1209 Citations
Membrane proteins are indispensable for every living organism yet their structural organization remains underexplored Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography which have significantly increased the structural coverage of membrane proteins across various kingdoms certain scientific methods such as time-resolved crystallography still mostly rely on crystallization techniques such as lipidic cubic phase LCP or in meso crystallization In this study we present an open-access blueprint for a humidity control chamber designed for LCP in meso crystallization experiments using a Gryphon crystallization robot Using this chamber we have obtained crystals of a transmembrane aspartate transporter ... More
Membrane proteins are indispensable for every living organism, yet their structural organization remains underexplored. Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography, which have significantly increased the structural coverage of membrane proteins across various kingdoms, certain scientific methods, such as time-resolved crystallography, still mostly rely on crystallization techniques, such as lipidic cubic phase (LCP) or in meso crystallization. In this study, we present an open-access blueprint for a humidity control chamber designed for LCP/in meso crystallization experiments using a Gryphon crystallization robot. Using this chamber, we have obtained crystals of a transmembrane aspartate transporter GltTk from Thermococcus kodakarensis in a lipidic environment using in meso crystallization. The data collected from these crystals allowed us to perform an analysis of lipids bound to the transporter. With this publication of our open-access design of a humidity chamber, we aim to improve the accessibility of in meso protein crystallization for the scientific community. Less
StayGold is an exceptionally bright and stable fluorescent protein that is highly resistant to photobleaching Despite favorable fluorescence properties use of StayGold as a fluorescent tag is limited because it forms a natural dimer Here we report the structure of StayGold and generate a derivative mStayGold that retains the brightness and photostability of the original protein while being fully monomeric
Microbial natural products are specialized metabolites that are sources of many bioactive compounds including antibiotics antifungals antiparasitics anticancer agents and probes of biology The assembly of libraries of producers of natural products has traditionally been the province of the pharmaceutical industry This sector has gathered significant historical collections of bacteria and fungi to identify new drug leads with outstanding outcomes - upwards of of drug scaffolds originate from such libraries Despite this success the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds ... More
Microbial natural products are specialized metabolites that are sources of many bioactive
compounds including antibiotics, antifungals, antiparasitics, anticancer agents, and probes of
biology. The assembly of libraries of producers of natural products has traditionally been the
province of the pharmaceutical industry. This sector has gathered significant historical
collections of bacteria and fungi to identify new drug leads with outstanding outcomes - upwards of 60% of drug scaffolds originate from such libraries. Despite this success, the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds in the drug industry. The advent of advanced mass spectrometry tools, along with rapid whole genome sequencing and in silico identification of biosynthetic gene clusters that encode the machinery necessary for the synthesis of specialized metabolites, offers the opportunity to revisit microbial natural product libraries with renewed vigor. Assembling a suitable library of microbes and extracts for screening requires the investment of resources and the development of methods that have customarily been the proprietary purview of large pharmaceutical companies. Here, we
report a perspective on our efforts to assemble a library of natural product-producing microbes
and the establishment of methods to extract and fractionate bioactive compounds using resources available to most academic labs. We validate the library and approach through a series of screens for antimicrobial and cytotoxic agents. This work serves as a blueprint for establishing libraries of microbial natural product producers and bioactive extract fractions suitable for screens of bioactive compounds. Less
compounds including antibiotics, antifungals, antiparasitics, anticancer agents, and probes of
biology. The assembly of libraries of producers of natural products has traditionally been the
province of the pharmaceutical industry. This sector has gathered significant historical
collections of bacteria and fungi to identify new drug leads with outstanding outcomes - upwards of 60% of drug scaffolds originate from such libraries. Despite this success, the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds in the drug industry. The advent of advanced mass spectrometry tools, along with rapid whole genome sequencing and in silico identification of biosynthetic gene clusters that encode the machinery necessary for the synthesis of specialized metabolites, offers the opportunity to revisit microbial natural product libraries with renewed vigor. Assembling a suitable library of microbes and extracts for screening requires the investment of resources and the development of methods that have customarily been the proprietary purview of large pharmaceutical companies. Here, we
report a perspective on our efforts to assemble a library of natural product-producing microbes
and the establishment of methods to extract and fractionate bioactive compounds using resources available to most academic labs. We validate the library and approach through a series of screens for antimicrobial and cytotoxic agents. This work serves as a blueprint for establishing libraries of microbial natural product producers and bioactive extract fractions suitable for screens of bioactive compounds. Less
For optimal bioperformance the drug in an amorphous solid dispersion ASD should ideally not undergo crystallization in the solid dosage form during storage or from the supersaturated solution generated upon dissolution Incomplete processing during hot melt extrusion HME can lead to residual crystallinity Commonly residual crystallinity is evaluated using techniques such as powder X-ray diffraction pXRD However residual crystallinity at levels below the detection limit of pXRD can be detrimental to the ASD performance The goal of this study was to evaluate the impact of different levels of residual crystallinity in an ASD containing the fast-crystallizing drug posaconazole PCZ and ... More
For optimal bioperformance, the drug in an amorphous solid dispersion (ASD) should ideally not undergo crystallization in the solid dosage form during storage or from the supersaturated solution generated upon dissolution. Incomplete processing during hot melt extrusion (HME) can lead to residual crystallinity. Commonly, residual crystallinity is evaluated using techniques such as powder X-ray diffraction (pXRD). However, residual crystallinity at levels below the detection limit of pXRD can be detrimental to the ASD performance. The goal of this study was to evaluate the impact of different levels of residual crystallinity in an ASD containing the fast-crystallizing drug posaconazole (PCZ) and hydroxypropyl methylcellulose acetate succinate (HPMCAS) on dissolution and additional crystallization. ASDs with and without residual crystallinity at 10, 25, and 50 wt % drug loadings were prepared using HME, processing at temperatures below and above the critical temperature, which was calculated using the Flory–Huggins theory. Some of the ASDs contained levels of residual crystallinity that were below the quantification limit of pXRD, requiring the use of second harmonic generation (SHG) imaging. The impact of residual crystallinity on dissolution was studied by using two-stage dissolution. Additional characterization in support of dissolution measurements included SHG imaging and particle size evolution with focused beam reflectance measurement (FBRM) using pH-shift experiments. The 10 wt % ASD processed below the critical solution temperature contained residual crystallinity of 0.3%, which promoted rapid crystallization when the ASD was in a solution environment. Real-time monitoring of both the solid and solution phases revealed that PCZ in ASDs containing residual crystals underwent crystallization both in the matrix and from solution. The study supports the need to select a sufficiently sensitive crystallinity estimation technique, a suitable discriminatory dissolution technique, and appropriate HME processing conditions in order to optimize and achieve successful performance of ASDs of fast-crystallizing drugs. Less
Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins yet have very similar spectra when in the oxidized state Recently we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue Q V or red I V A Q shifts of fluorescence emission maxima Here we use time-resolved and low-temperature spectroscopy to show that whereas the chromophore environment is static in Q V an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I V A Q variant Consequently in Q V excitation emission and phosphorescence spectra are shifted whereas in I V A ... More
Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins, yet have very similar spectra when in the oxidized state. Recently, we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue (Q148V) or red (I52V A85Q) shifts of fluorescence emission maxima. Here, we use time-resolved and low-temperature spectroscopy to show that whereas the chromophore environment is static in Q148V, an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I52V A85Q variant. Consequently, in Q148V, excitation, emission, and phosphorescence spectra are shifted, whereas in I52V A85Q, excitation and low-temperature phosphorescence spectra are relatively unchanged, while emission spectrum is altered. We also determine the x-ray structures of the two variants to reveal the flavin environment and complement the spectroscopy data. Our findings illustrate two distinct color-tuning mechanisms of flavin-binding proteins and could be helpful for the engineering of new variants with improved optical properties. Less
NADH cytochrome b oxidoreductase Ncb or is a cytosolic ferric reductase implicated in diabetes and neurological conditions Ncb or comprises cytochrome b b and cytochrome b reductase b R domains separated by a CHORD-Sgt CS linker domain Ncb or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme While full-length human Ncb or has proven resistant to crystallization we have succeeded in obtaining high-resolution atomic structures of the b domain and a construct containing the CS and b R domains CS b R Ncb or also contains an N-terminal intrinsically ... More
NADH cytochrome b5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5) and cytochrome b5 reductase (b5R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b5 domain and a construct containing the CS and b5R domains (CS/b5R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L34MDWIRL40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25–129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp120 (corresponding to invariant Trp37 in Ncb5or) to be part of an 11-residue α-helix (S116QMDWLKLTRT126) packing against two of the four helices in the b5 domain that surround heme (α2 and α5). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr85 and Tyr88 (α2), Cys124 (α5), and Leu47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp37 in nucleating the formation of the N-terminal helix, whose location in the N/b5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b5 module as reflected by its existence in a class of cytochrome b5 proteins from three kingdoms among eukaryotes. Less
Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection It is responsible for US deaths annually Neutrophils are an integral part of the innate immune response and rapidly clear pathogens from circulation using neutrophil extracellular traps NETs which are released through a process called NETosis NETs prevent dissemination of pathogens by entrapment in externalized chromatin containing deactivating enzymes While we have learned much about the mechanisms underlying NETosis we are yet to translate it to improved therapies or patient outcomes This gap may be attributable to the models used to study NETosis Current models used ... More
Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection (1). It is responsible for ~370,000 US deaths annually (2). Neutrophils are an integral part of the innate immune response and rapidly clear pathogens from circulation using neutrophil extracellular traps (NETs), which are released through a process called NETosis (3). NETs prevent dissemination of pathogens by entrapment in externalized chromatin containing deactivating enzymes. While we have learned much about the mechanisms underlying NETosis, we are yet to translate it to improved therapies or patient outcomes. This gap may be attributable to the models used to study NETosis. Current models used to investigate NETosis are limited and routinely employ unnatural triggers such as phorbol 12-myristate 13-acetate (PMA). PMA is not a physiological trigger present in the immune system and may bypass the natural pathways that regulate NETs production. Mouse models that use isolated neutrophils and neutrophil-like cells induced from immortalized cell lines do not completely reflect the complex cellular and molecular biology underlying neutrophil activation and NETosis, especially in a whole-blood environment. Therefore, it is crucial to study how specific factors, known to be upregulated in disease, interact and potentially induce NETosis. Here we use high-throughput screening and natural NETosis triggers to develop a more biologically relevant ex vivo NETosis (Synthetic-Sepsis™) model.
Whole blood was collected from healthy donors and aliquoted into a 384 well plate using a Formulatrix Mantis liquid handler. This plate contained small molecules associated with neutrophils or NETosis activation, such as interleukins: Il-1b, IL-5, IL-6, IL-8, IL-15, IL-17, IL-18 and other molecules TNF- α, LT-α, IFN-γ, G-CSF, GM-CSF, E-selectin, PAF-16, CXCL1, CXCL2, LTB4, CXCL5, CCL2, CCL3, fMLP, Ferritin, HMGB1, C5a and LPS. We used a combinatorial pooling strategy designed using JMP software to identify which combinations of small molecules could stimulate NET formation. NETosis was assessed using Sytox green intercalation at 5 minute intervals for up to 24 hours using a Molecular Devices plate reader. PMA was utilized as a positive control for NETosis induction at varying concentrations.
Using our combinatorial pooling approach of the various factors, we successfully induced NETosis in an ex vivo whole blood system using naturally occurring cytokines and chemokines at physiologically relevant concentrations. We found that different combinations of factors evoke distinct neutrophil responses both in the time of NET generation and/or magnitude of NET-associated intercalation signal. We observed inter-donor variability in response time and amplitude however, similar small molecule pools induced consistent responses across donors. Furthermore, our findings suggest that at least four naturally occurring factors are necessary to induce NETosis in our system. Although some factors activate similar pathways, they are unable to induce a signal alone and as the number of factors increased beyond four, there was an enhanced NET response. Interestingly, we found either TNF-α or LT-α was required to cause a NETosis response, underlining the potentially significant roles these factors play in inflammatory disease. These results suggest an underlying master regulatory mechanism, such that certain factors are essential but not individually sufficient to trigger NETosis.
To our knowledge, we report the first ex-vivo model using naturally occurring cytokines and chemokines to induce NETosis in whole blood. These findings emphasize the importance of expanding our understanding of neutrophil physiology in a biologically relevant context with physiological triggers to induce NETosis. This approach could reveal new dimensions in our understanding of disease pathology and risk factors and might unearth potential therapeutic targets providing novel strategies for disease intervention and treatment. Further investigation of these factors is underway to further understand the release of NETs in natural and pathological states. Less
Whole blood was collected from healthy donors and aliquoted into a 384 well plate using a Formulatrix Mantis liquid handler. This plate contained small molecules associated with neutrophils or NETosis activation, such as interleukins: Il-1b, IL-5, IL-6, IL-8, IL-15, IL-17, IL-18 and other molecules TNF- α, LT-α, IFN-γ, G-CSF, GM-CSF, E-selectin, PAF-16, CXCL1, CXCL2, LTB4, CXCL5, CCL2, CCL3, fMLP, Ferritin, HMGB1, C5a and LPS. We used a combinatorial pooling strategy designed using JMP software to identify which combinations of small molecules could stimulate NET formation. NETosis was assessed using Sytox green intercalation at 5 minute intervals for up to 24 hours using a Molecular Devices plate reader. PMA was utilized as a positive control for NETosis induction at varying concentrations.
Using our combinatorial pooling approach of the various factors, we successfully induced NETosis in an ex vivo whole blood system using naturally occurring cytokines and chemokines at physiologically relevant concentrations. We found that different combinations of factors evoke distinct neutrophil responses both in the time of NET generation and/or magnitude of NET-associated intercalation signal. We observed inter-donor variability in response time and amplitude however, similar small molecule pools induced consistent responses across donors. Furthermore, our findings suggest that at least four naturally occurring factors are necessary to induce NETosis in our system. Although some factors activate similar pathways, they are unable to induce a signal alone and as the number of factors increased beyond four, there was an enhanced NET response. Interestingly, we found either TNF-α or LT-α was required to cause a NETosis response, underlining the potentially significant roles these factors play in inflammatory disease. These results suggest an underlying master regulatory mechanism, such that certain factors are essential but not individually sufficient to trigger NETosis.
To our knowledge, we report the first ex-vivo model using naturally occurring cytokines and chemokines to induce NETosis in whole blood. These findings emphasize the importance of expanding our understanding of neutrophil physiology in a biologically relevant context with physiological triggers to induce NETosis. This approach could reveal new dimensions in our understanding of disease pathology and risk factors and might unearth potential therapeutic targets providing novel strategies for disease intervention and treatment. Further investigation of these factors is underway to further understand the release of NETs in natural and pathological states. Less
The phospholipase D PLD family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids PLD is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes yielding a soluble active form The deficiency of PLD leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors While the mechanism of PLD phospholipase activity has been extensively characterized not much is known about how PLDs bind and hydrolyze nucleic acids Here we determined the high-resolution crystal structure of the luminal N-glycosylated domain of ... More
The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3. Less
The pathogen Paracoccidioides lutzii Pb is found in South America countries Colombia Ecuador Venezuela and Brazil especially in the central west and north regions of the latter It belongs to the Ajellomycetaceae family Onygenales order and is typically thermodimorphic presenting yeast cells when it grows in animal tissues but mycelia when in the environment where it produces the infectious propagule This fungus is one of the etiologic agents of Paracoccidioidomycosis PCM the most important endemic fungal infection in Latin America Investigations on its genome have contributed to a better understanding about its metabolism and revealed the complexity of several metabolic ... More
The pathogen Paracoccidioides lutzii (Pb01) is found in South America countries Colombia, Ecuador, Venezuela and Brazil, especially in the central, west, and north regions of the latter. It belongs to the Ajellomycetaceae family, Onygenales order, and is typically thermodimorphic, presenting yeast cells when it grows in animal tissues, but mycelia when in the environment, where it produces the infectious propagule. This fungus is one of the etiologic agents of Paracoccidioidomycosis (PCM), the most important endemic fungal infection in Latin America. Investigations on its genome have contributed to a better understanding about its metabolism and revealed the complexity of several metabolic glycolytic pathways. Glyceraldehyde-3-Phosphate Dehydrogenase from Paracoccidioides lutzii (PlGAPDH) is considered a moonlighting protein and participates in several biological processes of this pathogen. The enzyme was expressed and purified, as seen in SDS-PAGE gel, crystallized and had its three dimensional structure (3D) determined in complex with NAD+, a sulphate ion and d-galactonic acid, therefore, a type of ‘GAA site’. It is the first GAPDH structure to show this chemical type in this site and how this protein can bind an acid derived from oxidation of a linear hexose. Less
The efficacy of aminoglycoside antibiotics is waning due to the acquisition of diverse resistance mechanisms by bacteria Among the most prevalent are aminoglycoside acetyltransferases AACs that inactivate the antibiotics through acetyl coenzyme A-mediated modification Most AACs are members of the GCN superfamily of acyltransferases which lack conserved active site residues that participate in catalysis ApmA is the first reported AAC belonging to the left-handed -helix superfamily These enzymes are characterized by an essential active site histidine that acts as an active site base Here we show that ApmA confers broad-spectrum aminoglycoside resistance with a molecular mechanism that diverges from other ... More
The efficacy of aminoglycoside antibiotics is waning due to the acquisition of diverse resistance mechanisms by bacteria. Among the most prevalent are aminoglycoside acetyltransferases (AACs) that inactivate the antibiotics through acetyl coenzyme A-mediated modification. Most AACs are members of the GCN5 superfamily of acyltransferases which lack conserved active site residues that participate in catalysis. ApmA is the first reported AAC belonging to the left-handed β-helix superfamily. These enzymes are characterized by an essential active site histidine that acts as an active site base. Here we show that ApmA confers broad-spectrum aminoglycoside resistance with a molecular mechanism that diverges from other detoxifying left-handed β-helix superfamily enzymes and canonical GCN5 AACs. We find that the active site histidine plays different functions depending on the acetyl-accepting aminoglycoside substrate. This flexibility in the mechanism of a single enzyme underscores the plasticity of antibiotic resistance elements to co-opt protein catalysts in the evolution of drug detoxification. Less
This study aimed to identify inhibitors of the translocated intimin receptor Tir of enteropathogenic Escherichia coli EPEC EPEC is an intestinal pathogen that causes diarrhea and is a major health concern worldwide Because Tir is a key virulence factor involved in EPEC pathogenesis inhibiting its function is a potential strategy for controlling EPEC infections Virtual screening was applied to chemical libraries to search for compounds that inhibit Tir-mediated bacterial adherence to host cells Three sites were targeted using the cocrystal structure published earlier A selection of compounds was then assessed in a cell-based infection model and fluorescence microscopy assay The ... More
This study aimed to identify inhibitors of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC). EPEC is an intestinal pathogen that causes diarrhea and is a major health concern worldwide. Because Tir is a key virulence factor involved in EPEC pathogenesis, inhibiting its function is a potential strategy for controlling EPEC infections. Virtual screening was applied to chemical libraries to search for compounds that inhibit Tir-mediated bacterial adherence to host cells. Three sites were targeted using the cocrystal structure published earlier. A selection of compounds was then assessed in a cell-based infection model and fluorescence microscopy assay. The results of this study provide a basis for further optimization and testing of Tir inhibitors as potential therapeutic agents for EPEC infections. Less
Spatial tissue proteomics combining microscopy-based cell phenotyping with ultra-sensitive mass spectrometry MS -based proteomics is an emerging and powerful concept for the study of cell function and heterogeneity in health and disease However optimized workflows that preserve morphological information for image-based phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected archival tissue are currently lacking Here we report a robust and scalable workflow for the proteomic analysis of ultra-low input formalin-fixed paraffin-embedded FFPE material Benchmarking in the murine liver resulted in up to quantified proteins from single hepatocyte contours and nearly proteins from -cell ... More
Spatial tissue proteomics combining microscopy-based cell phenotyping with ultra-sensitive mass spectrometry (MS)-based proteomics is an emerging and powerful concept for the study of cell function and heterogeneity in health and disease. However, optimized workflows that preserve morphological information for image-based phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected archival tissue, are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low input formalin-fixed, paraffin-embedded (FFPE) material. Benchmarking in the murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions with high quantitative reproducibility. Applied to human tonsil, we profiled 146 microregions including spatially defined T and B lymphocyte niches and quantified cell type specific markers, cytokines, immune cell regulators and transcription factors. These rich data also highlighted proteome dynamics in spatially defined zones of activated germinal centers, illuminating sites undergoing active B-cell proliferation and somatic hypermutation. Our results demonstrate the power of spatially-resolved proteomics for tissue phenotyping by integrating high-content imaging, laser microdissection, and ultra-sensitive mass spectrometry. This approach has broad implications for a wide range of biomedical applications, including early disease profiling, drug target discovery and biomarker research. Less
Cryptophyte algae have a unique phycobiliprotein light-harvesting antenna that fills a spectral gap in chlorophyll absorption from photosystems However it is unclear how the antenna transfers energy efficiently to these photosystems We show that the cryptophyte Hemiselmis andersenii expresses an energetically complex antenna comprising three distinct spectrotypes of phycobiliprotein each composed of two protomers but with different quaternary structures arising from a diverse subunit family We report crystal structures of the major phycobiliprotein from each spectrotype Two-thirds of the antenna consists of open quaternary form phycobiliproteins acting as primary photon acceptors These are supplemented by a newly discovered open-braced form ... More
Cryptophyte algae have a unique phycobiliprotein light-harvesting antenna that fills a spectral gap in chlorophyll absorption from photosystems. However, it is unclear how the antenna transfers energy efficiently to these photosystems. We show that the cryptophyte Hemiselmis andersenii expresses an energetically complex antenna comprising three distinct spectrotypes of phycobiliprotein, each composed of two αβ protomers but with different quaternary structures arising from a diverse α subunit family. We report crystal structures of the major phycobiliprotein from each spectrotype. Two-thirds of the antenna consists of open quaternary form phycobiliproteins acting as primary photon acceptors. These are supplemented by a newly discovered open-braced form (~15%), where an insertion in the α subunit produces ~10 nm absorbance red-shift. The final components (~15%) are closed forms with a long wavelength spectral feature due to substitution of a single chromophore. This chromophore is present on only one β subunit where asymmetry is dictated by the corresponding α subunit. This chromophore creates spectral overlap with chlorophyll, thus bridging the energetic gap between the phycobiliprotein antenna and the photosystems. We propose that the macromolecular organization of the cryptophyte antenna consists of bulk open and open-braced forms that transfer excitations to photosystems via this bridging closed form phycobiliprotein. Less
Gonorrhea a prevalent sexually transmitted infection affecting millions annually worldwide is caused by Neisseria gonorrhoeae Ngo a superbug resistant to all antibiotic classes Compounding the challenge the absence of protective immunity upon infection allows for reinfection and a viable vaccine against gonococcal infection remains elusive In response to infection the human host deploys nutritional immunity sequestering essential metals like iron from invading bacteria using metal binding proteins To overcome this Ngo employs outer-membrane TonB-dependent transporters TdTs like HpuAB to acquire iron from host metal binding proteins such as hemoglobin Hb Part of our study focused on HpuA the lipoprotein component ... More
Gonorrhea, a prevalent sexually transmitted infection affecting millions annually worldwide, is caused by Neisseria gonorrhoeae (Ngo), a "superbug" resistant to all antibiotic classes. Compounding the challenge, the absence of protective immunity upon infection allows for reinfection, and a viable vaccine against gonococcal infection remains elusive.
In response to infection, the human host deploys nutritional immunity, sequestering essential metals like iron from invading bacteria, using metal binding proteins. To overcome this, Ngo employs outer-membrane TonB-dependent transporters (TdTs), like HpuAB, to acquire iron from host metal binding proteins, such as hemoglobin (Hb).
Part of our study focused on HpuA, the lipoprotein component of the HpuAB system. Mutations targeting hydrophobic residues crucial for Hb interaction were studied. Results demonstrated that without HpuB, strains failed to grow, emphasizing HpuB's role in iron internalization. Notably, when HpuB is produced, deletion and insertion mutations in loop 2 of HpuA affected growth and binding to Hb.
Further investigations into HpuB, the transmembrane protein of the HpuAB system, uncovered essential loop regions for binding and growth on Hb as a sole iron source. Deletion mutations in loops 2, 3, and 4 facilitated binding and growth independently of HpuA production. Intriguingly, mutations in loop 7 abrogated binding and impaired growth in the absence of HpuA, but partial growth and full binding recovery occurred when HpuA was present. This highlighted the importance of loop 7 in iron acquisition and suggested a potential role for both HpuA and HpuB in the binding Hb.
As a combination of non-binding TdT mutants is hypothesized to have the potential to improve vaccine efficacy and provide protection, identifying non-binding HpuB mutants could be important. In summary, this research sheds light on the intricacies of the HpuAB system, contributing valuable insights that could inform the development of an effective gonorrhea vaccine. Less
In response to infection, the human host deploys nutritional immunity, sequestering essential metals like iron from invading bacteria, using metal binding proteins. To overcome this, Ngo employs outer-membrane TonB-dependent transporters (TdTs), like HpuAB, to acquire iron from host metal binding proteins, such as hemoglobin (Hb).
Part of our study focused on HpuA, the lipoprotein component of the HpuAB system. Mutations targeting hydrophobic residues crucial for Hb interaction were studied. Results demonstrated that without HpuB, strains failed to grow, emphasizing HpuB's role in iron internalization. Notably, when HpuB is produced, deletion and insertion mutations in loop 2 of HpuA affected growth and binding to Hb.
Further investigations into HpuB, the transmembrane protein of the HpuAB system, uncovered essential loop regions for binding and growth on Hb as a sole iron source. Deletion mutations in loops 2, 3, and 4 facilitated binding and growth independently of HpuA production. Intriguingly, mutations in loop 7 abrogated binding and impaired growth in the absence of HpuA, but partial growth and full binding recovery occurred when HpuA was present. This highlighted the importance of loop 7 in iron acquisition and suggested a potential role for both HpuA and HpuB in the binding Hb.
As a combination of non-binding TdT mutants is hypothesized to have the potential to improve vaccine efficacy and provide protection, identifying non-binding HpuB mutants could be important. In summary, this research sheds light on the intricacies of the HpuAB system, contributing valuable insights that could inform the development of an effective gonorrhea vaccine. Less
MbtI from Mycobacterium tuberculosis Mtb is a Mg -dependent salicylate synthase belonging to the chorismate-utilizing enzyme CUE family As a fundamental player in iron acquisition MbtI promotes the survival and pathogenicity of Mtb in the infected host Hence it has emerged in the last decade as an innovative potential target for the anti-virulence therapy of tuberculosis In this context -phenylfuran- -carboxylic acids have been identified as potent MbtI inhibitors The first co-crystal structure of MbtI in complex with a member of this class was described in showing the enzyme adopting an open configuration Due to the high mobility of the ... More
MbtI from Mycobacterium tuberculosis (Mtb) is a Mg2+-dependent salicylate synthase, belonging to the chorismate-utilizing enzyme (CUE) family. As a fundamental player in iron acquisition, MbtI promotes the survival and pathogenicity of Mtb in the infected host. Hence, it has emerged in the last decade as an innovative, potential target for the anti-virulence therapy of tuberculosis. In this context, 5-phenylfuran-2-carboxylic acids have been identified as potent MbtI inhibitors. The first co-crystal structure of MbtI in complex with a member of this class was described in 2020, showing the enzyme adopting an open configuration. Due to the high mobility of the loop adjacent to the binding pocket, large portions of the amino acid chain were not defined in the electron density map, hindering computational efforts aimed at structure-driven ligand optimization. Herein, we report a new, high-resolution co-crystal structure of MbtI with a furan-based derivative, in which the closed configuration of the enzyme allowed tracing the entirety of the active site pocket in the presence of the bound inhibitor. Moreover, we describe a new crystal structure of MbtI in open conformation and in complex with the known inhibitor methyl-AMT, suggesting that in vitro potency is not related to the observed enzyme conformation. These findings will prove fundamental to enhance the potency of this series via rational structure-based drug-design approaches. Less
The mammalian Voltage-gated sodium Nav channel is composed of a single subunit kDa a multi-pass membrane protein that renders ion selectivity and two or more Nav subunits kDa that are Type I single-pass membrane proteins and regulate Nav subunit function These subunits are assembled on the plasma membrane of electrically-excitable cells as an intrinsic membrane protein complex and help to initiate and propagate the action potential The four major mammalian Nav -subunit isoforms Nav proteins possess an N-terminal extracellular Immunoglobulin Ig domain ECD a single transmembrane -helix and an intracellular C-terminal region ICD This thesis is mainly focused on the ... More
The mammalian Voltage-gated sodium (Nav) channel is composed of a single α subunit (~ 260 kDa), a multi-pass membrane protein that renders ion selectivity and two or more Navβ subunits (25‒40 kDa), that are Type I single-pass membrane proteins and regulate Navα subunit function. These subunits are assembled on the plasma membrane of electrically-excitable cells as an intrinsic membrane protein complex and help to initiate and propagate the action potential. The four major mammalian Navβ-subunit isoforms, Navβ1‒4 proteins possess an N-terminal extracellular Immunoglobulin (Ig) domain (ECD), a single transmembrane α-helix, and an intracellular C-terminal region (ICD).
This thesis is mainly focused on the structural biology aspects of the human Navβ3 subunit. It reports the atomic structure of the Navβ3-Ig domain as determined by X-ray crystallography. Interestingly, the Navβ3-Ig domain is observed as a trimer in the crystal structure. The homo-trimer assembly interface lies at the N-terminus and is constrained by a disulphide bond not normally present in Ig domains. The Navβ3 subunit Ig domain is known to be glycosylated and contains four potential N-linked glycosylation sites. However, the X-ray crystallography was conducted on deglycosylated protein. Using computational modelling, it is shown that glycan addition would not interfere with Navβ3-Ig domain trimerization. Independent evidence gathered using Analytical Ultracentrifugation (crosslinked, glycosylated Navβ3-Ig domain, in vitro), Proximity Ligation Assay (full-length Navβ3, in vivo), Atomic Force Microscopy (isolated full-length Navβ3, in vitro) and Photo-activated Localisation Microscopic experiments (full-length Navβ3, in situ) support the view that the Navβ3 subunit can form trimers when expressed in cells. The biological significance of Navβ3 subunit trimerization is discussed.
Strategies to express and purify the Navβ1/β2/β4-Ig domains were made. Wild type Navβ2- and Navβ4-Ig domains exist as monomers and dimers, simultaneously in solution, although crystals that diffracted to the necessary resolution were not produced. Less
This thesis is mainly focused on the structural biology aspects of the human Navβ3 subunit. It reports the atomic structure of the Navβ3-Ig domain as determined by X-ray crystallography. Interestingly, the Navβ3-Ig domain is observed as a trimer in the crystal structure. The homo-trimer assembly interface lies at the N-terminus and is constrained by a disulphide bond not normally present in Ig domains. The Navβ3 subunit Ig domain is known to be glycosylated and contains four potential N-linked glycosylation sites. However, the X-ray crystallography was conducted on deglycosylated protein. Using computational modelling, it is shown that glycan addition would not interfere with Navβ3-Ig domain trimerization. Independent evidence gathered using Analytical Ultracentrifugation (crosslinked, glycosylated Navβ3-Ig domain, in vitro), Proximity Ligation Assay (full-length Navβ3, in vivo), Atomic Force Microscopy (isolated full-length Navβ3, in vitro) and Photo-activated Localisation Microscopic experiments (full-length Navβ3, in situ) support the view that the Navβ3 subunit can form trimers when expressed in cells. The biological significance of Navβ3 subunit trimerization is discussed.
Strategies to express and purify the Navβ1/β2/β4-Ig domains were made. Wild type Navβ2- and Navβ4-Ig domains exist as monomers and dimers, simultaneously in solution, although crystals that diffracted to the necessary resolution were not produced. Less
Norovirus is the leading cause of viral gastroenteritis worldwide and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI noroviruses despite most global outbreaks being caused by GII noroviruses Here we determined the crystal structures of the GII Sydney ligand-free norovirus protease at and at with a covalently bound high-affinity IC M protease inhibitor NV- We show that the active sites of the ligand-free protease structure are present in both open and closed conformations as determined by their Arg side ... More
Norovirus is the leading cause of viral gastroenteritis worldwide, and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections. The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI.1 noroviruses, despite most global outbreaks being caused by GII.4 noroviruses. Here, we determined the crystal structures of the GII.4 Sydney 2012 ligand-free norovirus protease at 2.79 Å and at 1.83 Å with a covalently bound high-affinity (IC50 = 0.37 µM) protease inhibitor (NV-004). We show that the active sites of the ligand-free protease structure are present in both open and closed conformations, as determined by their Arg112 side chain orientation. A comparative analysis of the ligand-free and ligand-bound protease structures reveals significant structural differences in the active site cleft and substrate-binding pockets when an inhibitor is covalently bound. We also report a second molecule of NV-004 non-covalently bound within the S4 substrate binding pocket via hydrophobic contacts and a water-mediated hydrogen bond. These new insights can guide structure-aided drug design against the GII.4 genogroup of noroviruses. Less
Kinases are important therapeutic targets and their inhibitors are classified according to their mechanism of action which range from blocking ATP binding to covalent inhibition Here a mechanism of inhibition is highlighted by capturing p -activated kinase PAK in an intermediate state of activation using an Affimer reagent that binds in the P pocket PAK was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells Silencing of PAK resulted in reduced cell number G S arrest and enlargement of cells suggesting it to be important in urothelial cancer cell line survival and proliferation Affimer reagents were isolated ... More
Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible. Less
Poly ADP-ribose polymerase PARP inhibitors are used in the clinic to treat BRCA-deficient breast ovarian and prostate cancers As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH there is considerable interest in the development of highly specific small molecule DNPH inhibitors Here we present X-ray crystal structures of dimeric DNPH bound to its substrate hydroxymethyl deoxyuridine monophosphate hmdUMP Direct interaction with the hydroxymethyl group is important for substrate positioning while conserved residues surrounding the base facilitate target discrimination Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation ... More
Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the clinic to treat BRCA-deficient breast, ovarian and prostate cancers. As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH1 there is considerable interest in the development of highly specific small molecule DNPH1 inhibitors. Here, we present X-ray crystal structures of dimeric DNPH1 bound to its substrate hydroxymethyl deoxyuridine monophosphate (hmdUMP). Direct interaction with the hydroxymethyl group is important for substrate positioning, while conserved residues surrounding the base facilitate target discrimination. Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation and subsequent disruption of a covalent glycosyl-enzyme intermediate. Mutation of a previously uncharacterised yet conserved glutamate traps the intermediate in the active site, demonstrating its role in the hydrolytic step. These observations define the enzyme’s catalytic site and mechanism of hydrolysis, and provide important insights for inhibitor discovery. Less
Transglutaminases TGases are a family of calcium-dependent enzymes primarily known for their ability to cross-link proteins Transglutaminase TG is one isozyme in this family whose role is multifaceted TG can act not only as a typical transamidase through its catalytic core but also as a G-protein via its GTP binding site These two discrete activities are tightly regulated by both environmental stimuli and redox reactions Ubiquitously expressed in humans TG has been implicated in numerous disease pathologies that require extensive investigation The catalytic activity of TG can be monitored through various mechanisms including hydrolysis transamidation or cleavage of isopeptide bonds ... More
Transglutaminases (TGases) are a family of calcium-dependent enzymes primarily known for their ability to cross-link proteins. Transglutaminase 2 (TG2) is one isozyme in this family whose role is multifaceted. TG2 can act not only as a typical transamidase through its catalytic core but also as a G-protein via its GTP binding site. These two discrete activities are tightly regulated by both environmental stimuli and redox reactions. Ubiquitously expressed in humans, TG2 has been implicated in numerous disease pathologies that require extensive investigation. The catalytic activity of TG2 can be monitored through various mechanisms, including hydrolysis, transamidation, or cleavage of isopeptide bonds. Activity assays are required to monitor the activity of this isozyme not only for studying its transamidation reaction but also for validation of therapeutics designed to abolish this activity. Herein, we present the design, synthesis, and evaluation of a new TG2 activity substrate based on a previously optimized inhibitor scaffold. The substrate APH7 exhibits excellent affinity, selectivity, and reactivity with TG2 (KM = 3.0 μM). Furthermore, its application also allowed the discovery of unique hysteresis at play within the catalytic activity and inhibition reactivity of TG2. Less
Acute gastroenteritis caused by human noroviruses HuNoVs is a significant global health and economic burden and is without licensed vaccines or antiviral drugs The GII HuNoV causes most epidemics worldwide This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens HBGA the determinants of cell attachment and susceptibility hampering the development of immunotherapeutics Here we show that a llama-derived nanobody M neutralizes multiple GII variants with high potency in human intestinal enteroids The crystal structure of M complexed with the protruding domain of the GII capsid protein VP revealed ... More
Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide. This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens (HBGA), the determinants of cell attachment and susceptibility, hampering the development of immunotherapeutics. Here, we show that a llama-derived nanobody M4 neutralizes multiple GII.4 variants with high potency in human intestinal enteroids. The crystal structure of M4 complexed with the protruding domain of the GII.4 capsid protein VP1 revealed a conserved epitope, away from the HBGA binding site, fully accessible only when VP1 transitions to a “raised” conformation in the capsid. Together with dynamic light scattering and electron microscopy of the GII.4 VLPs, our studies suggest a mechanism in which M4 accesses the epitope by altering the conformational dynamics of the capsid and triggering its disassembly to neutralize GII.4 infection. Less
Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin
Moose Alces alces recombinant chymosin with a milk-clotting activity of AU mL was synthesized in the Kluyveromyces lactis expression system After precipitation with ammonium sulfate and chromatographic purification a sample of genetically engineered moose chymosin with a specific milk-clotting activity of AU mg was obtained which was used for extensive biochemical characterization of the enzyme The threshold of the thermal stability of moose chymosin was C its complete inactivation occurred after heating at C The total proteolytic activity of moose chymosin was A units The ratio of milk-clotting and total proteolytic activities of the enzyme was The Km kcat and ... More
Moose (Alces alces) recombinant chymosin with a milk-clotting activity of 86 AU/mL was synthesized in the Kluyveromyces lactis expression system. After precipitation with ammonium sulfate and chromatographic purification, a sample of genetically engineered moose chymosin with a specific milk-clotting activity of 15,768 AU/mg was obtained, which was used for extensive biochemical characterization of the enzyme. The threshold of the thermal stability of moose chymosin was 55 °C; its complete inactivation occurred after heating at 60 °C. The total proteolytic activity of moose chymosin was 0.332 A280 units. The ratio of milk-clotting and total proteolytic activities of the enzyme was 0.8. The Km, kcat and kcat/Km values of moose chymosin were 4.7 μM, 98.7 s−1, and 21.1 μM−1 s−1, respectively. The pattern of change in the coagulation activity as a function of pH and Ca2+ concentration was consistent with the requirements for milk coagulants for cheese making. The optimum temperature of the enzyme was 50–55 °C. The introduction of Mg2+, Zn2+, Co2+, Ba2+, Fe2+, Mn2+, Ca2+, and Cu2+ into milk activated the coagulation ability of moose chymosin, while Ni ions on the contrary inhibited its activity. Using previously published data, we compared the biochemical properties of recombinant moose chymosin produced in bacterial (Escherichia coli) and yeast (K. lactis) producers. Less
Objectives In order to treat a rat model of rotator cuff rupture this work concentrated on the expression of TNMD and RUNX followed by rotator cuff repair and secretome-hMSCs Methods A total of thirty -weeks-old male Sprague Dawley rats were separated into five groups randomly RC on week lesion treated with a rotator cuff repair and saline RC NaCl group n for and weeks and lesion treated with a rotator cuff repair and secretome-hMSCs RC secretome-hMSC group n for and weeks The supraspinatus and infraspinatus muscle tendon units were obtained for histological and biomechanical investigation at and weeks following injury ... More
Objectives: In order to treat a rat model of rotator cuff rupture, this work concentrated on the expression
of TNMD and RUNX2, followed by rotator cuff repair and secretome-hMSCs.
Methods: A total of thirty 10-weeks-old male Sprague–Dawley rats were separated into five groups randomly, RC
on week 0, lesion treated with a rotator cuff repair and saline (RC + NaCl group, n = 6) for 2 and 8 weeks, and lesion
treated with a rotator cuff repair and secretome-hMSCs (RC + secretome-hMSC group, n = 6) for 2 and 8 weeks.
The supraspinatus and infraspinatus muscle–tendon units were obtained for histological and biomechanical
investigation at 0, 2 and 8 weeks following injury.
Results: The findings showed that, in comparison with the RC + NaCl group, secretome-hMSCs significantly
improved tendon repair by upregulating TNMD and RUNX2 expression and histology score.
Conclusion: Combining Secretome-hypoxia MSCs with RC healing may help rats with rotator cuff tears.
Level of evidence: IV Less
of TNMD and RUNX2, followed by rotator cuff repair and secretome-hMSCs.
Methods: A total of thirty 10-weeks-old male Sprague–Dawley rats were separated into five groups randomly, RC
on week 0, lesion treated with a rotator cuff repair and saline (RC + NaCl group, n = 6) for 2 and 8 weeks, and lesion
treated with a rotator cuff repair and secretome-hMSCs (RC + secretome-hMSC group, n = 6) for 2 and 8 weeks.
The supraspinatus and infraspinatus muscle–tendon units were obtained for histological and biomechanical
investigation at 0, 2 and 8 weeks following injury.
Results: The findings showed that, in comparison with the RC + NaCl group, secretome-hMSCs significantly
improved tendon repair by upregulating TNMD and RUNX2 expression and histology score.
Conclusion: Combining Secretome-hypoxia MSCs with RC healing may help rats with rotator cuff tears.
Level of evidence: IV Less
Multidrug efflux is a well-established mechanism of drug resistance in bacterial pathogens like Salmonella Typhi styMdtM locus name STY is a multidrug efflux transporter of the major facilitator superfamily expressed in S Typhi Functional assays identified several residues important for its transport activity Here we used an AlphaFold model to identify additional residues for analysis by mutagenesis Mutation of peripheral residue Cys had no effect on the structure or function of the transporter However substitution of channel-lining residues Tyr and Tyr completely abolished transport function Finally mutation of Gln which faces peripheral helices of the transporter resulted in the loss ... More
Multidrug efflux is a well-established mechanism of drug resistance in bacterial pathogens like Salmonella Typhi. styMdtM (locus name; STY4874) is a multidrug efflux transporter of the major facilitator superfamily expressed in S. Typhi. Functional assays identified several residues important for its transport activity. Here, we used an AlphaFold model to identify additional residues for analysis by mutagenesis. Mutation of peripheral residue Cys185 had no effect on the structure or function of the transporter. However, substitution of channel-lining residues Tyr29 and Tyr231 completely abolished transport function. Finally, mutation of Gln294, which faces peripheral helices of the transporter, resulted in the loss of transport of some substrates. Crystallization studies yielded diffraction data for the wild-type protein at 4.5 Å resolution and allowed the unit cell parameters to be established as a = b = 64.3 Å, c = 245.4 Å, α = β = γ = 90°, in space group P4. Our studies represent a further stepping stone towards a mechanistic understanding of the clinically important multidrug transporter styMdtM. Less
The thermophilic bacterium Clostridium thermocellum efficiently degrades polysaccharides into oligosaccharides The metabolism of - -linked cello-oligosaccharides is initiated by three enzymes i e the cellodextrin phosphorylase Cdp the cellobiose phosphorylase Cbp and the -glucosidase A BglA in C thermocellum In comparison how the oligosaccharides containing other kinds of linkage are utilized is rarely understood In this study we found that BglA could hydrolyze the - -disaccharide laminaribiose with much higher activity than that against the - -disaccharide cellobiose The structural basis of the substrate specificity was analyzed by crystal structure determination and molecular docking Genetic deletions of BglA and Cbp ... More
The thermophilic bacterium Clostridium thermocellum efficiently degrades polysaccharides into oligosaccharides. The metabolism of β-1,4-linked cello-oligosaccharides is initiated by three enzymes, i.e., the cellodextrin phosphorylase (Cdp), the cellobiose phosphorylase (Cbp), and the β-glucosidase A (BglA), in C. thermocellum. In comparison, how the oligosaccharides containing other kinds of linkage are utilized is rarely understood. In this study, we found that BglA could hydrolyze the β-1,3-disaccharide laminaribiose with much higher activity than that against the β-1,4-disaccharide cellobiose. The structural basis of the substrate specificity was analyzed by crystal structure determination and molecular docking. Genetic deletions of BglA and Cbp, respectively, and enzymatic analysis of cell extracts demonstrated that BglA is the key enzyme responsible for laminaribiose metabolism. Furthermore, the deletion of BglA can suppress the expression of Cbp and the deletion of Cbp can up-regulate the expression of BglA, indicating that BglA and Cbp have cross-regulation and BglA is also critical for cellobiose metabolism. These insights pave the way for both a fundamental understanding of metabolism and regulation in C. thermocellum and emphasize the importance of the degradation and utilization of polysaccharides containing β-1,3-linked glycosidic bonds in lignocellulose biorefinery. Less
Septins are membrane-associated GTP-binding proteins that are present in most eukaryotes They polymerize to play important roles as scaffolds and or diffusion barriers as part of the cytoskeleton -Helical coiled-coil domains are believed to contribute to septin assembly and those observed in both human SEPT and SEPT form antiparallel homodimers These are not compatible with their parallel heterodimeric organization expected from the current model for protofilament assembly but they could explain the interfilament cross-bridges observed by microscopy Here the first structure of a heterodimeric septin coiled coil is presented that between SEPT and SEPT the former is a SEPT SEPT ... More
Septins are membrane-associated, GTP-binding proteins that are present in most eukaryotes. They polymerize to play important roles as scaffolds and/or diffusion barriers as part of the cytoskeleton. α-Helical coiled-coil domains are believed to contribute to septin assembly, and those observed in both human SEPT6 and SEPT8 form antiparallel homodimers. These are not compatible with their parallel heterodimeric organization expected from the current model for protofilament assembly, but they could explain the interfilament cross-bridges observed by microscopy. Here, the first structure of a heterodimeric septin coiled coil is presented, that between SEPT14 and SEPT7; the former is a SEPT6/SEPT8 homolog. This new structure is parallel, with two long helices that are axially shifted by a full helical turn with reference to their sequence alignment. The structure also has unusual knobs-into-holes packing of side chains. Both standard seven-residue (heptad) and the less common 11-residue (hendecad) repeats are present, creating two distinct regions with opposite supercoiling, which gives rise to an overall straight coiled coil. Part of the hendecad region is required for heterodimerization and therefore may be crucial for selective septin recognition. These unconventional sequences and structural features produce a metastable heterocomplex that nonetheless has enough specificity to promote correct protofilament assembly. For instance, the lack of supercoiling may facilitate unzipping and transitioning to the antiparallel homodimeric state. Less
Analytical theory is proposed predicting remarkably large and fully electric-dipole-allowed circular dichroism CD in electronic ultraviolet-visible UV-vis absorbance spectroscopy of uniaxial surface assemblies Partial depolarization of the transmitted beam provides a pathway for surface-specific and chiral-specific dissymmetry parameters that are orders of magnitude greater than those from analogous measurements of isotropic systems Predictions of the model generated using ab initio quantum chemical calculations with no adjustable parameters agreed with UV-vis absorbance CD measurements of naproxen microcrystals prepared on hydrophilic substrates Notably these calculations correctly predicted i the key spectroscopic features ii the relative magnitudes of chiral-specific peaks in the CD ... More
Analytical theory is proposed predicting remarkably large and fully electric-dipole-allowed circular dichroism (CD) in electronic ultraviolet-visible (UV-vis) absorbance spectroscopy of uniaxial surface assemblies. Partial depolarization of the transmitted beam provides a pathway for surface-specific and chiral-specific dissymmetry parameters that are orders of magnitude greater than those from analogous measurements of isotropic systems. Predictions of the model generated using ab initio quantum chemical calculations with no adjustable parameters agreed with UV-vis absorbance CD measurements of naproxen microcrystals prepared on hydrophilic substrates. Notably, these calculations correctly predicted (i) the key spectroscopic features, (ii) the relative magnitudes of chiral-specific peaks in the CD spectrum, (iii) the absolute CD sign, and (iv) the reciprocal CD sign inversion arising from sample reorientation in the instrument. These results connect the molecular structure and orientation to large CD observable in oriented thin-film assemblies, with the potential for further extension to broad classes of chiral-specific spectral analyses. Less
We have adapted the cell painting assay developed by Carpenter and colleagues on cultured U OS cells to human spermatozoa In Sperm Cell Painting SCP we assemble an image-based quantitative fingerprint of the functional state of sperm We use this assay to gain insight into the mechanism of action of compounds that modify sperm function and as a platform for contraceptive discovery
Isopentenyl diphosphate IDP and dimethylallyl diphosphate DMADP are synthesized as the final step of the -C-methyl-D-erythritol -phosphate MEP pathway by E - - hydroxy- -methylbut- -en- -yl diphosphate reductase HDR and serve as the fundamental precursors in the biosynthesis of isoprenoids Previous studies have determined distinct activities among HDR homologous originating from the same woody plants This study aims to via crystallization determine the structure for two Picea abies HDR isoforms to shed light on the observed variation in enzymatic activity Crystals for both of the HDr isoforms have been achieved in this study However time constraints have prevented any ... More
Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP) are synthesized
as the final step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway by (E)-4-
hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR) and serve as the fundamental
precursors in the biosynthesis of isoprenoids. Previous studies have determined distinct
activities among HDR homologous originating from the same woody plants. This study
aims to, via crystallization, determine the structure for two Picea abies HDR isoforms
to shed light on the observed variation in enzymatic activity. Crystals for both of the
HDr isoforms have been achieved in this study. However, time constraints have prevented
any further analysis, leaving their structures unresolved. Nonetheless, future endeavors
dedicated to exploring the HDR building upon these results are likely to result in solved
structures. Less
as the final step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway by (E)-4-
hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR) and serve as the fundamental
precursors in the biosynthesis of isoprenoids. Previous studies have determined distinct
activities among HDR homologous originating from the same woody plants. This study
aims to, via crystallization, determine the structure for two Picea abies HDR isoforms
to shed light on the observed variation in enzymatic activity. Crystals for both of the
HDr isoforms have been achieved in this study. However, time constraints have prevented
any further analysis, leaving their structures unresolved. Nonetheless, future endeavors
dedicated to exploring the HDR building upon these results are likely to result in solved
structures. Less
The COVID- pandemic caused by severe acute respiratory syndrome coronavirus SARS-CoV- virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV- variants or novel CoVs Here we describe small molecule inhibitors for SARS-CoV- Mac which counters ADP-ribosylation mediated innate immune responses The compounds inhibiting Mac were discovered through high-throughput screening HTS using a protein FRET-based competition assay and the best hit compound had an IC of M Three validated HTS hits have the same -amide- -methylester thiophene scaffold and the scaffold was selected for structure-activity relationship SAR studies through commercial and synthesized ... More
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV-2 variants or novel CoVs. Here, we describe small molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation mediated innate immune responses. The compounds inhibiting Mac1 were discovered through high-throughput screening (HTS) using a protein FRET-based competition assay and the best hit compound had an IC50 of 14 µM. Three validated HTS hits have the same 2-amide-3-methylester thiophene scaffold and the scaffold was selected for structure-activity relationship (SAR) studies through commercial and synthesized analogs. We studied the compound binding mode in detail using X-ray crystallography and this allowed us to focus on specific features of the compound and design analogs. Compound 27 (MDOLL-0229) had an IC50 of 2.1 µM and was generally selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human ADP-ribose binding proteins. The improved potency allowed testing of its effect on virus replication and indeed, 27 inhibited replication of a mouse hepatitis virus, a prototype CoV. Compound 27 is the first Mac1 targeted small molecule demonstrated to inhibit coronavirus replication in a cell model. This, together with its well-defined binding mode, makes 27 a good candidate for further hit/lead-optimization efforts. Less
RAF dimer inhibitors offer therapeutic potential in RAF- and RAS-driven cancers The utility of such drugs is predicated on their capacity to occupy both RAF protomers in the RAS-RAF signaling complex Here we describe a method to conditionally quantify drug-target occupancy at selected RAF protomers within an active RAS-RAF complex in cells RAF target engagement can be measured in the presence or absence of any mutant KRAS allele enabling the high-affinity state of RAF dimer inhibitors to be quantified in the cellular milieu The intracellular protomer selectivity of clinical-stage type II RAF inhibitors revealed that ARAF protomer engagement but not ... More
RAF dimer inhibitors offer therapeutic potential in RAF- and RAS-driven cancers. The utility of such drugs is predicated on their capacity to occupy both RAF protomers in the RAS-RAF signaling complex. Here we describe a method to conditionally quantify drug-target occupancy at selected RAF protomers within an active RAS-RAF complex in cells. RAF target engagement can be measured in the presence or absence of any mutant KRAS allele, enabling the high-affinity state of RAF dimer inhibitors to be quantified in the cellular milieu. The intracellular protomer selectivity of clinical-stage type II RAF inhibitors revealed that ARAF protomer engagement, but not engagement of BRAF or CRAF, is commensurate with inhibition of MAPK signaling in various mutant RAS cell lines. Our results support a fundamental role for ARAF in mutant RAS signaling and reveal poor ARAF protomer vulnerability for a cohort of RAF inhibitors undergoing clinical evaluation. Less
Biomolecular condensates are membrane-less cellular foci formed via liquid-liquid-phase separation LLPS of specific biological macromolecules to provide specialized compartments for regulating cellular functions Many viral proteins undergo LLPS to form such condensates to support viral replication and evade host antiviral responses and thus these condensates are potential targets for designing antivirals Human noroviruses HuNoV cause epidemic and sporadic gastroenteritis worldwide and are of significant health and economic burden Here we show that the RNA-dependent-RNA polymerase RdRp of the pandemic GII HuNoV which is essential for viral replication forms distinct condensates capable of recruiting other viral replication components Confocal microscopy and ... More
Biomolecular condensates are membrane-less cellular foci formed via liquid-liquid-phase separation (LLPS) of specific biological macromolecules to provide specialized compartments for regulating cellular functions. Many viral proteins undergo LLPS to form such condensates to support viral replication and evade host antiviral responses, and thus, these condensates are potential targets for designing antivirals. Human noroviruses (HuNoV) cause epidemic and sporadic gastroenteritis worldwide and are of significant health and economic burden. Here, we show that the RNA-dependent-RNA polymerase (RdRp) of the pandemic GII.4 HuNoV, which is essential for viral replication, forms distinct condensates capable of recruiting other viral replication components. Confocal microscopy and light scattering experiments show that RdRp phase separates into dynamic liquid-like condensates at physiological conditions. These condensates exhibit all the signature features of LLPS, including fluorescence recovery after photo-bleaching, droplet-fusion, surface wetting, and dripping in vitro and in live cells. More importantly, within these condensates, the RdRp exhibits a significant time-dependent increase in its enzymatic activity and recruits other components, such as RNA and the viral genome-linked protein (VPg), which are essential for viral replication. Such condensates, recognized by anti-RdRp antibodies, are observed in HuNoV-infected human intestinal enteroid cultures. Together, our studies demonstrate a hitherto unsuspected activity of HuNoV RdRp to form LLPS, which we suggest provides distinct cellular sites for efficient viral replication and its regulation. Less
Wastewater-based SARS-CoV- epidemiology WBE has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants However for a timely transmission of results sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis comparative quality control of the analytical procedures is essential to report reliable and comparable results In this study we performed an interlaboratory comparison at laboratories ... More
Wastewater-based SARS-CoV-2 epidemiology (WBE) has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies. WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants. However, for a timely transmission of results, sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required. Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis, comparative quality control of the analytical procedures is essential to report reliable and comparable results.In this study, we performed an interlaboratory comparison at laboratories specialized for PCR and high-throughput-sequencing (HTS)-based WBE analysis. Frozen reserve samples from low COVID-19 incidence periods were spiked with different inactivated authentic SARS-CoV-2 variants in graduated concentrations and ratios. Samples were sent to the participating laboratories for analysis using laboratory specific methods and the reported viral genome copy numbers and the detection of viral variants were compared with the expected values.Despite the different procedures, a high concordance regarding the SARS-CoV-2 PCR quantification could be achieved with low variation between the workflows. PCR-based genotyping was, in dependence of the underlying PCR-assay performance, able to predict the relative amount of variant specific substitutions even in samples with low spike-in amount. The identification of variants by HTS, however, required >100 copies/mL wastewater and had limited predictive value when analyzing at a genome coverage below 60%.This interlaboratory test demonstrates that despite different extraction and analysis methods, a high agreement of the SARS-CoV-2 genome copy equivalents could be achieved. Hence, decentralized SARS-CoV-2 wastewater monitoring is feasible to generate comparable analysis results. However, since not all assays detected the correct variant, prior evaluation of PCR and sequencing workflows as well as sustained quality control such as interlaboratory comparisons are mandatory for correct variant detection. Less
Background Immunosuppression in sepsis is hypothesized to result from the increased expression of the immune checkpoint molecules programmed death- PD- and pro grammed death ligand- PD-L PD- and PD-L blockade therapies have been reported to increase survival in septic animals Currently the interleukin IL - within mesenchymal stem cell MSC secretome is known for its immunomodulatory capacity Objective To study the effect of IL- within MSC secretome on the expression of immune check points in the rat model of sepsis Methods We used male Rattus norvegicus rats in this research and divided them into four groups sham rats without sepsis ... More
Background: Immunosuppression in sepsis is hypothesized to result from the increased
expression of the immune checkpoint molecules programmed death-1 (PD-1) and pro
grammed death ligand-1 (PD-L1). PD-1 and PD-L1 blockade therapies have been reported
to increase survival in septic animals. Currently, the interleukin (IL)-10 within mesenchymal
stem cell (MSC) secretome is known for its immunomodulatory capacity. Objective: To
study the effect of IL-10 within MSC secretome on the expression of immune check
points in the rat model of sepsis. Methods: We used 48 male Rattus norvegicus rats
in this research and divided them into four groups: sham (rats without sepsis induction
and treatment), control (sepsis-induced rats without treatment), T1 (sepsis-induced rats
treated with 150 µL of secreted IL-10 from MSC), and T2 (sepsis-induced rats treated
with 300 µL of secreted IL-10 from MSC). Forty-eight hours after sepsis induction, we
terminated the rats and collected the blood to examine the PD-1 and PD-L1 expression
levels. Results: We found a decrease in the relative expression of PD-1 in the septic rat
group given 150 µL and 300 µL of secreted IL-10 from MSC compared to the control
group, but the decrease was not significant. We also found a decrease in the relative
expression of PD-L1 mRNA in the septic rat group given 150 µL and 300 µL of secreted
IL-10 from MSC compared to the control group. Conclusion: Administering secreted IL-10
from MSC reduces the expression of PD-1 and PD-L1 in sepsis. These findings suggest
that MSC secretome can improve the immunosuppression in sepsis. Less
expression of the immune checkpoint molecules programmed death-1 (PD-1) and pro
grammed death ligand-1 (PD-L1). PD-1 and PD-L1 blockade therapies have been reported
to increase survival in septic animals. Currently, the interleukin (IL)-10 within mesenchymal
stem cell (MSC) secretome is known for its immunomodulatory capacity. Objective: To
study the effect of IL-10 within MSC secretome on the expression of immune check
points in the rat model of sepsis. Methods: We used 48 male Rattus norvegicus rats
in this research and divided them into four groups: sham (rats without sepsis induction
and treatment), control (sepsis-induced rats without treatment), T1 (sepsis-induced rats
treated with 150 µL of secreted IL-10 from MSC), and T2 (sepsis-induced rats treated
with 300 µL of secreted IL-10 from MSC). Forty-eight hours after sepsis induction, we
terminated the rats and collected the blood to examine the PD-1 and PD-L1 expression
levels. Results: We found a decrease in the relative expression of PD-1 in the septic rat
group given 150 µL and 300 µL of secreted IL-10 from MSC compared to the control
group, but the decrease was not significant. We also found a decrease in the relative
expression of PD-L1 mRNA in the septic rat group given 150 µL and 300 µL of secreted
IL-10 from MSC compared to the control group. Conclusion: Administering secreted IL-10
from MSC reduces the expression of PD-1 and PD-L1 in sepsis. These findings suggest
that MSC secretome can improve the immunosuppression in sepsis. Less
In sepsis simultaneously elevated levels of pro-inflammatory cytokines and interleukin IL - indicate immune response dysregulation increasing the mortality of the host As mesenchymal stem cell MSC secretome is known to have immunomodulatory effects we aim to assess the role of MSC secretome in the inflammatory mediators NF- B p and p TNF- IL- and the survival rate of a rat model of sepsis In this study forty-eight male Rattus norvegicus rats were divided into one sham group and three groups with sepsis induction the control group and the sepsis-induced rat groups treated with L T and L T of ... More
In sepsis, simultaneously elevated levels of pro-inflammatory cytokines and interleukin (IL)-10 indicate immune response dysregulation, increasing the mortality of the host. As mesenchymal stem cell (MSC) secretome is known to have immunomodulatory effects, we aim to assess the role of MSC secretome in the inflammatory mediators (NF-κB p65 and p50, TNF-α, IL-10) and the survival rate of a rat model of sepsis. In this study, forty-eight male Rattus norvegicus rats were divided into one sham group and three groups with sepsis induction: the control group and the sepsis-induced rat groups treated with 150 μL (T1) and 300 μL (T2) of secretome. The survival rate was observed per 6 h for 48 h and plotted using the Kaplan–Meier method. Compared to the control group, T2 showed a significant decrease in the relative expression of NF-κB and the serum TNF-α level, and a significant increase in the serum IL-10 level. Meanwhile, T1 showed a significant decrease in the serum TNF-α level compared to the control group. The Kaplan–Meier Log Rank test did not show significance in the distribution of survival between T1, T2, and the control group. However, from the 18th to the 36th hour, the survival rate of T2 was lower than the survival rate of the control group and T1, with a noticeable difference between T2 and the control group, as well as T1 at the 36th hour. At the 42nd hour, the survival rate of T2 was the same as the control group and remained lower than T1. In conclusion, MSC secretome regulated the inflammatory mediators in rat model of sepsis, with a dose of 150 μL being more effective. Less
Next-generation sequencing NGS technology advancements continue to reduce the cost of high-throughput genome-wide genotyping for breeding and genetics research Skim sequencing which surveys the entire genome at low coverage has become feasible for quantitative trait locus QTL mapping and genomic selection in various crops However the genome complexity of allopolyploid crops such as wheat Triticum aestivum L still poses a significant challenge for genome-wide genotyping Targeted sequencing of the protein-coding regions i e exome reduces sequencing costs compared to whole genome re-sequencing and can be used for marker discovery and genotyping We developed a method called skim exome capture SEC ... More
Next-generation sequencing (NGS) technology advancements continue to reduce the cost of high-throughput genome-wide genotyping for breeding and genetics research. Skim sequencing, which surveys the entire genome at low coverage, has become feasible for quantitative trait locus (QTL) mapping and genomic selection in various crops. However, the genome complexity of allopolyploid crops such as wheat (Triticum aestivum L.) still poses a significant challenge for genome-wide genotyping. Targeted sequencing of the protein-coding regions (i.e., exome) reduces sequencing costs compared to whole genome re-sequencing and can be used for marker discovery and genotyping. We developed a method called skim exome capture (SEC) that combines the strengths of these existing technologies and produces targeted genotyping data while decreasing the cost on a per-sample basis compared to traditional exome capture. Specifically, we fragmented genomic DNA using a tagmentation approach, then enriched those fragments for the low-copy genic portion of the genome using commercial wheat exome baits and multiplexed the sequencing at different levels to achieve desired coverage. We demonstrated that for a library of 48 samples, ∼7–8× target coverage was sufficient for high-quality variant detection. For higher multiplexing levels of 528 and 1056 samples per library, we achieved an average coverage of 0.76× and 0.32×, respectively. Combining these lower coverage SEC sequencing data with genotype imputation using a customized wheat practical haplotype graph database that we developed, we identified hundreds of thousands of high-quality genic variants across the genome. The SEC method can be used for high-resolution QTL mapping, genome-wide association studies, genomic selection, and other downstream applications. Less
Single-cell analysis has clearly established itself in biology and biomedical fields as an invaluable tool that allows one to comprehensively understand the relationship between cells including their types states transitions trajectories and spatial position Scientific methods such as fluorescence labeling nanoscale super-resolution microscopy advances in single cell RNAseq and proteomics technologies provide more detailed information about biological processes which were not evident with the analysis of bulk material This new era of single-cell biology provides a better understanding of such complex biological systems as cancer inflammation immunity mechanism and aging processes and opens the door into the field of drug ... More
Single-cell analysis has clearly established itself in biology and biomedical fields as an invaluable tool that allows one to comprehensively understand the relationship between cells, including their types, states, transitions, trajectories, and spatial position. Scientific methods such as fluorescence labeling, nanoscale super-resolution microscopy, advances in single cell RNAseq and proteomics technologies, provide more detailed information about biological processes which were not evident with the analysis of bulk material. This new era of single-cell biology provides a better understanding of such complex biological systems as cancer, inflammation, immunity mechanism and aging processes, and opens the door into the field of drug response heterogeneity. The latest discoveries of cellular heterogeneity gives us an unique understanding of complex biological processes, such as disease mechanism, and will lead to new strategies for better and personalized treatment strategies. Recently, single-cell proteomics techniques that allow quantification of thousands of proteins from single mammalian cells have been introduced. Here we present an improved single-cell mass spectrometry-based proteomics platform called SCREEN (Single Cell pRotEomE aNalysis) for deep and high-throughput single-cell proteome coverage with high efficiency, less turnaround time and with an improved ability for protein quantitation across more cells than previously achieved. We applied this new platform to analyze the single-cell proteomic landscape under different drug treatment over time to uncover heterogeneity in cancer cell response, which for the first time, to our knowledge, has been achieved by mass spectrometry based analytical methods. We discuss challenges in single-cell proteomics, future improvements and general trends with the goal to encourage forthcoming technical developments. Less
Schistosomiasis is caused by parasites of the genus Schistosoma which infect more than million people While praziquantel PZQ has been the main drug for controlling schistosomiasis for over four decades PZQ drug resistant strains have already been reported highlighting the need to search for new schistosomicidal drugs S mansoni survival relies on the redox enzyme thioredoxin glutathione reductase Sm TGR a validated target for the development of new anti-schistosomal drugs Here we report a fragment screening campaign of compounds against Sm TGR using X-ray crystallography and our efforts to optimize the hits found into potent inhibitors We observed binding events ... More
Schistosomiasis is caused by parasites of the genus Schistosoma , which infect more than 200 million people. While praziquantel (PZQ) has been the main drug for controlling schistosomiasis for over four decades, PZQ drug resistant strains have already been reported, highlighting the need to search for new schistosomicidal drugs. S. mansoni survival relies on the redox enzyme thioredoxin glutathione reductase ( Sm TGR), a validated target for the development of new anti-schistosomal drugs. Here we report a fragment screening campaign of 768 compounds against Sm TGR using X-ray crystallography and our efforts to optimize the hits found into potent inhibitors. We observed 49 binding events involving 35 distinct molecular fragments which were found to be distributed across 16 binding sites. Most sites are described for the first time within Sm TGR, a noteworthy exception being the “door stop pocket” near the NADPH binding site. Fragments binding to the latter were prioritized to undergo a “SAR by catalog” strategy for optimization into potential inhibitors. A search for compounds containing any of the prioritized fragments as a substructure was made in commercial databases. The ability of these compounds to inhibit Sm TGR was predicted based on a binary ML classification model, followed by an analysis of the putative binding mode by molecular docking. The 38 best ranked compounds were purchased and experimentally evaluated for Sm TGR inhibition. Compound 14 inhibited 63.6% of enzyme activity at 100 µM and presented an estimated IC 50 of 33 µM against Sm TGR. Less
Several hydrolases have been described to degrade polyethylene terephthalate PET at moderate temperatures ranging from C to C These mesophilic PET hydrolases PETases are less efficient in degrading this plastic polymer than their thermophilic homologs and have therefore been the subject of many protein engineering campaigns However enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations Additionally evaluating the improvements in both activity and stability requires screening numerous variants either individually or using high-throughput screening methods Here we utilize instead the design of chimeras ... More
Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25°C to 40°C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic homologs and have, therefore, been the subject of many protein engineering campaigns. However, enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations. Additionally, evaluating the improvements in both activity and stability requires screening numerous variants, either individually or using high-throughput screening methods. Here, we utilize instead the design of chimeras as a protein engineering strategy to increase the activity and stability of Mors1, an Antarctic PETase active at 25°C. First, we obtained the crystal structure of Mors1 at 1.6 Å resolution, which we used as a scaffold for structure- and sequence-based chimeric design. Then, we designed a Mors1 chimera via loop exchange of a highly divergent active site loop from the thermophilic leaf-branch compost cutinase (LCC) into the equivalent region in Mors1. After restitution of an active site disulfide bond into this chimera, the enzyme exhibited a shift in optimal temperature for activity to 45°C and an increase in fivefold in PET hydrolysis when compared with wild-type Mors1 at 25°C. Our results serve as a proof of concept of the utility of chimeric design to further improve the activity and stability of PETases active at moderate temperatures. Less
Receptor-interacting protein kinases and RIPK and RIPK are considered attractive therapeutic enzyme targets for the treatment of a multitude of inflammatory diseases and cancers In this study we developed three interrelated series of novel quinazoline-based derivatives to investigate the effects of extensive modifications of positions and of the central core on the inhibitory activity and the selectivity against these RIPKs The design of the derivatives was inspired by analyses of available literary knowledge on both RIPK and RIPK in complex with known quinazoline or quinoline inhibitors Enzymatic investigations for bioactivity of the prepared molecules against purified RIPKs RIPK - shed ... More
Receptor-interacting protein kinases 2 and 3 (RIPK2 and RIPK3) are considered attractive therapeutic enzyme targets for the treatment of a multitude of inflammatory diseases and cancers. In this study, we developed three interrelated series of novel quinazoline-based derivatives to investigate the effects of extensive modifications of positions 6 and 7 of the central core on the inhibitory activity and the selectivity against these RIPKs. The design of the derivatives was inspired by analyses of available literary knowledge on both RIPK2 and RIPK3 in complex with known quinazoline or quinoline inhibitors. Enzymatic investigations for bioactivity of the prepared molecules against purified RIPKs (RIPK1-4) shed light on multiple potent and selective RIPK2 and dual RIPK2/3 inhibitors. Furthermore, evaluations in living cells against the RIPK2-NOD1/2-mediated signaling pathways, identified as the potential primary targets, demonstrated nanomolar inhibition for a majority of the compounds. In addition, we have demonstrated overall good stability of various lead inhibitors in both human and mouse microsomes and plasma. Several of these compounds also were evaluated for selectivity across 58 human kinases other than RIPKs, exhibiting outstanding specificity profiles. We have thus clearly demonstrated that tuning appropriate substitutions at positions 6 and 7 of the developed quinazoline derivatives may lead to interesting potency and specificities against RIPK2 and RIPK3. This knowledge might therefore be employed for the targeted preparation of new, highly potent and selective tools against these RIPKs, which could be of utility in biological and clinical research. Less
Laboratory production of recombinant mammalian proteins particularly antibodies requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications Transient gene expression TGE in the suspension-adapted Chinese Hamster Ovary CHO cell lines has become the method of choice for this task The antibodies can be secreted into the media which facilitates subsequent purification and can be glycosylated However in general protein production in CHO cells is expensive and may provide variable outcomes namely in laboratories without previous experience While achievable yields may be influenced by the nucleotide sequence there are other aspects of the process which offer ... More
Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes. Less
The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte TIL therapy We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors TCRs following successful TIL therapy for stage IV malignant melanoma Remarkably individual TCRs could target multiple different tumor types via the HLA A -restricted epitopes EAAGIGILTV LLLGIGILVL and NLSALGIFST from Melan A BST and IMP respectively Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A G-I L-G-I-x-x-x recognition motif Multi-epitope targeting allows individual ... More
The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such “multipronged” T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies. Less
The Dictyostelium discoideum dye-decolorizing peroxidase DdDyP is a newly discovered peroxidase which belongs to a unique class of heme peroxidase family that lacks homology to the known members of plant peroxidase superfamily DdDyP catalyzes the H O -dependent oxidation of a wide-spectrum of substrates ranging from polycyclic dyes to lignin biomass holding promise for potential industrial and biotechnological applications To study the molecular mechanism of DdDyP highly pure and functional protein with a natively incorporated heme is required however obtaining a functional DyP-type peroxidase with a natively bound heme is challenging and often requires addition of expensive biosynthesis precursors Alternatively ... More
The Dictyostelium discoideum dye-decolorizing peroxidase (DdDyP) is a newly discovered peroxidase, which belongs to a unique class of heme peroxidase family that lacks homology to the known members of plant peroxidase superfamily. DdDyP catalyzes the H2O2-dependent oxidation of a wide-spectrum of substrates ranging from polycyclic dyes to lignin biomass, holding promise for potential industrial and biotechnological applications. To study the molecular mechanism of DdDyP, highly pure and functional protein with a natively incorporated heme is required, however, obtaining a functional DyP-type peroxidase with a natively bound heme is challenging and often requires addition of expensive biosynthesis precursors. Alternatively, a heme in vitro reconstitution approach followed by a chromatographic purification step to remove the excess heme is often used. Here, we show that expressing the DdDyP peroxidase in ×2 YT enriched medium at low temperature (20°C), without adding heme supplement or biosynthetic precursors, allows for a correct native incorporation of heme into the apo-protein, giving rise to a stable protein with a strong Soret peak at 402 nm. Further, we crystallized and determined the native structure of DdDyP at a resolution of 1.95 Å, which verifies the correct heme binding and its geometry. The structural analysis also reveals a binding of two water molecules at the distal site of heme plane bridging the catalytic residues (Arg239 and Asp149) of the GXXDG motif to the heme-Fe(III) via hydrogen bonds. Our results provide new insights into the geometry of native DdDyP active site and its implication on DyP catalysis. Less
The use of antibiotics has undoubtedly been a boon for humanity in combating infections and microbial threats However their widespread utilization has contributed to the emergence and spread of antibiotic resistance which now poses a significant public health challenge Streptomyces bacterium produce diverse secondary metabolites with antibacterial antifungal antiviral antitumoral and immunosuppressant activities Among these compounds is echinomycin a nonribosomal peptide antibiotic synthesized by Streptomyces lasalocidi which inhibits DNA replication and transcription by intercalating the DNA duplex at CpG steps A gene called ecm was identified in the echinomycin biosynthetic gene cluster which provides echinomycin self-resistance Ecm recognizes DNA duplexes ... More
The use of antibiotics has undoubtedly been a boon for humanity in combating infections and microbial threats. However, their widespread utilization has contributed to the emergence and spread of antibiotic resistance, which now poses a significant public health challenge. Streptomyces bacterium, produce diverse secondary metabolites with antibacterial, antifungal, antiviral, antitumoral, and immunosuppressant activities. Among these compounds is echinomycin, a nonribosomal peptide antibiotic synthesized by Streptomyces lasalocidi, which inhibits DNA replication and transcription by intercalating the DNA duplex at CpG steps. A gene called ecm16 was identified in the echinomycin biosynthetic gene cluster, which provides echinomycin self-resistance. Ecm16 recognizes DNA duplexes that contain echinomycin, and neutralizes its toxicity through a yet undetermined mechanism. To shed light on this process, we are determining the crystal structure of Ecm16 containing ATP. We introduced strategic mutations in the nucleotide binding site of Ecm16 to prevent ATP hydrolysis. We have successfully cloned, expressed, and purified the recombinant Ecm16 E399Q,E708Q double mutant protein. We have solved the structure of this protein using X-ray Crystallography at a resolution of 2.07 Å. However, our crystal structure contained ADP instead of the expected ATP. We propose and alternative experimental strategies for structure determination of ATP-containing Ecm16. Less
Aim Type diabetes mellitus T DM is an autoimmune disease characterized by the chronic inflammation of the pancreatic islets of Langerhans Hyperglycaemia leads to suppressed antioxidant enzyme and increased inflammation in the pancreatic cell resul ting in pancreatic cell death Hypoxic secretome mesenchymal stem cells HS-MSCs are soluble molecules secreted by MSCS that have the antiinflammation ability by secreting various cytoki nes including IL- and TGF- which potent as a promising the rapeutic modality for T DM This study aims to investigate the role of HS-MSCs in regulating superoxide dismutase SOD and caspase- gene expression in T DM model Methods ... More
Aim Type 1 diabetes mellitus (T1DM) is an autoimmune disease
characterized by the chronic inflammation of the pancreatic islets
of Langerhans. Hyperglycaemia leads to suppressed antioxidant
enzyme and increased inflammation in the pancreatic cell, resul
ting in pancreatic cell death. Hypoxic secretome mesenchymal
stem cells (HS-MSCs) are soluble molecules secreted by MSCS
that have the antiinflammation ability by secreting various cytoki
nes including IL-10 and TGF-β which potent as a promising the
rapeutic modality for T1DM. This study aims to investigate the
role of HS-MSCs in regulating superoxide dismutase (SOD) and
caspase-3 gene expression in T1DM model.
Methods Twenty male Wistar rats (6 to 8 weeks old) were rando
mly divided into four groups (sham, control, HS-MSCs 0.5 mL
and HS-MSCs 1 mL intraperitoneal treatment group). Streptozo
tocin (STZ) 60mg/kgBB was conducted once on day 1, HS-MSCs
0.5mL (T1) and HS-MSCs 1 mL (T2) were administrated intrape
ritoneally on day 7, 14, and 21 after STZ administration. The rats
were sacrificed on day 28; the gene expression of SOD and IL-6
was analysed by qRT-PCR.
Results This study showed that the ratio of SOD significantly
increased in HS-MSCs treatment associated with suppression of
IL-6 gene expression.
Conclusion HS-MSCs administration suppresses oxidative stre
ss and inflammation by up regulating SOD and inhibiting IL-6 to
control T1DM. Less
characterized by the chronic inflammation of the pancreatic islets
of Langerhans. Hyperglycaemia leads to suppressed antioxidant
enzyme and increased inflammation in the pancreatic cell, resul
ting in pancreatic cell death. Hypoxic secretome mesenchymal
stem cells (HS-MSCs) are soluble molecules secreted by MSCS
that have the antiinflammation ability by secreting various cytoki
nes including IL-10 and TGF-β which potent as a promising the
rapeutic modality for T1DM. This study aims to investigate the
role of HS-MSCs in regulating superoxide dismutase (SOD) and
caspase-3 gene expression in T1DM model.
Methods Twenty male Wistar rats (6 to 8 weeks old) were rando
mly divided into four groups (sham, control, HS-MSCs 0.5 mL
and HS-MSCs 1 mL intraperitoneal treatment group). Streptozo
tocin (STZ) 60mg/kgBB was conducted once on day 1, HS-MSCs
0.5mL (T1) and HS-MSCs 1 mL (T2) were administrated intrape
ritoneally on day 7, 14, and 21 after STZ administration. The rats
were sacrificed on day 28; the gene expression of SOD and IL-6
was analysed by qRT-PCR.
Results This study showed that the ratio of SOD significantly
increased in HS-MSCs treatment associated with suppression of
IL-6 gene expression.
Conclusion HS-MSCs administration suppresses oxidative stre
ss and inflammation by up regulating SOD and inhibiting IL-6 to
control T1DM. Less
Mutagens often prefer specific nucleotides or oligonucleotide motifs that can be revealed by studying the hypermutation spectra in single-stranded ss DNA We utilized a yeast model to explore mutagenesis by glycidamide a simple epoxide formed endogenously in humans from the environmental toxicant acrylamide Glycidamide caused ssDNA hypermutation in yeast predominantly in cytosines and adenines The most frequent mutations in adenines occurred in the nAt nGt trinucleotide motif Base substitutions A G in this motif relied on Rev translesion polymerase activity Inactivating Rev did not alter the nAt trinucleotide preference suggesting it may be an intrinsic specificity of the chemical reaction ... More
Mutagens often prefer specific nucleotides or oligonucleotide motifs that can be revealed by studying the hypermutation spectra in single-stranded (ss) DNA. We utilized a yeast model to explore mutagenesis by glycidamide, a simple epoxide formed endogenously in humans from the environmental toxicant acrylamide. Glycidamide caused ssDNA hypermutation in yeast predominantly in cytosines and adenines. The most frequent mutations in adenines occurred in the nAt→nGt trinucleotide motif. Base substitutions A→G in this motif relied on Rev1 translesion polymerase activity. Inactivating Rev1 did not alter the nAt trinucleotide preference, suggesting it may be an intrinsic specificity of the chemical reaction between glycidamide and adenine in the ssDNA. We found this mutational motif enriched in published sequencing data from glycidamide-treated mouse cells and ubiquitous in human cancers. In cancers, this motif was positively correlated with the single base substitution (SBS) smoking-associated SBS4 signature, with the clock-like signatures SBS1, SBS5, and was strongly correlated with smoking history and with age of tumor donors. Clock-like feature of the motif was also revealed in cells of human skin and brain. Given its pervasiveness, we propose that this mutational motif reflects mutagenic lesions to adenines in ssDNA from a potentially broad range of endogenous and exogenous agents. Less
Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents However to fully realise the potential of these molecules selectivity remains a limiting challenge Herein we addressed the issue of selectivity in the design of CRL CRBN recruiting PROteolysis TArgeting Chimeras PROTACs Thalidomide derivatives used to generate CRL CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates such as GSPT Ikaros and Aiolos We leveraged structural insights from known CRL CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known ... More
Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC-induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation. Less
Gene therapies offer promising therapeutic alternatives for many disorders that currently lack efficient treatment options Due to their chemical nature and physico-chemical properties delivery of polynucleic acids into target cells and subcellular compartments remains a significant challenge Adeno associated viruses AAV have gained a lot of interest for the efficient delivery of therapeutic single-stranded DNA ssDNA genomes over the past decades More than a hundred products have been tested in clinical settings and three products have received market authorization by the US FDA in recent years A lot of effort is being made to generate potent recombinant AAV rAAV vectors ... More
Gene therapies offer promising therapeutic alternatives for many disorders that currently lack efficient treatment options. Due to their chemical nature and physico-chemical properties, delivery of polynucleic acids into target cells and subcellular compartments remains a significant challenge. Adeno associated viruses (AAV) have gained a lot of interest for the efficient delivery of therapeutic single-stranded DNA (ssDNA) genomes over the past decades. More than a hundred products have been tested in clinical settings and three products have received
market authorization by the US FDA in recent years. A lot of effort is being made to generate potent recombinant AAV (rAAV) vectors that show favorable safety and immunogenicity profiles for either local or systemic administration. Manufacturing processes are gradually being optimized to deliver a consistently high product quality and to serve potential market needs beyond rare indications. In contrast to protein therapeutics, most rAAV products are still supplied as frozen liquids within rather simple formulation buffers to enable sufficient product shelf life, significantly hampering global distribution and access. In this review, we aim to outline the hurdles of rAAV drug product development and discuss critical formulation and composition aspects of rAAV
products under clinical evaluation. Further, we highlight recent development efforts in order to achieve stable liquid or lyophilized products. This review therefore provides a comprehensive overview on current state-of-the- art rAAV formulations and can further serve as a map for rational formulation development activities in the
future. Less
market authorization by the US FDA in recent years. A lot of effort is being made to generate potent recombinant AAV (rAAV) vectors that show favorable safety and immunogenicity profiles for either local or systemic administration. Manufacturing processes are gradually being optimized to deliver a consistently high product quality and to serve potential market needs beyond rare indications. In contrast to protein therapeutics, most rAAV products are still supplied as frozen liquids within rather simple formulation buffers to enable sufficient product shelf life, significantly hampering global distribution and access. In this review, we aim to outline the hurdles of rAAV drug product development and discuss critical formulation and composition aspects of rAAV
products under clinical evaluation. Further, we highlight recent development efforts in order to achieve stable liquid or lyophilized products. This review therefore provides a comprehensive overview on current state-of-the- art rAAV formulations and can further serve as a map for rational formulation development activities in the
future. Less
TP is the most frequently mutated gene in cancer yet key target genes for p -mediated tumor suppression remain unidentified Here we characterize a rare African-specific germline variant of TP in the DNA-binding domain Tyr His Y H Nuclear magnetic resonance and crystal structures reveal that Y H is structurally similar to wild-type p Consistent with this we find that Y H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p target genes this includes the epigenetic modifier PADI which deiminates arginine to the nonnatural amino acid citrulline Surprisingly we show ... More
TP53 is the most frequently mutated gene in cancer, yet key target genes for p53-mediated tumor suppression remain unidentified. Here, we characterize a rare, African-specific germline variant of TP53 in the DNA-binding domain Tyr107His (Y107H). Nuclear magnetic resonance and crystal structures reveal that Y107H is structurally similar to wild-type p53. Consistent with this, we find that Y107H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p53 target genes; this includes the epigenetic modifier PADI4, which deiminates arginine to the nonnatural amino acid citrulline. Surprisingly, we show that Y107H mice develop spontaneous cancers and metastases and that Y107H shows impaired tumor suppression in two other models. We show that PADI4 is itself tumor suppressive and that it requires an intact immune system for tumor suppression. We identify a p53–PADI4 gene signature that is predictive of survival and the efficacy of immune-checkpoint inhibitors. Less
The small size and flexibility of G protein-coupled receptors GPCRs have long posed a significant challenge to determining their structures for research and therapeutic applications Single particle cryogenic electron microscopy cryoEM is often out of reach due to the small size of the receptor without a signaling partner Crystallization of GPCRs in lipidic cubic phase LCP often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers Here we determine the previously unknown structure of the human vasopressin B receptor V BR ... More
The small size and flexibility of G protein-coupled receptors (GPCRs) have long posed a significant challenge to determining their structures for research and therapeutic applications. Single particle cryogenic electron microscopy (cryoEM) is often out of reach due to the small size of the receptor without a signaling partner. Crystallization of GPCRs in lipidic cubic phase (LCP) often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers. Here, we determine the previously unknown structure of the human vasopressin 1B receptor (V1BR) using microcrystal electron diffraction (MicroED). To achieve this, we grew V1BR microcrystals in LCP and transferred the material directly onto electron microscopy grids. The protein was labeled with a fluorescent dye prior to crystallization to locate the microcrystals using cryogenic fluorescence microscopy, and then the surrounding material was removed using a plasma-focused ion beam to thin the sample to a thickness amenable to MicroED. MicroED data from 14 crystalline lamellae were used to determine the 3.2 Å structure of the receptor in the crystallographic space group P 1. These results demonstrate the use of MicroED to determine previously unknown GPCR structures that, despite significant effort, were not tractable by other methods. Less