compared with brain tumors. Flank and brain tumors showed minimal differences in expression of immune checkpoint receptors/ligands or Mhc-1. Cytokine productions were similar in left flank and brain tumors in untreated mice. Following ISV+α-CTLA-4, production of immune-stimulatory cytokines was greater in left flank compared with brain tumor grafts. Conclusion ISV augmented response to ICIs in murine melanoma at brain and extracranial tumor sites. Although baseline tumor-immune microenvironments were similar at brain and extracranial tumor sites, response to ISV+α-CTLA-4 was divergent with reduced infiltration and activation of immune cells in brain tumors. Additional therapies may be needed for effective antitumor immune response against melanoma brain metastases. Less
Background Radiation therapy RT has the potential to enhance the efficacy of immunotherapy such as checkpoint inhibitors which has dramatically altered the landscape of treatments for many cancers but not yet for pancreatic ductal adenocarcinoma PDAC Our prior studies demonstrated that PD ligand- and indoleamine -dioxygenase IDO were induced on tumor epithelia of PDACs following neoadjuvant therapy including RT suggesting RT may prime PDAC for PD- blockade antibody PD- or IDO inhibitor IDO i treatments In this study we investigated the antitumor efficacy of the combination therapies with radiation and PD- blockade or IDO inhibition or both Methods We developed ... More
Background Radiation therapy (RT) has the potential to enhance the efficacy of immunotherapy, such as checkpoint inhibitors, which has dramatically altered the landscape of treatments for many cancers, but not yet for pancreatic ductal adenocarcinoma (PDAC). Our prior studies demonstrated that PD ligand-1 and indoleamine 2,3-dioxygenase 1 (IDO1) were induced on tumor epithelia of PDACs following neoadjuvant therapy including RT, suggesting RT may prime PDAC for PD-1 blockade antibody (αPD-1) or IDO1 inhibitor (IDO1i) treatments. In this study, we investigated the antitumor efficacy of the combination therapies with radiation and PD-1 blockade or IDO1 inhibition or both. Methods We developed and used a mouse syngeneic orthotopic model of PDAC suitable for hypofractionated RT experiments. Results The combination therapy of αPD-1 and RT improved survival. The dual combination of RT/IDO1i and triple combination of RT/αPD-1/IDO1i did not improve survival compared with RT/αPD-1, although all of these combinations offer similar local tumor control. RT/αPD-1 appeared to result in the best systemic interferon-γ response compared with other treatment groups and the highest local expression of immune-activation genes, including Cd28 and Icos. Conclusion Our RT model allows examining the immune-modulatory effects of RT alone and in combination with immune-checkpoint inhibitors in the pancreas/local microenvironment. This study highlights the importance of choosing the appropriate immune-modulatory agents to be combined with RT to tip the balance toward antitumor adaptive immune responses. Less
Sirtuins are NAD -dependent lysine deacylases which regulate various cell signaling pathways and are associated with lifespan extension through caloric restriction The human isoforms Sirt - are linked to diverse age-related diseases such as Alzheimer s Parkinson s but also cancer and AIDS Sirtuins are therefore emerging targets for therapeutic approaches and regulating sirtuin activity contextually is important While current pharmacological modulation of sirtuin activity is almost exclusively restricted to inhibitors which often lack isoform-specificity potency or bioavailability the physiological modulation of sirtuin activity by other proteins is incompletely understood This study covers important and new aspects for the physiological ... More
Sirtuins are NAD+-dependent lysine deacylases, which regulate various cell signaling pathways and are associated with lifespan extension through caloric restriction. The human isoforms Sirt1-7 are linked to diverse age-related diseases such as Alzheimer’s, Parkinson’s, but also cancer and AIDS. Sirtuins are therefore emerging targets for therapeutic approaches and regulating sirtuin activity contextually is important. While current pharmacological modulation of sirtuin activity is almost exclusively restricted to inhibitors, which often lack isoform-specificity, potency, or bioavailability, the physiological modulation of sirtuin activity by other proteins is incompletely understood. This study covers important and new aspects for the physiological and pharmacological regulation of human Sirtuin 1. Concerning regulation by small molecules, the activation of hSirt1 by dehydroabietic acid was characterized providing potential for pharmacological modulation with a new nature-derived activator scaffold. In addition, the proposed anti-tumor potential of tranilast was linked to inhibition of hSirt1. Regarding the physiological modulation of hSirt1 activity by other proteins, conflictive results were available for hAROS. Within this thesis, hAROS was validated as hSirt1 inhibitor. Furthermore, a stable core was suggested for the intrinsically disordered protein hAROS, which will be used for future interaction analyses. Conversely, an hSirt1-activity modulating effect for human Hic1 has not been described yet. Focusing on the interaction between hSirt1 and the BTB/POZ domain of Hic1, this study showed that the interaction is limited to the catalytic domain of hSirt1 and does not require phosphorylation of hSirt1, nor the presence of NAD+ or sirtuin substrate. Hic1-BTB might even have a dual and concentration-dependent effect on hSirt1 activity, which will be subject to future studies. In addition, the first crystal structure of Hic1 was solved demonstrating a typical BTB fold and offering an analysis of putative interacting regions with hSirt1. Finally, the region of hSirt1 interacting with HIV1-Tat was also identified as the catalytic domain and an additional in vitro inhibition of the structurally similar catalytic domains of hSirt2 and hSirt3 was observed. Tat binding to hSirt1-3 was competitive to the sirtuin substrate, but not to NAD+. In line with this observation, several complex structures of hSirt3 with Tat peptides could demonstrate that acetylated or deacetylated Tat binds to the sirtuin substrate binding cleft with its basic region. Using structural superpositions and crosslinking, the major contribution to binding was found to be mediated by HIV1-Tat amino acids 49-52, while Tat amino acids 53-59 provide the additional high potency needed for physiological inhibition of hSirt1 through disruption of an important salt bridge between the hSirt1 SBD and catalytic core. Less
G-protein-coupled receptors GPCRs the largest family of cell-surface membrane proteins mediate the intracellular signal transduction of many external ligands Thus GPCRs have become important drug targets X-ray crystal structures of GPCRs are very useful for structure-based drug design SBDD Herein we produced a new antibody SRP targeting the thermostabilised apocytochrome b from Escherichia coli M W H I R L BRIL We found that a fragment of this antibody SRP Fab facilitated the crystallisation of the BRIL-tagged ligand bound GPCRs HT B and AT R Furthermore the electron densities of the ligands were resolved suggesting that SPR Fab is versatile ... More
G-protein-coupled receptors (GPCRs)�the largest family of cell-surface membrane proteins�mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT1B and AT2R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them. Less
T cell recognition of peptides presented by human leukocyte antigens HLAs is mediated by the highly variable T cell receptor TCR Despite this built-in TCR variability individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD T cells Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA However few examples exist for CD T cells Here we investigate CD T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity We identify five internal epitopes that are ... More
T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies. Less
Emergent nanoscale order in materials such as self-assembled lipid phases colloidal materials and metal-organic frameworks is often characterized by small-angle X-ray scattering SAXS Frequently residual disorder in these materials prevents high-resolution D structural characterization Here we demonstrate that angular intensity variations in SAXS patterns can provide previously inaccessible information about local D structure via a rich real-space distribution of three- and four-body statistics We present the many-body characterisation of a monoolein-based hexagonal phase doped with a phospholipid revealing non-uniform curvature in the lipid channels likely due to phase separation of the lipids in the membrane Our many-body technique has general ... More
Emergent nanoscale order in materials such as self-assembled lipid phases, colloidal materials and metal-organic frameworks is often characterized by small-angle X-ray scattering (SAXS). Frequently, residual disorder in these materials prevents high-resolution 3D structural characterization. Here we demonstrate that angular intensity variations in SAXS patterns can provide previously inaccessible information about local 3D structure via a rich, real-space distribution of three- and four-body statistics. We present the many-body characterisation of a monoolein-based hexagonal phase doped with a phospholipid, revealing non-uniform curvature in the lipid channels, likely due to phase separation of the lipids in the membrane. Our many-body technique has general applicability to nanomaterials with order in the range 10 nm−1 μm currently targeted by synchrotron SAXS and has the potential to impact diverse research areas within chemistry, biology and materials science. Less
Chemokines and their receptors mediate cell migration which influences multiple fundamental biological processes and disease conditions such as inflammation and cancer Although ample effort has been invested into the structural investigation of the chemokine receptors and receptor chemokine recognition less is known about endogenous chemokine-induced receptor activation and G-protein coupling Here we present the cryo-electron microscopy structures of interleukin- IL- also known as CXCL -activated human CXC chemokine receptor CXCR in complex with Gi protein along with a crystal structure of CXCR bound to a designed allosteric antagonist Our results reveal a unique shallow mode of binding between CXCL and ... More
Chemokines and their receptors mediate cell migration, which influences multiple fundamental biological processes and disease conditions such as inflammation and cancer1. Although ample effort has been invested into the structural investigation of the chemokine receptors and receptor–chemokine recognition2,3,4, less is known about endogenous chemokine-induced receptor activation and G-protein coupling. Here we present the cryo-electron microscopy structures of interleukin-8 (IL-8, also known as CXCL8)-activated human CXC chemokine receptor 2 (CXCR2) in complex with Gi protein, along with a crystal structure of CXCR2 bound to a designed allosteric antagonist. Our results reveal a unique shallow mode of binding between CXCL8 and CXCR2, and also show the interactions between CXCR2 and Gi protein. Further structural analysis of the inactive and active states of CXCR2 reveals a distinct activation process and the competitive small-molecule antagonism of chemokine receptors. In addition, our results provide insights into how a G-protein-coupled receptor is activated by an endogenous protein molecule, which will assist in the rational development of therapeutics that target the chemokine system for better pharmacological profiles. Less
Ketol-acid reductoisomerase KARI the second enzyme in the branched-chain amino acid biosynthesis pathway is a potential drug target for bacterial infections including Mycobacterium tuberculosis Here we have screened the Medicines for Malaria Venture Pathogen Box against purified M tuberculosis Mt KARI and identified two compounds that have Ki values below nm In Mt cell susceptibility assays one of these compounds exhibited an IC value of m Co-crystallization of this compound - methylsulfonyl methyl - H-benzo b oxazin- -one MMV in complex with Staphylococcus aureus KARI which has identity with Mt KARI NADPH and Mg yielded a structure to resolution However ... More
Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway, is a potential drug target for bacterial infections including Mycobacterium tuberculosis. Here, we have screened the Medicines for Malaria Venture Pathogen Box against purified M. tuberculosis (Mt) KARI and identified two compounds that have Ki values below 200 nm. In Mt cell susceptibility assays one of these compounds exhibited an IC50 value of 0.8 μm. Co-crystallization of this compound, 3-((methylsulfonyl)methyl)-2H-benzo[b][1,4]oxazin-2-one (MMV553002), in complex with Staphylococcus aureus KARI, which has 56 % identity with Mt KARI, NADPH and Mg2+ yielded a structure to 1.72 Å resolution. However, only a hydrolyzed product of the inhibitor (i.e. 3-(methylsulfonyl)-2-oxopropanic acid, missing the 2-aminophenol attachment) is observed in the active site. Surprisingly, Mt cell susceptibility assays showed that the 2-aminophenol product is largely responsible for the anti-TB activity of the parent compound. Thus, 3-(methylsulfonyl)-2-oxopropanic acid was identified as a potent KARI inhibitor that could be further explored as a potential biocidal agent and we have shown 2-aminophenol, as an anti-TB drug lead, especially given it has low toxicity against human cells. The study highlights that careful analysis of broad screening assays is required to correctly interpret cell-based activity data. Less
Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses Viral replication requires a C-like cysteine protease CLpro which processes the kDa viral polyprotein into six functional proteins The CLpro has attracted much interest due to its potential as a target for antiviral drugs A system for growing high-quality crystals of native Southampton norovirus CLpro SV CP has been established allowing the ligand-free crystal structure to be determined to in a tetrameric state This also allowed crystal-based fragment screening to be performed with various compound libraries ultimately to guide drug discovery for SV CP A total of fragments were ... More
Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses. Viral replication requires a 3C-like cysteine protease (3CLpro) which processes the 200 kDa viral polyprotein into six functional proteins. The 3CLpro has attracted much interest due to its potential as a target for antiviral drugs. A system for growing high-quality crystals of native Southampton norovirus 3CLpro (SV3CP) has been established, allowing the ligand-free crystal structure to be determined to 1.3 Å in a tetrameric state. This also allowed crystal-based fragment screening to be performed with various compound libraries, ultimately to guide drug discovery for SV3CP. A total of 19 fragments were found to bind to the protease out of the 844 which were screened. Two of the hits were located at the active site of SV3CP and showed good inhibitory activity in kinetic assays. Another 5 were found at the enzyme’s putative RNA-binding site and a further 11 were located in the symmetric central cavity of the tetramer. Less
The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states However current RNA-seq methods are unable to simultaneously monitor both short and long poly A and poly A -transcripts at the single-cell level and thus deliver only a partial snapshot of the cellular RNAome Here we describe Smart-seq-total a method capable of assaying a broad spectrum of coding and non-coding RNA from a single cell Built upon the template-switch mechanism Smart-seq-total bears the key feature of its predecessor Smart-seq namely the ability to capture full-length transcripts ... More
The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states. However, current RNA-seq methods are unable to simultaneously monitor both short and long, poly(A)+ and poly(A)-transcripts at the single-cell level, and thus deliver only a partial snapshot of the cellular RNAome. Here, we describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and non-coding RNA from a single cell. Built upon the template-switch mechanism, Smart-seq-total bears the key feature of its predecessor, Smart-seq2, namely, the ability to capture full-length transcripts with high yield and quality. It also outperforms current poly(A)–independent total RNA-seq protocols by capturing transcripts of a broad size range, thus, allowing us to simultaneously analyze protein-coding, long non-coding, microRNA and other non-coding RNA transcripts from single cells. We used Smart-seq-total to analyze the total RNAome of human primary fibroblasts, HEK293T and MCF7 cells as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. We show that simultaneous measurement of non-coding RNA and mRNA from the same cell enables elucidation of new roles of non-coding RNA throughout essential processes such as cell cycle or lineage commitment. Moreover, we show that cell types can be distinguished based on the abundance of non-coding transcripts alone. Less
N -Methyladenosine m A is the most widespread internal messenger RNA modification in humans Despite recent progress in understanding the biological roles of m A the inability to install m A site specifically in individual transcripts has hampered efforts to elucidate causal relationships between the presence of a specific m A and phenotypic outcomes In the present study we demonstrate that nucleus-localized dCas fusions with a truncated METTL methyltransferase domain and cytoplasm-localized fusions with a modified METTL METTL methyltransferase complex can direct site-specific m A incorporation in distinct cellular compartments with the former fusion protein having particularly low off-target activity ... More
N6-Methyladenosine (m6A) is the most widespread internal messenger RNA modification in humans. Despite recent progress in understanding the biological roles of m6A, the inability to install m6A site specifically in individual transcripts has hampered efforts to elucidate causal relationships between the presence of a specific m6A and phenotypic outcomes. In the present study, we demonstrate that nucleus-localized dCas13 fusions with a truncated METTL3 methyltransferase domain and cytoplasm-localized fusions with a modified METTL3:METTL14 methyltransferase complex can direct site-specific m6A incorporation in distinct cellular compartments, with the former fusion protein having particularly low off-target activity. Independent cellular assays across multiple sites confirm that this targeted RNA methylation (TRM) system mediates efficient m6A installation in endogenous RNA transcripts with high specificity. Finally, we show that TRM can induce m6A-mediated changes to transcript abundance and alternative splicing. These findings establish TRM as a tool for targeted epitranscriptome engineering that can reveal the effect of individual m6A modifications and dissect their functional roles. Less
ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes This posttranslational modification is carried out by ADP-ribosyltransferases converting -NAD into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR The reverse reaction release of ADP-ribose from the acceptor molecule is catalyzed by ADP-ribosylhydrolases Several hydrolases contain a macrodomain fold and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation Macrodomains have been linked to diseases such as cancer making them potential drug targets Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases and here we describe ... More
ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes. This posttranslational modification is carried out by ADP-ribosyltransferases converting β-NAD+ into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR. The reverse reaction, release of ADP-ribose from the acceptor molecule, is catalyzed by ADP-ribosylhydrolases. Several hydrolases contain a macrodomain fold, and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation. Macrodomains have been linked to diseases such as cancer, making them potential drug targets. Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases, and here we describe an inhibitor screening assay against mono-ADP-ribosylhydrolyzing enzymes. The activity-based assay uses an α-NAD+, anomer of β-NAD+, which is accepted as a substrate by MacroD1, MacroD2, and ARH3 due to its resemblance to the protein-linked ADP-ribose. The amount of α-NAD+ present after hydrolysis is measured by chemically converting it on a microtiter plate to a fluorescent compound. We optimized the assay for MacroD2 and performed a proof-of-concept compound screening. Three compounds were identified as screening hits with micromolar potency. However, further characterization of the compounds identified them as protein destabilizers, excluding further follow-up studies. Validation and screening demonstrated the usability of the in vitro assay for MacroD2, and we also demonstrate the applicability of the assay as a tool for other human ADP-ribosylhydrolases. Less
The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology these effectors are known as ROPs and GRAs respectively To examine the individual impacts of ROPs and GRAs on host gene expression we developed a robust novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected U-I cells which Toxoplasma injects with ROPs but subsequently fails to invade We then performed single-cell transcriptomic analysis at to h postinfection on U-I cells as well as on uninfected and ... More
The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single-cell transcriptomic analysis at 1 to 3 h postinfection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild-type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host’s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent proinflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggest that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes. Less
The pharmaceutical industry is continuing to face high research and development R D costs and low overall success rates of clinical compounds during drug development There is an increasing demand for development and validation of healthy or disease-relevant and physiological human cellular models that can be implemented in early-stage discovery thereby shifting attrition of future therapeutics to a point in discovery at which the costs are significantly lower There needs to be a paradigm shift in the early drug discovery phase which is lengthy and costly away from simplistic cellular models that show an inability to effectively and efficiently reproduce ... More
The pharmaceutical industry is continuing to face high research and development (R&D) costs and low overall success rates of clinical compounds during drug development. There is an increasing demand for development and validation of healthy or disease-relevant and physiological human cellular models that can be implemented in early-stage discovery, thereby shifting attrition of future therapeutics to a point in discovery at which the costs are significantly lower. There needs to be a paradigm shift in the early drug discovery phase (which is lengthy and costly), away from simplistic cellular models that show an inability to effectively and efficiently reproduce healthy or human disease-relevant states to steer target and compound selection for safety, pharmacology, and efficacy questions. This perspective article covers the various stages of early drug discovery from target identification (ID) and validation to the hit/lead discovery phase, lead optimization, and preclinical safety. We outline key aspects that should be considered when developing, qualifying, and implementing complex in vitro models (CIVMs) during these phases, because criteria such as cell types (e.g., cell lines, primary cells, stem cells, and tissue), platform (e.g., spheroids, scaffolds or hydrogels, organoids, microphysiological systems, and bioprinting), throughput, automation, and single and multiplexing endpoints will vary. The article emphasizes the need to adequately qualify these CIVMs such that they are suitable for various applications (e.g., context of use) of drug discovery and translational research. The article ends looking to the future, in which there is an increase in combining computational modeling, artificial intelligence and machine learning (AI/ML), and CIVMs. Less
Interleukin- IL- is a key cytokine implicated in the pathogenesis of autoimmune disorders including psoriasis and ulcerative colitis Although targeted IL- antibody therapeutics are used clinically there are no small-molecule therapeutics that selectively inhibit IL- signaling To address this gap we developed a high-throughput screening strategy employing an IL- -responsive cell-based luciferase reporter gene assay as the primary screen with cellular cytotoxicity and off-target counter screening assays to identify IL- pathway-specific inhibitors The primary screening assay utilized avian DT cells genetically engineered to overexpress IL- R IL- R STAT and firefly luciferase in a -well format Treatment of these cells ... More
Interleukin-23 (IL-23) is a key cytokine implicated in the pathogenesis of autoimmune disorders, including psoriasis and ulcerative colitis. Although targeted IL-23 antibody therapeutics are used clinically, there are no small-molecule therapeutics that selectively inhibit IL-23 signaling. To address this gap, we developed a high-throughput screening strategy employing an IL-23-responsive cell-based luciferase reporter gene assay as the primary screen, with cellular cytotoxicity and off-target counter screening assays to identify IL-23 pathway-specific inhibitors. The primary screening assay utilized avian DT40 cells, genetically engineered to overexpress IL-23R, IL-12Rβ1, STAT5, and firefly luciferase, in a 1536-well format. Treatment of these cells with IL-23 resulted in the phosphorylation and activation of STAT5, which was completely inhibited by the pan-JAK inhibitor tofacitinib. Assay performance was robust, with signal-to-background >7-fold and Z′ > 0.5 over 40 screening plates (approximately 24,000 compounds), with a hit rate of 5% (>66.9% activity cutoff). Of these 1288 hits, 66% were identified as cytotoxic by incubating the IL-23 reporter cells with compound overnight and measuring cell viability. Further assessment of specificity via examination of impact on off-target IFN-γ signaling eliminated an additional 230 compounds, leaving 209 that were evaluated for dose–response activity. Of these compounds, 24 exhibited IC50 values of <7 µM and ≥80% inhibition of IL-23 activity, with >3-fold selectivity over IFN-γ inhibition, thus representing promising starting points for prospective IL-23 pathway small-molecule inhibitors. Less
Of the eighteen hemagglutinin HA subtypes H H that have been identified in bats and aquatic birds many HA subtypes have been structurally characterized However several subtypes H H and H still require characterization To better understand all of these HA subtypes at the molecular level HA structures from an A H N A swine Missouri A an A H N A turkey Ontario an A H N A duck Memphis an A H N A mallard Gurjev and an A H N A wedge-tailed shearwater Western Australia were determined by X-ray crystallography at and resolution respectively The interactions between ... More
Of the eighteen hemagglutinin (HA) subtypes (H1–H18) that have been identified in bats and aquatic birds, many HA subtypes have been structurally characterized. However, several subtypes (H8, H11 and H12) still require characterization. To better understand all of these HA subtypes at the molecular level, HA structures from an A(H4N6) (A/swine/Missouri/A01727926/2015), an A(H8N4) (A/turkey/Ontario/6118/1968), an A(H11N9) (A/duck/Memphis/546/1974), an A(H14N5) A/mallard/Gurjev/263/1982, and an A(H15N9) (A/wedge-tailed shearwater/Western Australia/2576/1979 were determined by X-ray crystallography at 2.2Å, 2.3Å, 2.8Å, 3.0Å and 2.5Å resolution, respectively. The interactions between these viruses and host receptors were studied utilizing glycan-binding analyses with their recombinant HA. The data show that all avian HAs retain their strict binding preference to avian receptors, whereas swine H4 has a weak human receptor binding. The molecular characterization and structural analyses of the HA from these zoonotic influenza viruses not only provide a deeper appreciation and understanding of the structure of all HA subtypes, but also re-iterate why continuous global surveillance is needed. Less
The enzyme pyridoxal kinase PdxK catalyzes the conversion of pyridoxal to pyridoxal- -phosphate PLP using ATP as the co-factor The product pyridoxal- -phosphate plays a key role in several biological processes such as transamination decarboxylation and deamination In the present study full-length ORF of PdxK from Leishmania donovani LdPdxK was cloned and then purified using affinity chromatography LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH Biochemical analysis of LdPdxK with pyridoxal pyridoxamine pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK ... More
The enzyme pyridoxal kinase (PdxK) catalyzes the conversion of pyridoxal to pyridoxal-5′-phosphate (PLP) using ATP as the co-factor. The product pyridoxal-5′-phosphate plays a key role in several biological processes such as transamination, decarboxylation and deamination. In the present study, full-length ORF of PdxK from Leishmania donovani (LdPdxK) was cloned and then purified using affinity chromatography. LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH. Biochemical analysis of LdPdxK with pyridoxal, pyridoxamine, pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates. The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK to be predominantly α-helical in organization which tends to decline at lower and higher pH. Simultaneously, LdPdxK was crystallized and its three-dimensional structure in complex with ADP and different substrates were determined. Crystal structure of LdPdxK delineated that it has a central core of β-sheets surrounded by α-helices with a conserved GTGD ribokinase motif. The structures of LdPdxK disclosed no major structural changes between ADP and ADP- substrate bound structures. In addition, comparative structural analysis highlighted the key differences between the active site pockets of leishmanial and human PdxK, rendering LdPdxK an attractive candidate for the designing of novel and specific inhibitors. Less
NS is a kDa major nonstructural protein of avian reoviruses which cause significant economic losses in the poultry industry They replicate inside viral factories in host cells and the NS protein has been suggested to be the minimal viral factor required for factory formation Thus determining the structure of NS is of great importance for understanding its role in viral infection In the study presented here a fragment consisting of residues - of NS was expressed as an EGFP fusion protein in Sf insect cells EGFP- NS - crystallization in Sf cells was monitored and verified by several imaging techniques ... More
μNS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the �NS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of �NS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448-605 of �NS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP-�NS(448-605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP-�NS(448-605) baculovirus formed rod-shaped microcrystals (5-15 �m in length) which were reconstituted in high-viscosity media (LCP and agarose) and investigated by serial femtosecond X-ray diffraction using viscous jets at an X-ray free-electron laser (XFEL). The crystals diffracted to 4.5 � resolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit-cell parameters a = 109.29, b = 110.29, c = 324.97 �. The final data set was merged and refined to 7.0 � resolution. Preliminary electron-density maps were obtained. While more diffraction data are required to solve the structure of �NS(448-605), the current experimental strategy, which couples high-viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the �NS protein. Less
Synthetic lethal screens have the potential to identify new vulnerabilities incurred by specific cancer mutations but have been hindered by lack of agreement between studies In the case of KRAS we identify that published synthetic lethal screen hits significantly overlap at the pathway rather than gene level Analysis of pathways encoded as protein networks could identify synthetic lethal candidates that are more reproducible than those previously reported Lack of overlap likely stems from biological rather than technical limitations as most synthetic lethal phenotypes are strongly modulated by changes in cellular conditions or genetic context the latter determined using a pairwise ... More
Synthetic lethal screens have the potential to identify new vulnerabilities incurred by specific cancer mutations but have been hindered by lack of agreement between studies. In the case of KRAS, we identify that published synthetic lethal screen hits significantly overlap at the pathway rather than gene level. Analysis of pathways encoded as protein networks could identify synthetic lethal candidates that are more reproducible than those previously reported. Lack of overlap likely stems from biological rather than technical limitations as most synthetic lethal phenotypes are strongly modulated by changes in cellular conditions or genetic context, the latter determined using a pairwise genetic interaction map that identifies numerous interactions that suppress synthetic lethal effects. Accounting for pathway, cellular and genetic context nominates a DNA repair dependency in KRAS-mutant cells, mediated by a network containing BRCA1. We provide evidence for why most reported synthetic lethals are not reproducible which is addressable using a multi-faceted testing framework. Less
We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime FLT detection to perform high-throughput screening HTS in living cells for discovery of potential heart-failure drugs Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump SERCA a often due to excessive inhibition by phospholamban PLB a small transmembrane protein We sought to discover small molecules that restore SERCA a activity by disrupting this inhibitory interaction between PLB and SERCA a Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer FRET to detect changes in binding or structure of the complex To ... More
We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime (FLT) detection, to perform high-throughput screening (HTS) in living cells for discovery of potential heart-failure drugs. Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump (SERCA2a), often due to excessive inhibition by phospholamban (PLB), a small transmembrane protein. We sought to discover small molecules that restore SERCA2a activity by disrupting this inhibitory interaction between PLB and SERCA2a. Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer (FRET) to detect changes in binding or structure of the complex. To optimize sensitivity to these changes, we engineered a biosensor that concatenates the two fluorescently labeled proteins on a single polypeptide chain. This SERCA2a-PLB FRET biosensor construct is functionally active and effective for HTS. By implementing 2-wavelength FLT detection at extremely high speed during primary HTS, we culled fluorescent compounds as false-positive Hits. In pilot screens, we identified Hits that alter the SERCA2a-PLB interaction, and a newly developed secondary calcium uptake assay revealed both activators and inhibitors of Ca-transport. We are implementing this approach for large-scale screens to discover new drug-like modulators of SERCA2a-PLB interactions for heart failure therapeutic development. Less
We present a deep learning-based framework to design and quantify point-of-care sensors As a use-case we demonstrated a low-cost and rapid paper-based vertical flow assay VFA for high sensitivity C-Reactive Protein hsCRP testing commonly used for assessing risk of cardio-vascular disease CVD A machine learning-based framework was developed to determine an optimal configuration of immunoreaction spots and conditions spatially-multiplexed on a sensing membrane and to accurately infer target analyte concentration Using a custom-designed handheld VFA reader a clinical study with human samples showed a competitive coefficient-of-variation of and linearity of R among blindly-tested VFAs in the hsCRP range i e ... More
We present a deep learning-based framework to design and quantify point-of-care sensors. As a use-case, we demonstrated a low-cost and rapid paper-based vertical flow assay (VFA) for high sensitivity C-Reactive Protein (hsCRP) testing, commonly used for assessing risk of cardio-vascular disease (CVD). A machine learning-based framework was developed to (1) determine an optimal configuration of immunoreaction spots and conditions, spatially-multiplexed on a sensing membrane, and (2) to accurately infer target analyte concentration. Using a custom-designed handheld VFA reader, a clinical study with 85 human samples showed a competitive coefficient-of-variation of 11.2% and linearity of R2 = 0.95 among blindly-tested VFAs in the hsCRP range (i.e., 0–10 mg/L). We also demonstrated a mitigation of the hook-effect due to the multiplexed immunoreactions on the sensing membrane. This paper-based computational VFA could expand access to CVD testing, and the presented framework can be broadly used to design cost-effective and mobile point-of-care sensors. Less
Treatments for cognitive deficits associated with central nervous system CNS disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system The nicotinic acetylcholine receptor nAChR has garnered substantial attention as a target for cognitive deficits based on receptor localization robust preclinical effects genetics implicating its involvement in cognitive disorders and encouraging albeit mixed clinical data with nAChR orthosteric agonists Importantly previous orthosteric agonists at this receptor suffered from off-target activity receptor desensitization and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility To overcome the ... More
Treatments for cognitive deficits associated with central nervous system (CNS) disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system. The α7 nicotinic acetylcholine receptor (nAChR) has garnered substantial attention as a target for cognitive deficits based on receptor localization, robust preclinical effects, genetics implicating its involvement in cognitive disorders, and encouraging, albeit mixed, clinical data with α7 nAChR orthosteric agonists. Importantly, previous orthosteric agonists at this receptor suffered from off-target activity, receptor desensitization, and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility. To overcome the challenges with orthosteric agonists, we have identified a novel selective α7 positive allosteric modulator (PAM), BNC375. This compound is selective over related receptors and potentiates acetylcholine-evoked α7 currents with only marginal effect on the receptor desensitization kinetics. In addition, BNC375 enhances long-term potentiation of electrically evoked synaptic responses in rat hippocampal slices and in vivo. Systemic administration of BNC375 reverses scopolamine-induced cognitive deficits in rat novel object recognition and rhesus monkey object retrieval detour (ORD) task over a wide range of exposures, showing no evidence of an inverted U-shaped dose-effect curve. The compound also improves performance in the ORD task in aged African green monkeys. Moreover, ex vivo 13C-NMR analysis indicates that BNC375 treatment can enhance neurotransmitter release in rat medial prefrontal cortex. These findings suggest that α7 nAChR PAMs have multiple advantages over orthosteric α7 nAChR agonists for the treatment of cognitive dysfunction associated with CNS diseases. Less
The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing Co-crystallization with proteins is a straight forward approach to overcome these challenges The human RNA-binding protein U A has previously been established as crystallization module however the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U A system Here we report three crystal structures of tryptophan-containing U A variants which expand the crystallization toolbox for nucleic acids Analysis of the structures complemented by SAXS NMR spectroscopy and optical spectroscopy allow for insights into the potential of the U ... More
The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing. Co-crystallization with proteins is a straight forward approach to overcome these challenges. The human RNA-binding protein U1A has previously been established as crystallization module, however, the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U1A system. Here, we report three crystal structures of tryptophan-containing U1A variants, which expand the crystallization toolbox for nucleic acids. Analysis of the structures complemented by SAXS, NMR spectroscopy, and optical spectroscopy allow for insights into the potential of the U1A variants to serve as crystallization modules for nucleic acids. In addition, we report a fast and efficient protocol for crystallization of RNA by soaking and present a fluorescence-based approach for detecting RNA-binding in crystallo. Our results provide a new tool set for the crystallization of RNA and RNA:DNA complexes. Less
Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea They possess highly diverse biological functions Explosion of research on microbial rhodopsins led to breakthroughs in their applications in particular in neuroscience An unexpected new discovery was a Na -pumping KR rhodopsin from Krokinobacter eikastus the first light-driven non-proton cation pump A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and therefore Na pumping cannot be understood in the framework ... More
Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea. They possess highly diverse biological functions. Explosion of research on microbial rhodopsins led to breakthroughs in their applications, in particular, in neuroscience. An unexpected new discovery was a Na+-pumping KR2 rhodopsin from Krokinobacter eikastus, the first light-driven non-proton cation pump. A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and, therefore, Na+ pumping cannot be understood in the framework of classical proton pump, like bacteriorhodopsin. Extensive studies on the molecular mechanism of KR2 failed to reveal mechanism of pumping. The existing high-resolution structures relate only to the ground state of the protein and revealed no Na+ inside the protein, which is unusual for active ion transporters. KR2 is only known non proton cation transporter with demonstrated remarkable potential for optogenetic applications and, therefore, elucidation of the mechanism of cation transport is important. To understand conception of cation pumping we solved crystal structures of the functionally key O-intermediate state of physiologically relevant pentameric form of KR2 and its D116N and H30A key mutants at high resolution and performed additional functional studies. The structure of the O-state reveals a sodium ion near the retinal Schiff base coordinated by N112 and D116 residues of the characteristic (for the whole family) NDQ triad. The structural and functional data show that cation uptake and release are driven by a switching mechanism. Surprisely, Na+ pathway in KR2 is lined with the chain of polar pores/cavities, similarly to the channelrhodopsin-2. Using Parinello fast molecular dynamics approach we obtained a molecular movie of a probable ion release. Our data provides insight into the mechanism of a non-proton cation light-driven pumping, strongly suggest close relation of sodium pumps to channel rhodopsins and, we believe, expand the present knowledge of rhodopsin world. Certainly they might be used for engineering of cation pumps and ion channels for optogenetics. Less
Membrane-embedded sensor histidine kinases HKs and chemoreceptors are used ubiquitously by bacteria and archaea to percept the environment and are often crucial for their survival and pathogenicity The proteins can transmit the signal from the sensor domain to the catalytic kinase domain reliably over the span of several hundreds of angstroms and regulate the activity of the cognate response regulator proteins with which they form two-component signaling systems TCSs Several mechanisms of transmembrane signal transduction in TCS receptors have been proposed dubbed swinging piston helical rotation and diagonal scissoring Yet despite decades of studies there is no consensus on whether ... More
Membrane-embedded sensor histidine kinases (HKs) and chemoreceptors are used ubiquitously by bacteria and archaea to percept the environment, and are often crucial for their survival and pathogenicity. The proteins can transmit the signal from the sensor domain to the catalytic kinase domain reliably over the span of several hundreds of angstroms, and regulate the activity of the cognate response regulator proteins, with which they form two-component signaling systems (TCSs). Several mechanisms of transmembrane signal transduction in TCS receptors have been proposed, dubbed (swinging) piston, helical rotation, and diagonal scissoring. Yet, despite decades of studies, there is no consensus on whether these mechanisms are common for all TCS receptors. Here, we extend our previous work on Escherichia coli nitrate/nitrite sensor kinase NarQ. We determined a crystallographic structure of the sensor-TM-HAMP fragment of the R50S mutant, which, unexpectedly, was found in a ligand-bound-like conformation, despite an inability to bind nitrate. Subsequently, we reanalyzed the structures of the ligand-free and ligand-bound NarQ and NarX sensor domains, and conducted extensive molecular dynamics simulations of ligand-free and ligand-bound wild type and mutated NarQ. Based on the data, we show that binding of nitrate to NarQ causes, first and foremost, helical rotation and diagonal scissoring of the α-helices at the core of the sensor domain. These conformational changes are accompanied by a subtle piston-like motion, which is amplified by a switch in the secondary structure of the linker between the sensor and TM domains. We conclude that helical rotation, diagonal scissoring, and piston are simply different degrees of freedom in coiled-coil proteins and are not mutually exclusive in NarQ, and likely in other nitrate sensors and TCS proteins as well. Less
Tumor-associated peptide human leukocyte antigen complexes pHLAs represent the largest pool of cell surface expressed cancer-specific epitopes making them attractive targets for cancer therapies Soluble bispecific molecules that incorporate an anti-CD effector function are being developed to redirect T cells against these targets using different approaches The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs e g immune-mobilizing monoclonal T cell receptors against cancer ImmTAC molecules whereas the second harnesses an antibody-based format TCR-mimic antibodies For both classes of reagent target specificity is vital considering the vast universe of potential pHLA molecules that can be presented on healthy ... More
Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents. Less
Ruthenium-catalysed azide alkyne cycloaddition RuAAC provides access to -disubstituted -triazole motifs in peptide engineering applications However investigation of this motif as a disulfide mimetic in cyclic peptides has been limited and the structural consequences remain to be studied We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor- NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations RMSD of the triazole linkages compared to the parent disulfide molecules The triazole-bridged peptides also displayed ... More
Ruthenium-catalysed azide–alkyne cycloaddition (RuAAC) provides access to 1,5-disubstituted 1,2,3-triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor-1. NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole-bridged peptides also displayed superior half-lives in liver S9 stability assays compared to disulfide-bridged peptides. This work establishes a foundation for the application of 1,5-disubstituted 1,2,3-triazoles as disulfide mimetics. Less
Single-cell RNA sequencing scRNA-seq is the leading technique for characterizing the transcriptomes of individual cells in a sample The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues organs and organisms However the protocols differ substantially with respect to their RNA capture efficiency bias scale and costs and their relative advantages for different applications are unclear In the present study we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states We performed a multicenter study comparing commonly used scRNA-seq and single-nucleus ... More
Single-cell RNA sequencing (scRNA-seq) is the leading technique for characterizing the transcriptomes of individual cells in a sample. The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues, organs and organisms. However, the protocols differ substantially with respect to their RNA capture efficiency, bias, scale and costs, and their relative advantages for different applications are unclear. In the present study, we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states. We performed a multicenter study comparing 13 commonly used scRNA-seq and single-nucleus RNA-seq protocols applied to a heterogeneous reference sample resource. Comparative analysis revealed marked differences in protocol performance. The protocols differed in library complexity and their ability to detect cell-type markers, impacting their predictive value and suitability for integration into reference cell atlases. These results provide guidance both for individual researchers and for consortium projects such as the Human Cell Atlas. Less
Cellular signaling via binding of the cytokines IL- and along with binding of the accessory protein IL- RAcP to their cognate receptor IL- R is believed to play a major role in epithelial and immune cell-mediated inflammation responses Antagonizing the signaling cascade that results from these binding events via a directed monoclonal antibody provides an opportunity to suppress such immune responses We report here the molecular structure of a complex between an extracellular portion of human IL- R and a Fab derived from a high affinity anti-IL- R neutralizing monoclonal antibody at resolution This structure the first of IL- R ... More
Cellular signaling via binding of the cytokines IL-36α, β, and γ along with binding of the accessory protein IL-36RAcP, to their cognate receptor IL-36R is believed to play a major role in epithelial and immune cell-mediated inflammation responses. Antagonizing the signaling cascade that results from these binding events via a directed monoclonal antibody provides an opportunity to suppress such immune responses. We report here the molecular structure of a complex between an extracellular portion of human IL-36R and a Fab derived from a high affinity anti-IL-36R neutralizing monoclonal antibody at 2.3 Å resolution. This structure, the first of IL-36R, reveals similarities with other structurally characterized IL-1R family members and elucidates the molecular determinants leading to the high affinity binding of the monoclonal antibody. The structure of the complex reveals that the epitope recognized by the Fab is remote from both the putative ligand and accessory protein binding interfaces on IL-36R, suggesting that the functional activity of the antibody is noncompetitive for these binding events. Less