1052 Citations
Pathogenic bacteria utilize specialized macromolecular secretion systems to transport virulence factors across membrane s and manipulate their infected host To date secretion systems have been identified including the type IX secretion system T SS associated with human avian and farmed-fish diseases As a bacterial secretion system the T SS also facilitates gliding motility and the degradation of different macromolecules by the secretion of metabolic enzymes in nonpathogenic bacteria PorX is a highly conserved protein that regulates the transcription of essential T SS components and additionally mediates the function of T SS via direct interaction with PorL the rotary motor protein ... More
Pathogenic bacteria utilize specialized macromolecular secretion systems to transport virulence factors across membrane(s) and manipulate their infected host. To date, 11 secretion systems have been identified, including the type IX secretion system (T9SS) associated with human, avian and farmed-fish diseases. As a bacterial secretion system, the T9SS also facilitates gliding motility and the degradation of different macromolecules by the secretion of metabolic enzymes in nonpathogenic bacteria. PorX is a highly conserved protein that regulates the transcription of essential T9SS components and additionally mediates the function of T9SS via direct interaction with PorL, the rotary motor protein of the T9SS. PorX is also a member of a two-component system regulatory cascade, where it serves as the response regulator that relays a signal transduced from a conserved sensor histidine kinase, PorY, to a designated sigma factor. Here, the recombinant expression and purification of PorX homologous proteins from the pathogenic bacterium Porphyromonas gingivalis and the nonpathogenic bacterium Flavobacterium johnsoniae are reported. A bioinformatical characterization of the different domains comprising the PorX protein is also provided, and the crystallization and X-ray analysis of PorX from F. johnsoniae are reported. Less
Intrinsically disordered proteins IDPs often coordinate transient interactions with multiple proteins to mediate complex signals within large protein networks Among these the IDP hub protein G BP can form complexes with cytoplasmic phosphoprotein Caprin and ubiquitin peptidase USP the resulting control of USP activity contributes to a pathogenic virulence system that targets endocytic recycling of the ion channel CFTR However while the identities of protein interactors are known for many IDP hub proteins the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood Here we describe in vitro analysis of these G BP ... More
Intrinsically disordered proteins (IDPs) often coordinate transient interactions with multiple proteins to mediate complex signals within large protein networks. Among these, the IDP hub protein G3BP1 can form complexes with cytoplasmic phosphoprotein Caprin1 and ubiquitin peptidase USP10; the resulting control of USP10 activity contributes to a pathogenic virulence system that targets endocytic recycling of the ion channel CFTR. However, while the identities of protein interactors are known for many IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood. Here we describe in vitro analysis of these G3BP1 affinities, and show tryptophan substitutions of specific G3BP1 residues reduce its affinity for both USP10 and Caprin1. We show that these same mutations reduce the stability of complexes between the full-length proteins, suggesting that co-purification can serve as a surrogate measure of interaction strength. The crystal structure of G3BP1 TripleW (F15W/F33W/F124W) mutant reveals a clear reorientation of the side chain of W33, creating a steric clash with USP10 and Caprin1. Furthermore, an amino-acid scan of USP10 and Caprin1 peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific substitutions with the potential to create higher affinity peptides. Taken together, these data show that small changes in component binding affinities can have significant effects on the composition of cellular interaction hubs. These specific protein mutations can be harnessed to manipulate complex protein networks, informing future investigations into roles of these networks in cellular processes. Less
The multifunctional human Parkinson s disease protein PARK DJ is an attractive therapeutic target due to its link with early-onset Parkinson s disease upregulation in various cancers and contribution to chemoresistance However only a few compounds have been identified to bind PARK due to the lack of a dedicated chemical toolbox We report the creation of such a toolbox and showcase the application of each of its components The selective PARK submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys Installment of different dyes onto the inhibitor delivered two PARK probes The Rhodamine probe provides a ... More
The multifunctional human Parkinson’s disease protein 7 (PARK7/DJ1) is an attractive therapeutic target due to its link with early-onset Parkinson’s disease, upregulation in various cancers, and contribution to chemoresistance. However, only a few compounds have been identified to bind PARK7 due to the lack of a dedicated chemical toolbox. We report the creation of such a toolbox and showcase the application of each of its components. The selective PARK7 submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys106. Installment of different dyes onto the inhibitor delivered two PARK7 probes. The Rhodamine110 probe provides a high-throughput screening compatible FP assay, showcased by screening a compound library (8000 molecules). The SulfoCy5-equipped probe is a valuable tool to assess the effect of PARK7 inhibitors in a cell lysate. Our work creates new possibilities to explore PARK7 function in a physiologically relevant setting and develop new and improved PARK7 inhibitors. Less
Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets Despite recent advances in the field efforts to discover such compounds have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different PARP enzymes of the ARTD family We here expanded the methods and engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold Compared to the free enzymes the scaffold-based system results in an improved activity for the tested PARPs due to improved ... More
Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets. Despite recent advances in the field, efforts to discover such compounds have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different PARP enzymes of the ARTD family. We here expanded the methods and engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold. Compared to the free enzymes, the scaffold-based system results in an improved activity for the tested PARPs due to improved solubility, stability and the proximity of the catalytic domains, altogether boosting their activity beyond 10-fold in the case of PARP12. This allows us to measure their enhanced activity using a simple and easily accessible homogeneous NAD+ conversion assay, facilitating its automation to reduce the assay volume and lowering the assay costs. The approach will enable the discovery of more potent compounds due to increased assay sensitivity and it can be applied to compound screening campaigns as well as inhibitor profiling. Less
The main focus of this project was chicken protein MICAL which is involved in the semaphorin-plexin signalling pathway and has a significant effect on the rearrangement of the cytoskeleton The prominent role of the MICAL protein is primarily associated with axon guidance as it destabilizes actin filaments through its oxidative activity We focused on elucidating the molecular mechanisms of chicken MICAL autoinhibition using molecular and structural biology methods together with new protein structure prediction methods Chicken MICAL was produced in Sf insect cells using a baculovirus expression system and we produced both full-length and truncated versions of chicken MICAL protein ... More
The main focus of this project was chicken protein MICAL1, which is involved in the semaphorin-plexin signalling pathway and has a significant effect on the rearrangement of the cytoskeleton. The prominent role of the MICAL1 protein is primarily associated with axon guidance, as it destabilizes actin filaments through its oxidative activity. We focused on elucidating the molecular mechanisms of chicken MICAL1 autoinhibition using molecular and structural biology methods together with new protein structure prediction methods. Chicken MICAL1 was produced in Sf9 insect cells using a baculovirus expression system and we produced both full-length and truncated versions of chicken MICAL1 protein. We kinetically characterized the protein and determined its oligomeric state in solution. We made great efforts to solve the protein structure using crystallography, electron microscopy and protein structure prediction in Alphafold 2. Based on the results of these experiments and assays, we conclude that MICAL1 proteins are regulated through their C terminal domain, which interacts with the monooxygenase domain. The part of this interaction is the autoinhibition of chicken MICAL1. We excluded the possibility that chicken MICAL1 is regulated by changing its oligomeric state. The results of this master's thesis. Less
Autofluorescence-detected photothermal mid-infrared AF-PTIR microscopy was shown to enable parts-per-million detection of -indomethacin impurity in -indomethacin samples Subtle differences in the photothermal response of the UV-autofluorescence of two indomethacin crystal polymorphs were used for sub-micron chemical discrimination based on fingerprint region mid-IR spectroscopy The AF-PTIR assignment was independently confirmed by second harmonic generation SHG microscopy which was shown to reduce the total analysis time by rapidly identifying the suitable fields of view AF-PTIR microscopy has the potential to assist in the early identification of crystal form impurities in the solid dosage forms development pipeline
Modulators of the G protein-coupled A A adenosine receptor A AAR have been considered promising agents to treat Parkinson s disease inflammation cancer and central nervous system disorders Herein we demonstrate that a thiophene modification at the C position in the common adenine scaffold converted an A AAR agonist into an antagonist We synthesized and characterized a novel A AAR antagonist LJ- with Ki nM X-ray crystallographic structures of in complex with two thermostabilized A AAR constructs were solved at and resolutions In contrast to A AAR agonists which simultaneously interact with both Ser and His only transiently contacts His ... More
Modulators of the G protein-coupled A2A adenosine receptor (A2AAR) have been considered promising agents to treat Parkinson’s disease, inflammation, cancer, and central nervous system disorders. Herein, we demonstrate that a thiophene modification at the C8 position in the common adenine scaffold converted an A2AAR agonist into an antagonist. We synthesized and characterized a novel A2AAR antagonist, 2 (LJ-4517), with Ki = 18.3 nM. X-ray crystallographic structures of 2 in complex with two thermostabilized A2AAR constructs were solved at 2.05 and 2.80 Å resolutions. In contrast to A2AAR agonists, which simultaneously interact with both Ser2777.42 and His2787.43, 2 only transiently contacts His2787.43, which can be direct or water-mediated. The n-hexynyl group of 2 extends into an A2AAR exosite. Structural analysis revealed that the introduced thiophene modification restricted receptor conformational rearrangements required for subsequent activation. This approach can expand the repertoire of adenosine receptor antagonists that can be designed based on available agonist scaffolds. Less
The recent discovery of comammox complete ammonia oxidation Nitrospira has upended the long-held nitrification paradigm Although comammox Nitrospira have been identified in wastewater treatment systems the conditions for their dominance over canonical ammonia oxidizers remain unclear Here we report the dominance of comammox Nitrospira in a moving bed biofilm reactor MBBR fed with synthetic mainstream wastewater Integrated S rRNA gene amplicon sequencing fluorescence in situ hybridization FISH and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen DO concentration above mg O L The dominance of comammox Nitrospira over canonical ... More
The recent discovery of comammox (complete ammonia oxidation) Nitrospira has upended the long-held nitrification paradigm. Although comammox Nitrospira have been identified in wastewater treatment systems, the conditions for their dominance over canonical ammonia oxidizers remain unclear. Here, we report the dominance of comammox Nitrospira in a moving bed biofilm reactor (MBBR) fed with synthetic mainstream wastewater. Integrated 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization (FISH), and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen (DO) concentration above 6 mg O2/L. The dominance of comammox Nitrospira over canonical ammonia oxidizers (i.e., Nitrosomonas) was attributed to the low residual ammonium concentration (0.02–0.52 mg N/L) formed in the high-DO MBBR. Two clade A comammox Nitrospira were identified, which are phylogenetically close to Candidatus Nitrospira nitrosa. Interestingly, cryosectioning-FISH showed these two comammox species spatially distributed on the surface of the biofilm. Moreover, the ammonia-oxidizing activity of comammox Nitrospira-dominated biofilms was susceptible to the oxygen supply, which dropped by half with the DO concentration decrease from 6 to 2 mg O2/L. These features collectively suggest a low apparent oxygen affinity for the comammox Nitrospira-dominated biofilms in the high-DO nitrifying MBBR. Less
Chickpea is a crop that is known as a source of high-quality proteins CL-AI which belongs to the S globulin and cupin superfamily was initially identified in chickpea seeds CL-AI has recently been shown to inhibit various types of -amylases To determine its molecular mechanism the crystal structure of CL-AI was solved at a final resolution of Structural analysis indicated that each asymmetric unit contains three molecules with threefold symmetry and a head-to-tail association and each molecule is divided into an -chain and a -chain CL-AI has high structural similarity to other S globulins and canonical metal-dependent enzyme-related cupin proteins ... More
Chickpea is a crop that is known as a source of high-quality proteins. CL-AI, which belongs to the 11S globulin and cupin superfamily, was initially identified in chickpea seeds. CL-AI has recently been shown to inhibit various types of α-amylases. To determine its molecular mechanism, the crystal structure of CL-AI was solved at a final resolution of 2.2 Å. Structural analysis indicated that each asymmetric unit contains three molecules with threefold symmetry and a head-to-tail association, and each molecule is divided into an α-chain and a β-chain. CL-AI has high structural similarity to other 11S globulins and canonical metal-dependent enzyme-related cupin proteins, whereas its stimilarity to α-amylase inhibitor from Phaseolus vulgaris is quite low. The structure presented here will provide insight into the function of CL-AI. Less
Polyurethane-based hydrogels are relatively inexpensive and mechanically robust biomaterials with ideal properties for various applications including drug delivery prosthetics implant coatings soft robotics and tissue engineering In this report we present a simple method for synthesizing and casting biocompatible polyurethane-poly ethylene glycol PU-PEG hydrogels with tunable mechanical properties non-fouling characteristics and sustained tolerability as an implantable material or coating The hydrogels are synthesized via a simple one-pot method using commercially available precursors and low toxicity solvents and reagents yielding a consistent and biocompatible gel platform primed for long-term biomaterial applications The mechanical and physical properties of the gels are easily ... More
Polyurethane-based hydrogels are relatively inexpensive and mechanically robust biomaterials with ideal properties for various applications, including drug delivery, prosthetics, implant coatings, soft robotics, and tissue engineering. In this report, we present a simple method for synthesizing and casting biocompatible polyurethane-poly(ethylene glycol) (PU-PEG) hydrogels with tunable mechanical properties, non-fouling characteristics, and sustained tolerability as an implantable material or coating. The hydrogels are synthesized via a simple one-pot method using commercially available precursors and low toxicity solvents and reagents, yielding a consistent and biocompatible gel platform primed for long-term biomaterial applications. The mechanical and physical properties of the gels are easily controlled by varying the curing concentration, producing networks with complex shear moduli of 0.82 kPa – 190 kPa, similar to a range of human soft tissues. When evaluated against a mechanically-matched PDMS formulation the PU-PEG hydrogels demonstrated favorable non-fouling characteristics including comparable adsorption of plasma proteins (albumin and fibrinogen) and significantly reduced cellular adhesion. Moreover, preliminary murine implant studies reveal a mild foreign body response after 41 days. Due to the tunable mechanical properties, excellent biocompatibility, and sustained in vivo tolerability of these hydrogels, we propose that this method offers a simplified platform for fabricating soft PU-based biomaterials for a variety of applications. Less
Mice deficient for all ten-eleven translocation TET genes exhibit early gastrulation lethality However separating cause and effect in such embryonic failure is challenging To isolate cell-autonomous effects of TET loss we used temporal single-cell atlases from embryos with partial or complete mutant contributions Strikingly when developing within a wild-type embryo Tet-mutant cells retain near-complete differentiation potential whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential We map de-repressions of early epiblast factors e g Dppa and Gdf and failure to activate multiple signaling from nascent mesoderm Lefty FGF and Notch as likely ... More
Mice deficient for all ten-eleven translocation (TET) genes exhibit early gastrulation lethality. However, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions. Strikingly, when developing within a wild-type embryo, Tet-mutant cells retain near-complete differentiation potential, whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential. We map de-repressions of early epiblast factors (e.g., Dppa4 and Gdf3) and failure to activate multiple signaling from nascent mesoderm (Lefty, FGF, and Notch) as likely cell-intrinsic drivers of TET loss phenotypes. We further suggest loss of enhancer demethylation as the underlying mechanism. Collectively, our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications. Less
The bioactive lysophospholipid sphingosine- -phosphate S P acts via five different subtypes of S P receptors S PRs - S P - S P is predominantly expressed in nervous and immune systems regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders as well as carcinogenesis Several S PR therapeutic drugs have been developed to treat these diseases however they lack receptor subtype selectivity which leads to side effects In this article we describe a resolution room temperature crystal structure of the human S P receptor in complex with a selective ... More
The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PRs) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 Å resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric sub-pocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, our structure with S1P5-inverse agonist sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR family. Less
Acute myelogenous leukemia AML a disease of the blood and bone marrow is characterized by the inability of myeloblasts to differentiate into mature cell types Dihydroorotate dehydrogenase DHODH is an enzyme well-known in the pyrimidine biosynthesis pathway however small molecule DHODH inhibitors were recently shown to induce differentiation in multiple AML subtypes Using virtual screening and structure-based drug design approaches a new series of N-heterocyclic -pyridyl carboxamide DHODH inhibitors were discovered Two lead compounds and have potent biochemical and cellular DHODH activity favorable physicochemical properties and efficacy in a preclinical model of AML
We studied the prevalent cytotoxic CD T cell response mounted against severe acute respiratory syndrome coronavirus SARS-CoV- Spike glycoprotein - epitope sequence YLQPRTFLL via the most frequent human leukocyte antigen HLA class I worldwide HLA A The Spike P L mutation that has arisen in at least different SARS-CoV- lineages to date including in lineages classified as variants of concern was not recognized by the large CD T cell response seen across cohorts of HLA A convalescent patients and individuals vaccinated against SARS-CoV- despite these responses comprising of over different individual T cell receptors Viral escape at prevalent T cell ... More
We studied the prevalent cytotoxic CD8 T cell response mounted against severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent
human leukocyte antigen (HLA) class I worldwide, HLA A*02. The Spike P272L mutation that has arisen in at
least 112 different SARS-CoV-2 lineages to date, including in lineages classified as ‘‘variants of concern,’’
was not recognized by the large CD8 T cell response seen across cohorts of HLA A*02+ convalescent patients
and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different
individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may
be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike,
providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting
the need for monitoring T cell escape in new SARS-CoV-2 variants. Less
coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent
human leukocyte antigen (HLA) class I worldwide, HLA A*02. The Spike P272L mutation that has arisen in at
least 112 different SARS-CoV-2 lineages to date, including in lineages classified as ‘‘variants of concern,’’
was not recognized by the large CD8 T cell response seen across cohorts of HLA A*02+ convalescent patients
and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different
individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may
be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike,
providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting
the need for monitoring T cell escape in new SARS-CoV-2 variants. Less
A series of amino acid based H-pyrrolo d pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV- nsp macrodomain Mac an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis Structure activity studies identified compound c as a low-micromolar inhibitor of Mac in two ADP-ribose binding assays This compound also demonstrated inhibition in an enzymatic assay of Mac and displayed a thermal shift comparable to ADPr in the melting temperature of Mac supporting binding to the target protein A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network ... More
A series of amino acid based 7H-pyrrolo[2,3–d]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp22 and the amide backbone NH of Ile23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe157 and Asp156, part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low µM Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization. Less
Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of D protein structures which has recently been further augmented by revolutionary advances in cryoEM However a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts limiting the overview of inherent protein physiological dynamics which play a critical role in the biological functions of proteins Here a room-temperature X-ray crystallo graphic method using temperature as a trigger to record movie-like structural snapshots has been developed The method has been used to show how TL a Da fragment undergoes ... More
Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15 Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery. Less
The SARS-CoV- infection cycle is a multistage process that relies on functional interactions between the host and the pathogen Here we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA '-O-ribose cap needed for viral immune escape We find that the host cap '-O-ribose methyltransferase MTr can compensate for loss of viral NSP methyltransferase in facilitating virus replication Concomitant inhibition of MTr and NSP efficiently suppresses SARS-CoV- replication Using in silico target-based drug screening we identify a bispecific MTr NSP inhibitor with anti-SARS-CoV- activity in vitro and in vivo but with unfavorable ... More
The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19. Less
Microbial rhodopsins are light-sensitive transmembrane proteins evolutionary adapted by various organisms like archaea bacteria simple eukaryote and viruses to utilize solar energy for their survival A complete understanding of functional mechanisms of these proteins is not possible without the knowledge of their high-resolution structures which can be primarily obtained by X-ray crystallography This technique however requires high-quality crystals growing of which is a great challenge especially in case of membrane proteins In this chapter we summarize methods applied for crystallization of microbial rhodopsins with the emphasis on crystallization in lipidic mesophases also known as in meso approach In particular we ... More
Microbial rhodopsins are light-sensitive transmembrane proteins, evolutionary adapted by various organisms like archaea, bacteria, simple eukaryote, and viruses to utilize solar energy for their survival. A complete understanding of functional mechanisms of these proteins is not possible without the knowledge of their high-resolution structures, which can be primarily obtained by X-ray crystallography. This technique, however, requires high-quality crystals, growing of which is a great challenge especially in case of membrane proteins. In this chapter, we summarize methods applied for crystallization of microbial rhodopsins with the emphasis on crystallization in lipidic mesophases, also known as in meso approach. In particular, we describe in detail the methods of crystallization using lipidic cubic phase to grow both large crystals optimized for traditional crystallographic data collection and microcrystals for serial crystallography. Less
T-cell receptor TCR immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials The safety of patients is paramount so innovative cell engineering methods are being employed to exploit adaptive immunity while controlling the factors governing antigen receptor ie TCR specificity and cross-reactivity We recently reported a TCR engineering campaign and selectivity profiling assay X-scan targeting a melanoma antigen gene MAGE -A peptide This helped to distinguish between two well-performing TCRs based on cross-reactivity potential during preclinical drug evaluation allowing one to be advanced to T-cell immunotherapeutic clinical trials Here we present three-dimensional structural information on ... More
T-cell receptor (TCR) immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials. The safety of patients is paramount, so innovative cell engineering methods are being employed to exploit adaptive immunity while controlling the factors governing antigen receptor (ie, TCR) specificity and cross-reactivity. We recently reported a TCR engineering campaign and selectivity profiling assay (X-scan) targeting a melanoma antigen gene (MAGE)-A10 peptide. This helped to distinguish between two well-performing TCRs based on cross-reactivity potential during preclinical drug evaluation, allowing one to be advanced to T-cell immunotherapeutic clinical trials. Here, we present three-dimensional structural information on those TCRs, highlighting engineering improvements and molecular mechanisms likely underpinning differential selectivity. Less
Heavy-atom soaking has been a major method for experimental phasing but it has been difficult for membrane proteins partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding The lipid cubic phase LCP has proven to be a successful method for membrane protein crystallization but experimental phasing with LCP-grown crystals remains difficult and so far only such structures were phased experimentally Here the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion SAD phasing of crystals grown in LCP Using a single crystal the structure of the glycerol -phosphate acyltransferase ... More
Heavy-atom soaking has been a major method for experimental phasing, but it has been difficult for membrane proteins, partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding. The lipid cubic phase (LCP) has proven to be a successful method for membrane protein crystallization, but experimental phasing with LCP-grown crystals remains difficult, and so far, only 68 such structures were phased experimentally. Here, the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion (SAD) phasing of crystals grown in LCP. Using a single crystal, the structure of the glycerol 3-phosphate acyltransferase (PlsY, ~21 kDa), a very hydrophobic enzyme with 80% membrane-embedded residues, was solved. Remarkably, a total of 15 Se sites were found in the two monomers of PlsY, translating to one selenourea-binding site per every six residues in the accessible extramembrane protein. Structure analysis reveals that surface-exposed selenourea sites are mostly contributed by mainchain amides and carbonyls. This low-specificity binding pattern may explain its high loading ratio. Importantly, both the crystal diffraction quality and the LCP integrity were unaffected by selenourea soaking. Taken together, selenourea presents a promising and generally useful reagent for heavy-atom soaking of membrane protein crystals grown in LCP. Less
Clonogenic assays evaluate the ability of single cells to proliferate and form colonies This process approximates the regrowth and recurrence of tumors after treatment with radiation or chemotherapy and thereby provides a drug discovery platform for compounds that block this process However because of their labor-intensive and cumbersome nature adapting canonical clonogenic assays for high throughput screening HTS has been challenging We overcame these barriers by developing an integrated system that automates cell- and liquid-handling irradiation dosimetry drug administration and incubation Further we developed a fluorescent live-cell based automated colony scoring methodology that identifies and counts colonies precisely based upon ... More
Clonogenic assays evaluate the ability of single cells to proliferate and form colonies. This process approximates the regrowth and recurrence of tumors after treatment with radiation or chemotherapy, and thereby provides a drug discovery platform for compounds that block this process. However, because of their labor-intensive and cumbersome nature, adapting canonical clonogenic assays for high throughput screening (HTS) has been challenging. We overcame these barriers by developing an integrated system that automates cell- and liquid-handling, irradiation, dosimetry, drug administration, and incubation. Further, we developed a fluorescent live-cell based automated colony scoring methodology that identifies and counts colonies precisely based upon actual nuclei number rather than colony area, thereby eliminating errors in colony counts caused by radiation induced changes in colony morphology. We identified 13 cell lines from 7 cancer types, where radiation is a standard treatment module, that exhibit identical radiation and chemoradiation response regardless of well format and are amenable to miniaturization into small-well HTS formats. We performed pilot screens through a 1584 compound NCI Diversity Set library using two cell lines representing different cancer indications. Radiation modulators identified in the pilot screens were validated in traditional clonogenic assays, providing proof-of-concept for the screen. The integrated methodology, hereafter ‘clonogenic HTS’, exhibits excellent robustness (Z’ values >0.5) and shows high reproducibility (>95%). We propose that clonogenic HTS we developed can function as a drug discovery platform to identify compounds that inhibit tumor regrowth following radiation therapy, to identify new efficacious pair-wise combinations of known oncologic therapies, or to identify novel modulators of approved therapies. Less
Buffalo flies Haematobia irritans exigua are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas most commonly on the medial canthus of the eye along the lateral and ventral neck and on the abdomen of cattle For closely related horn flies Haematobia irritans irritans Staphylococcus aureus has been suggested as a contributing factor in the development of lesions To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions swabs were taken from ... More
Buffalo flies (Haematobia irritans exigua) are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds. Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas, most commonly on the medial canthus of the eye, along the lateral and ventral neck, and on the abdomen of cattle. For closely related horn flies (Haematobia irritans irritans), Staphylococcus aureus has been suggested as a contributing factor in the development of lesions. To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions, swabs were taken from lesions and normal skin, and bacteria were also isolated from surface washings of buffalo flies and surface-sterilized homogenized flies. Bacterial identification was conducted by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) and strain typing by repetitive sequence-based PCR (rep-PCR) and DNA sequencing to determine species similarity and virulence factors. Of 50 bacterial isolates collected from lesions, 38 were identified as Staphylococcus agnetis and 12 as Staphylococcus hyicus, whereas four isolates from normal skin were S. hyicus and one was Mammaliicoccus sciuri. Of the Staphylococcus isolates isolated from buffalo flies, five were identified as S. agnetis and three as S. hyicus. Fifty percent of the buffalo fly isolates had rep-PCR genotypic patterns identical to those of the lesion isolates. Genome sequencing of 16 S. agnetis and four S. hyicus isolates revealed closely similar virulence factor profiles, with all isolates possessing exfoliative toxin A and C genes. The findings from this study suggest the involvement of S. agnetis and S. hyicus in buffalo fly lesion pathogenesis. This should be taken into account in the development of effective treatment and control strategies for lesions. Less
Candida Als family adhesins mediate adhesion to biological and abiotic substrates as well as fungal cell aggregation fungal-bacterial co-aggregation and biofilm formation The activity of at least two family members Als and Als is dependent on amyloid-like protein aggregation that is initiated by shear force Each Als adhesin has a -residue N-terminal Ig-like invasin region The following -residue low complexity threonine-rich T domain unfolds under shear force to expose a critical amyloid-forming segment SNGIVIVATTRTV at the interface between the Ig-like invasin domain and the T domain of Candida albicans Als Amyloid prediction programs identified six potential amyloidogenic sequences in the ... More
Candida Als family adhesins mediate adhesion to biological and abiotic substrates, as well as fungal cell aggregation, fungal-bacterial co-aggregation and biofilm formation. The activity of at least two family members, Als5 and Als1, is dependent on amyloid-like protein aggregation that is initiated by shear force. Each Als adhesin has a ∼300-residue N-terminal Ig-like/invasin region. The following 108-residue, low complexity, threonine-rich (T) domain unfolds under shear force to expose a critical amyloid-forming segment 322SNGIVIVATTRTV334 at the interface between the Ig-like/invasin domain 2 and the T domain of Candida albicans Als5. Amyloid prediction programs identified six potential amyloidogenic sequences in the Ig-like/invasin region and three others in the T domain of C. albicans Als5. Peptides derived from four of these sequences formed fibrils that bound thioflavin T, the amyloid indicator dye, and three of these revealed atomic-resolution structures of cross-β spines. These are the first atomic-level structures for fungal adhesins. One of these segments, from the T domain, revealed kinked β-sheets, similarly to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. Based on the cross-β structures in Als proteins, we use evolutionary arguments to identify functional amyloidogenic sequences in other fungal adhesins, including adhesins from Candida auris. Thus, cross-β structures are often involved in fungal pathogenesis and potentially in antifungal therapy. Less
G protein-coupled receptors GPCRs play vital roles in human physiology and pathophysiology This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics However the production and crystallization of GPCRs for structure determination comes with many challenges In this chapter we provide a comprehensive protocol for expressing and purifying the thromboxane A receptor TPR an attractive therapeutic target for use in structure studies Guidelines for crystallizing the TPR are also included Together these procedures provide a template for generating crystal structures of the TPR and indeed other ... More
G protein-coupled receptors (GPCRs) play vital roles in human physiology and pathophysiology. This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics. However, the production and crystallization of GPCRs for structure determination comes with many challenges.
In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands. Less
In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands. Less
Background The consequences of past coronavirus disease COVID- infection for personal and population health are emerging but accurately identifying distant infection is a challenge Anti-spike antibodies rise after both vaccination and infection and anti-nucleocapsid antibodies rapidly decline Methods We evaluated anti-membrane antibodies in COVID- naive vaccinated and convalescent subjects to determine if they persist and accurately detect distant infection Results We found that anti-membrane antibodies persist for at least year and are a sensitive and specific marker of past COVID- infection Conclusions Thus anti-membrane and anti-spike antibodies together can differentiate between COVID- convalescent vaccinated and naive states to advance public ... More
Background The consequences of past coronavirus disease 2019 (COVID-19) infection for personal and population health are emerging, but accurately identifying distant infection is a challenge. Anti-spike antibodies rise after both vaccination and infection and anti-nucleocapsid antibodies rapidly decline. Methods We evaluated anti-membrane antibodies in COVID-19 naive, vaccinated, and convalescent subjects to determine if they persist and accurately detect distant infection. Results We found that anti-membrane antibodies persist for at least 1 year and are a sensitive and specific marker of past COVID-19 infection. Conclusions Thus, anti-membrane and anti-spike antibodies together can differentiate between COVID-19 convalescent, vaccinated, and naive states to advance public health and research. Less
As discussed in previous chapters the methylation of specific arginine and lysine side chains is carried out by two families of histone methyltransferases the Protein Arginine Methyltransferase PRMT family for arginine and the SET domain family for lysine The methylation of H K by Dot is a notable outlier In all cases X-ray crystallography has been a powerful technique that has provided the framework for understanding the enzyme mechanism kinetics regulation and specificity of these enzymes and is now a platform for the design of compounds aimed to inhibit their activity either to further understand their function or in a ... More
As discussed in previous chapters, the methylation of specific arginine and lysine side chains is carried out by two families of histone methyltransferases, the Protein Arginine Methyltransferase (PRMT) family for arginine, and the SET domain family for lysine. The methylation of H3K79 by Dot1 is a notable outlier. In all cases, X-ray crystallography has been a powerful technique that has provided the framework for understanding the enzyme mechanism, kinetics, regulation and specificity of these enzymes and is now a platform for the design of compounds aimed to inhibit their activity either to further understand their function or in a therapeutic setting. Notably, in combination with the structures of the complementary recognition domains that recognize their products, these structures have provided an important insight into how integral the number of methyl groups added to the acceptor amine is to making histone methylation a key process in epigenetic regulation of gene transcription. Here the concepts applied to determine their structure by X-ray crystallography are outlined, with particular emphasis on lysine methylation by the SET domain. Less
Chemical proteomics studies the effects of drugs upon a cellular proteome Due to the complexity and diversity of tumors the response of cancer cells to drugs is also heterogeneous and thus proteome analysis at the single-cell level is needed Here we demonstrate that single-cell proteomics techniques have become quantitative enough to tackle the drug effects on target proteins enabling single-cell chemical proteomics SCCP Using SCCP we studied here the time-resolved response of individual adenocarcinoma A cells to anticancer drugs methotrexate camptothecin and tomudex revealing the early emergence of cellular subpopulations committed and uncommitted to death As a novel and useful ... More
Chemical proteomics studies the effects of drugs upon a cellular proteome. Due to the complexity and diversity of tumors, the response of cancer cells to drugs is also heterogeneous, and thus, proteome analysis at the single-cell level is needed. Here, we demonstrate that single-cell proteomics techniques have become quantitative enough to tackle the drug effects on target proteins, enabling single-cell chemical proteomics (SCCP). Using SCCP, we studied here the time-resolved response of individual adenocarcinoma A549 cells to anticancer drugs methotrexate, camptothecin, and tomudex, revealing the early emergence of cellular subpopulations committed and uncommitted to death. As a novel and useful approach to exploring the heterogeneous response to drugs of cancer cells, SCCP may prove to be a breakthrough application for single-cell proteomics. Less
Advancements in technology and communication have revolutionised the twenty-first century with the introduction of mobile phones and smartphones These phones are known to be platforms harbouring microbes with recent research shedding light on the abundance and broad spectrum of organisms they harbour Mobile phone use in the community and in professional sectors including health care settings is a potential source of microbial dissemination To identify the diversity of microbial genetic signature present on mobile phones owned by hospital medical staff Twenty-six mobile phones of health care staff were swabbed DNA extraction for downstream next generation sequencing shotgun metagenomic microbial profiling ... More
Advancements in technology and communication have revolutionised the twenty-first century with the introduction of mobile phones and smartphones. These phones are known to be platforms harbouring microbes with recent research shedding light on the abundance and broad spectrum of organisms they harbour. Mobile phone use in the community and in professional sectors including health care settings is a potential source of microbial dissemination. To identify the diversity of microbial genetic signature present on mobile phones owned by hospital medical staff. Twenty-six mobile phones of health care staff were swabbed. DNA extraction for downstream next generation sequencing shotgun metagenomic microbial profiling was performed. Survey questionnaires were handed to the staff to collect information on mobile phone usage and users’ behaviours. Each of the 26 mobile phones of this study was contaminated with microbes with the detection of antibiotic resistance and virulent factors. Taken together the sum of microbes and genes added together across all 26 mobile phones totalised 11,163 organisms (5714 bacteria, 675 fungi, 93 protists, 228 viruses, 4453 bacteriophages) and 2096 genes coding for antibiotic resistance and virulent factors. The survey of medical staff showed that 46% (12/26) of the participants used their mobile phones in the bathroom. Mobile phones are vectors of microbes and can contribute to microbial dissemination and nosocomial diseases worldwide. As fomites, mobile phones that are not decontaminated may pose serious risks for public health and biosecurity. Less
The cysteine of HCD C in DYRK A is involved in disulfide bridge formation with a cysteine C in the DFGSSC sequence The purpose of this project was to investigate how the state of the disulfide bridge would affect enzyme catalytic and ligand binding properties of the protein kinase A mutant DYRK A C A was thus designed to eliminate the disulfide bridge The mutant was expressed and purified following the same protocol as for DYRK A wt including HisTrap purification TEV cleavage and size exclusion chromatography Crystallization trials were performed for both the wt and the mutant with the ... More
The cysteine of HCD (C286) in DYRK1A is involved in disulfide bridge formation with a cysteine (C312) in the DFGSSC sequence. The purpose of this project was to investigate how the state of the disulfide bridge would affect enzyme catalytic and ligand binding properties of the protein kinase. A mutant, DYRK1A C312A, was thus designed to eliminate the disulfide bridge. The mutant was expressed and purified following the same protocol as for DYRK1A wt, including HisTrap purification, TEV cleavage and size exclusion chromatography. Crystallization trials were performed for both the wt and the mutant with the kinase inhibitor Staurosporine. DYRK1A wt with STU crystallized and diffracted with at a resolution of 2.33 Å. The DYRK1A C312A mutant with STU crystallized and diffracted with a resolution of 2.59 Å. The structure was solved by molecular replacement in Molrep (CCP4) and refined by Refmac5 and Phenix. Molecular dynamics (MD) simulations (SCHRODINGER) were performed with the intent to compare diverse disulfide bridge states. Ligand binding and enzyme catalytic properties were analyzed using a combination of techniques, including activity assays, microscale thermophoresis, and isothermal calorimetry. The Thermofluor assay confirmed that both the wt and the mutant bind tightly to STU and AZ-191. It also showed that the mutant consistently has a slightly lower melting temperature than the wt, which would indicate that it is less stable. Solvent accessible surface area (SASA) analysis support the theory of accessibility to conserved cysteine residues. Less
Chitin is a biopolymer of N-acetyl-d-glucosamine with - -bond and is the main component of arthropod exoskeletons and the cell walls of many fungi Chitinase EC is an enzyme that hydrolyzes the - -bond in chitin and degrades chitin into oligomers It has been found in a wide range of organisms Chitinase from Gazyumaru Ficus microcarpa latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields However the enzyme s thermostability is an important factor chitinase is not thermostable enough to maintain its activity under the ... More
Chitin is a biopolymer of N-acetyl-d-glucosamine with β-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.1.14) is an enzyme that hydrolyzes the β-1,4-bond in chitin and degrades chitin into oligomers. It has been found in a wide range of organisms. Chitinase from Gazyumaru (Ficus microcarpa) latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields. However, the enzyme’s thermostability is an important factor; chitinase is not thermostable enough to maintain its activity under the actual application conditions. In addition to the fact that thermostable chitinases exhibiting antifungal activity can be used under various conditions, they have some advantages for the production process and long-term preservation, which are highly demanded in industrial use. We solved the crystal structure of chitinase to explore the target sites to improve its thermostability. We rationally introduced proline residues, a disulfide bond, and salt bridges in the chitinase using protein-engineering methods based on the crystal structure and sequence alignment among other chitinases. As a result, we successfully constructed the thermostable mutant chitinases rationally with high antifungal and specific activities. The results provide a useful strategy to enhance the thermostability of this enzyme family. Less
Antimicrobial resistance primarily caused by the overuse of antimicrobials such as antibiotics is becoming an increasing concern to public health To that end the global spread of the -lactamase OXA- is worrisome as it readily catalyzes the hydrolysis of -lactam drugs such as penicillins as well as our last resort carbapenems On the contrary OXA- exhibits only limited catalytic activity against rd generations cephalosporins like ceftazidime However naturally evolving variants and results from laboratory studies have shown that OXA- can expand its substrate profile conferring increased ceftazidime resistance Expansion of the substrate profile towards ceftazidime is seen to be accompanied ... More
Antimicrobial resistance, primarily caused by the overuse of antimicrobials such as antibiotics, is becoming an increasing concern to public health. To that end, the global spread of the -lactamase OXA-48 is worrisome, as it readily catalyzes the hydrolysis of -lactam drugs, such as penicillins as well as our “last resort” carbapenems. On the contrary, OXA-48 exhibits only limited catalytic activity against 3rd generations cephalosporins like ceftazidime. However, naturally evolving variants and results from laboratory studies have shown that OXA-48 can expand its substrate profile, conferring increased ceftazidime resistance. Expansion of the substrate profile towards ceftazidime is seen to be accompanied by a trade-off towards carbapenems and penicillins, greatly reducing OXA-48 ability to catalyze the hydrolysis of penicillins and carbapenems. Here, X-ray crystallography, steady-state enzyme kinetics and differential scanning fluorimetry were used to characterize and analyze wild type (wt) OXA-48:wt and two variants, OXA-48:F72L and OXA-48:A33V/K51E/F72L/S212A/T213A (OXA-48:Q5), where the latter two were evolved towards increased ceftazidime resistance. Steady-state enzyme kinetics revealed that the two mutants had increased catalytic ability to hydrolyze ceftazidime. Such increases in kcat/Km hypothesized to arise from increased flexibility of the -loop, which was observed in the OXA-48:Q5 X-ray crystal structure in complex with piperacillin, is in line with previous studies. Further supporting the hypothesis, urea dependent kinetics and thermostability measurements show that these mutants likely exhibit increased dynamical behavior that would aid ceftazidime binding. OXA-48:F72L showed a bigger urea dependence on the enzyme activity with no activity at 4 M urea, whereas OXA-48:wt and OXA-48:Q5 needed 6 M urea to become inactive. This suggested that OXA-48:F72L is more flexible, and that OXA-48:Q5 regains some resistance to chemical denaturing by urea. The pH dependency showed higher piperacillin activity at pH 7.2 compared to 5.2 and 9.2 for all three variants. The increase in ceftazidime activity came along with a functional trade-off against the penicillin piperacillin as well as reduced thermostability of (OXA-48:F72L: -6.5C/OXA-48:Q5: -6.4C) compared to wt OXA-48, which may be caused by sub-optimal substrate positioning within the active site of OXA-48:Q5. This work provides experimental evidence, that during evolution of OXA-48 towards increased ceftazidime activity, structural changes can arise, likely affecting the chemical environment within the active site, causing increased enzyme flexibility, and ultimately shaping functional trade-offs. Less
This work aims to determine a model of the autoinhibition mechanism of MICAL proteins using biochemical biophysical and bioinformatical approaches MICAL proteins are a group of flavin monooxygenases that play a key role in various cellular processes as they facilitate the reorganization of the actin cytoskeleton MICAL- has long been known for its vital role in axon guidance as an effector of repulsive signaling through oxidative destabilization of actin filaments However recent findings indicate that MICAL- can also serve as a signaling molecule using localized hydrogen peroxide production to regulate other downstream effectors Despite the consensus that MICAL- activity must ... More
This work aims to determine a model of the autoinhibition mechanism of MICAL proteins using biochemical, biophysical, and bioinformatical approaches. MICAL proteins are a group of flavin monooxygenases that play a key role in various cellular processes, as they facilitate the reorganization of the actin cytoskeleton. MICAL-1 has long been known for its vital role in axon guidance as an effector of repulsive signaling through oxidative destabilization of actin filaments. However, recent findings indicate that MICAL-1 can also serve as a signaling molecule, using localized hydrogen peroxide production to regulate other downstream effectors. Despite the consensus that MICAL-1 activity must be strictly regulated, the exact molecular mechanism of this regulation has not yet been described. In this work, we provide a novel model of MICAL-1 autoinibiton mechanism based on a comparison of steady-state kinetic experiments and molecular dynamics simulations between full-length MICAL-1 from Coturnix japonica and its truncated form lacking the C-terminal domain. In our model, we conclude that changes in MICAL-1 activity are the result of intramolecular protein interactions between the C-terminal and the monooxygenase domain. Furthermore, we rule out the role of MICAL-1 oligomerization in its activity regulation. Our work provides the basis for further research that will need to focus on a more detailed investigation of intramolecular interactions between the MICAL-1 domains. Less
Streptococcus suis is an emerging catalase-negative zoonotic pathogen that uses the peroxide resistance protein Dpr as a defensive mechanism against oxidative stress Dpr belongs to a family of proteins that form spherical dodecamers with a hollow cavity in the middle Dpr as other members of the family uses four pores found on the surface of the dodecamer and formed by the N-terminals of adjacent monomers N-terminal pores to take up Fe and deposit it inside the cavity after its oxidation to Fe in ferroxidase sites in the interior of the dodecamer In this way the generation of toxic hydroxyl radicals ... More
Streptococcus suis is an emerging catalase-negative zoonotic pathogen that uses the peroxide resistance protein (Dpr) as a defensive mechanism against oxidative stress. Dpr belongs to a family of proteins that form spherical dodecamers with a hollow cavity in the middle. Dpr, as other members of the family, uses four pores found on the surface of the dodecamer and formed by the N-terminals of adjacent monomers (N-terminal pores) to take up Fe2+ and deposit it inside the cavity after its oxidation to Fe3+ in ferroxidase sites in the interior of the dodecamer. In this way, the generation of toxic hydroxyl radicals via Fenton’s reaction is avoided. In this study, a new purification process and crystallization conditions for Dpr were found. Besides, the ligandability of Dpr for use as a drug target was investigated. 6xHis-tagged Dpr was successfully produced and purified. Crystallization screens yielded crystals in 10 conditions and further optimization led to crystals suitable for structural analysis. Synchrotron X-ray data were collected to 2.2 Å resolution. A novel ligand library design led to an initial library of 82 compounds that could act as possible N-terminal pore blockers. After a score threshold of -7, twenty (20) ligands remained. Similar, to the latter ones, marketed ligands were retrieved, and ten (10) of them were kept, all sharing the feature of having aromatic rings. Phe133 was found as the only residue responsible for Pi-pi interactions with the ligands. This is the first successful approach for 6xHis-tag Dpr crystal production and structure determination. It is also the first approach for ligand creation against the N-terminal pores of the Dpr, setting the basis for new possible future therapeutic approaches for S. suis-related infections treatment, avoiding the obstacle of antibiotic resistance. Less
Current single-cell RNA sequencing scRNA-seq methods with high cellular throughputs sacrifice full-transcript coverage and often sensitivity Here we describe Smart-seq xpress which miniaturizes and streamlines the Smart-seq protocol to substantially reduce reagent use and increase cellular throughput Smart-seq xpress analysis of peripheral blood mononuclear cells resulted in a granular atlas complete with common and rare cell types Compared with droplet-based single-cell RNA sequencing that sequences RNA ends the additional full-transcript coverage revealed cell-type-associated isoform variation
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In situ diffraction data collection using crystallization plates has been utilized for macromolecules to evaluate crystal quality without requiring additional sample treatment such as cryocooling Although it is difficult to collect complete data sets using this technique due to the mechanical limitation of crystal rotation recent advances in methods for data collection from multiple crystals have overcome this issue At SPring- an in situ diffraction measurement system was constructed consisting of a goniometer for a plate an articulated robot and plate storage Using this system complete data sets were obtained utilizing the small-wedge measurement method Combining this system with an ... More
In situ diffraction data collection using crystallization plates has been utilized for macromolecules to evaluate crystal quality without requiring additional sample treatment such as cryocooling. Although it is difficult to collect complete data sets using this technique due to the mechanical limitation of crystal rotation, recent advances in methods for data collection from multiple crystals have overcome this issue. At SPring-8, an in situ diffraction measurement system was constructed consisting of a goniometer for a plate, an articulated robot and plate storage. Using this system, complete data sets were obtained utilizing the small-wedge measurement method. Combining this system with an acoustic liquid handler to prepare protein-ligand complex crystals by applying fragment compounds to trypsin crystals for in situ soaking, binding was confirmed for seven out of eight compounds. These results show that the system functioned properly to collect complete data for structural analysis and to expand the capability for ligand screening in combination with a liquid dispenser. Less
The CRISPR-Cas type V-I is a family of Cas i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA Here we present an engineered Type V-I CRISPR system Cas i ABR- which utilizes a tracr-less guide RNA The compact Cas i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets which facilitates versatile delivery options and multiplexing respectively We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas i The engineered variant ABR- exhibits broad genome editing capability in human cell lines primary T cells and CD hematopoietic stem and ... More
The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy. Less
High-throughput experimentation HTE seeks to accelerate the exploration of materials space by uniting robotics combinatorial methods and parallel processing HTE is particularly relevant to metal halide perovskites MHPs a diverse class of optoelectronic materials with a large chemical space Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP methoxy-phenethylammonium lead iodide -MeO-PEAI -PbI We then synthesize MHP single crystals and measure their photoluminescence ... More
High-throughput experimentation (HTE) seeks to accelerate the exploration of materials space by uniting robotics, combinatorial methods, and parallel processing. HTE is particularly relevant to metal halide perovskites (MHPs), a diverse class of optoelectronic materials with a large chemical space. Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals, allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs. We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP, methoxy-phenethylammonium lead iodide ((4-MeO-PEAI)2-PbI2). We then synthesize 16 000 MHP single crystals and measure their photoluminescence to study the effects of synthesis parameters and compositional engineering on the emission intensity of 54 distinct MHPs: we achieve an acceleration factor of more than 100 times over previously reported HTE MHP synthesis and characterization methods. Using insights derived from this analysis, we screen an existing database for new, potentially emissive MHPs. On the basis of the Tanimoto similarity of the bright available emitters, we present our top candidates for future exploration. As a proof of concept, we use one of these (3,4-difluorophenylmethanamine) to synthesize an MHP which we find has a photoluminescence quantum yield of 10%. Less
High-throughput experimentation HTE seeks to accelerate the exploration of materials space by uniting robotics combinatorial methods and parallel processing HTE is particularly relevant to metal halide perovskites MHPs a diverse class of optoelectronic materials with a large chemical space Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP methoxy-phenethylammonium lead iodide -MeO-PEAI -PbI We then synthesize MHP single crystals and measure their photoluminescence ... More
High-throughput experimentation (HTE) seeks to accelerate the exploration of materials space by uniting robotics, combinatorial methods, and parallel processing. HTE is particularly relevant to metal halide perovskites (MHPs), a diverse class of optoelectronic materials with a large chemical space. Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals, allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs. We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP, methoxy-phenethylammonium lead iodide ((4-MeO-PEAI)2-PbI2). We then synthesize 16 000 MHP single crystals and measure their photoluminescence to study the effects of synthesis parameters and compositional engineering on the emission intensity of 54 distinct MHPs: we achieve an acceleration factor of more than 100 times over previously reported HTE MHP synthesis and characterization methods. Using insights derived from this analysis, we screen an existing database for new, potentially emissive MHPs. On the basis of the Tanimoto similarity of the bright available emitters, we present our top candidates for future exploration. As a proof of concept, we use one of these (3,4-difluorophenylmethanamine) to synthesize an MHP which we find has a photoluminescence quantum yield of 10%. Less
The receptor binding domain of spike protein of SARS-CoV- binds angitensin coverting enzyme on the surface of epithelial cells leading to fusion and entry of virus into the cell Based on our experience we described a work flow for expressing and purifying proteins and screening conditions for generating diffraction quality crystals of the complex Production and crystallization of protein complex take abut twelve days from construction of vectors to harvesting and freezing crystals for data collection
Broadly HIV- neutralizing VRC -class antibodies bind the CD -binding site of Env and contain VH - derived heavy chains paired with light chains expressing five amino acid long CDRL s Their unmutated germline forms do not recognize HIV- Env and their lack of elicitation in human clinical trials could be due to the absence of activation of the corresponding na ve B cells by the vaccine immunogens To address this point we examined Env-specific B cell receptor sequences from participants in the HVTN clinical trial Of all the sequences analyzed only one displayed homology to VRC -class antibodies but ... More
Broadly HIV-1–neutralizing VRC01-class antibodies bind the CD4-binding site of Env and contain VH1-2*02–derived heavy chains paired with light chains expressing five–amino acid–long CDRL3s. Their unmutated germline forms do not recognize HIV-1 Env, and their lack of elicitation in human clinical trials could be due to the absence of activation of the corresponding naïve B cells by the vaccine immunogens. To address this point, we examined Env-specific B cell receptor sequences from participants in the HVTN 100 clinical trial. Of all the sequences analyzed, only one displayed homology to VRC01-class antibodies, but the corresponding antibody (FH1) recognized the C1C2 gp120 domain. For FH1 to switch epitope recognition to the CD4-binding site, alterations in the CDRH3 and CDRL3 were necessary. Only germ line–targeting Env immunogens efficiently activated VRC01 B cells, even in the presence of FH1 B cells. Our findings support the use of these immunogens to activate VRC01 B cells in humans. Less
The dysregulation of the PRC complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype CBX a key component of the canonical PRC complex is an epigenetic reader recognizing trimethylated lysine on histone H K me and is overexpressed in metastatic neuroendocrine prostate cancer We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX binding to chromatin Construct design and phosphorylation state of CBX were critical for successful implementation and execution of an HTS library screen A rigorous screening funnel including counter and selectivity assays allowed us to ... More
The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively. Less
Chemically induced dimerization CID is used to induce proximity and result in artificial complex formation between a pair of proteins involved in biological processes in cells to investigate and regulate these processes The induced heterodimerization of FKBP fusion proteins by rapamycin and FK has been extensively exploited as a chemically induced dimerization system to regulate and understand highly dynamic cellular processes Here we report the crystal structure of the AtFKBP FKBD in complex with rapamycin The crystal packing reveals an unusual feature whereby two rapamycin molecules appear to mediate homodimerization of the FKBD The triene arm of rapamycin appears to ... More
Chemically induced dimerization (CID) is used to induce proximity and result in artificial complex formation between a pair of proteins involved in biological processes in cells to investigate and regulate these processes. The induced heterodimerization of FKBP fusion proteins by rapamycin and FK506 has been extensively exploited as a chemically induced dimerization system to regulate and understand highly dynamic cellular processes. Here, we report the crystal structure of the AtFKBP53 FKBD in complex with rapamycin. The crystal packing reveals an unusual feature whereby two rapamycin molecules appear to mediate homodimerization of the FKBD. The triene arm of rapamycin appears to play a significant role in forming this dimer. This forms the first structural report of rapamycin-mediated homodimerization of an FKBP. The structural information on the rapamycin-mediated FKBD dimerization may be employed to design and synthesize covalently linked dimeric rapamycin, which may subsequently serve as a chemically induced dimerization system for the regulation and characterization of cellular processes. Less
In the last decade zebrafish have accompanied the mouse as a robust animal model for cancer research The possibility of screening small-molecule inhibitors in a large number of zebrafish embryos makes this model particularly valuable However the dynamic visualization of fluorescently labeled tumor cells needs to be complemented by a more sensitive easy and rapid mode for evaluating tumor growth in vivo to enable high-throughput screening of clinically relevant drugs In this study we proposed and validated a pre-clinical screening model for drug discovery by utilizing bioluminescence as our readout for the determination of transplanted cancer cell growth and inhibition ... More
In the last decade, zebrafish have accompanied the mouse as a robust animal model for cancer research. The possibility of screening small-molecule inhibitors in a large number of zebrafish embryos makes this model particularly valuable. However, the dynamic visualization of fluorescently labeled tumor cells needs to be complemented by a more sensitive, easy, and rapid mode for evaluating tumor growth in vivo to enable high-throughput screening of clinically relevant drugs. In this study we proposed and validated a pre-clinical screening model for drug discovery by utilizing bioluminescence as our readout for the determination of transplanted cancer cell growth and inhibition in zebrafish embryos. For this purpose, we used NanoLuc luciferase, which ensured rapid cancer cell growth quantification in vivo with high sensitivity and low background when compared to conventional fluorescence measurements. This allowed us large-scale evaluation of in vivo drug responses of 180 kinase inhibitors in zebrafish. Our bioluminescent screening platform could facilitate identification of new small-molecules for targeted cancer therapy as well as for drug repurposing. Less
Although monoclonal antibodies mAbs have been shown to be extremely effective in treating a number of diseases they often suffer from poor developability attributes such as high viscosity and low solubility at elevated concentrations Since experimental candidate screening is often materials and labor intensive there is substantial interest in developing in silico tools for expediting mAb design Here we present a strategy using machine learning-based QSAR models for the a priori estimation of mAb solubility The extrapolated protein solubilities of a set of antibodies in a histidine buffer were determined using a high throughput PEG precipitation assay D homology models ... More
Although monoclonal antibodies (mAbs) have been shown to be extremely effective in treating a number of diseases, they often suffer from poor developability attributes, such as high viscosity and low solubility at elevated concentrations. Since experimental candidate screening is often materials and labor intensive, there is substantial interest in developing in silico tools for expediting mAb design. Here, we present a strategy using machine learning-based QSAR models for the a priori estimation of mAb solubility. The extrapolated protein solubilities of a set of 111 antibodies in a histidine buffer were determined using a high throughput PEG precipitation assay. 3D homology models of the antibodies were determined, and a large set of in house and commercially available molecular descriptors were then calculated. The resulting experimental and descriptor data were then used for the development of QSAR models of mAb solubilities. After feature selection and training with different machine learning algorithms, the models were evaluated with external test sets. The resulting regression models were able to estimate the solubility values of external test set data with R2 of 0.81 and 0.85 for the two regression models developed. In addition, three class and binary classification models were developed and shown to be good estimators of mAb solubility behavior, with overall test set accuracies of 0.70 and 0.95, respectively. The analysis of the selected molecular descriptors in these models was also found to be informative and suggested that several charge-based descriptors and isotype may play important roles in mAb solubility. The combination of high throughput relative solubility experimental techniques in concert with efficient machine learning QSAR models offers an opportunity to rapidly screen potential mAb candidates and to design therapeutics with improved solubility characteristics. Less
Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications Bilirubin oxidase BOD is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials BOD has been extensively investigated over the last few decades While the enzyme s internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated a crystal structure of BOD from bacterial origin has never been determined Here we present the first crystal structure of BOD from Bacillus pumilus BpBOD at resolution Overall BpBOD shows high homology with the fungal enzymes however it holds a ... More
Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications. Bilirubin oxidase (BOD) is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials. BOD has been extensively investigated over the last few decades. While the enzyme’s internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated, a crystal structure of BOD from bacterial origin has never been determined. Here we present the first crystal structure of BOD from Bacillus pumilus (BpBOD) at 3.5 Å resolution. Overall, BpBOD shows high homology with the fungal enzymes; however, it holds a unique surface-exposed disulfide bond between Cys229 and Cys322 residues. We present methodologies to orient the T1 site towards the electrode by coupling the reduced disulfide bond with maleimide moiety on the electrodes. The developed configurations were further investigated and revealed improved direct electron transfer rates with the electrodes. The work presented here may contribute to the construction of rationally designed bioanodes or biocathode configurations that are based on redox-active enzymes. Less
Understanding macrophage heterogeneity in tissue repair is a major challenge Here we describe a protocol that combines isolation of immune cells from skin wounds with subsequent flow-cytometry-based sorting of wound macrophages and single-cell RNA sequencing We use a modified version of the original Smart-seq protocol to increase speed and accuracy This protocol is useful for analyzing the pronounced heterogeneity of activation phenotypes in wound macrophages and might be adapted to other experimental models of skin inflammation
In this work we examine how small hydrophobic molecules such as inert gases interact with membrane proteins MPs at a molecular level High pressure atmospheres of argon and krypton were used to produce noble gas derivatives of crystals of three well studied MPs two different proton pumps and a sodium light-driven ion pump The structures obtained using X-ray crystallography showed that the vast majority of argon and krypton binding sites were located on the outer hydrophobic surface of the MPs a surface usually accommodating hydrophobic chains of annular lipids which are known structural and functional determinants for MPs In conformity ... More
In this work we examine how small hydrophobic molecules such as inert gases interact with membrane proteins (MPs) at a molecular level. High pressure atmospheres of argon and krypton were used to produce noble gas derivatives of crystals of three well studied MPs (two different proton pumps and a sodium light-driven ion pump). The structures obtained using X-ray crystallography showed that the vast majority of argon and krypton binding sites were located on the outer hydrophobic surface of the MPs – a surface usually accommodating hydrophobic chains of annular lipids (which are known structural and functional determinants for MPs). In conformity with these results, supplementary in silico molecular dynamics (MD) analysis predicted even greater numbers of argon and krypton binding positions on MP surface within the bilayer. These results indicate a potential importance of such interactions, particularly as related to the phenomenon of noble gas-induced anaesthesia. Less
Three betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past years The most current and widespread of these is SARS-CoV- The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks could occur Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-CoV protection We previously identified a neutralizing monoclonal antibody CV - that binds to the SARS-CoV- spike neutralizes the SARS-CoV- Beta variant comparably to the ancestral Wuhan Hu- ... More
Three betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks could occur. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-CoV protection. We previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 spike, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and binds to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is maintained against the Alpha, Delta, Gamma and Omicron variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region. Negative stain electron microscopy and a 1.74 Å crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs. Less
Synthetic biology efforts for cannabinoid research have seen a rapid expansion in recent years This is in response to the increasing awareness and legalization of the secondary metabolites from Cannabis sativa dubbed the green rush In transgenic synthetic biology applications NphB is a promiscuous prenyltransferase from Streptomyces sp often used as a replacement in the prenylation step producing the cannabinoid cannabigerolic acid CBGA the key precursor to many other cannabinoids However its application as a CBGA synthase replacement is limited by its nonspecific regioselectivity in producing a side product along with CBGA Herein we demonstrated a detailed and extensive computational ... More
Synthetic biology efforts for cannabinoid research have seen a rapid expansion in recent years. This is in response to the increasing awareness and legalization of the secondary metabolites from Cannabis sativa, dubbed the green rush. In transgenic synthetic biology applications, NphB is a promiscuous prenyltransferase from Streptomyces sp. often used as a replacement in the prenylation step producing the cannabinoid cannabigerolic acid (CBGA), the key precursor to many other cannabinoids. However, its application as a CBGA synthase replacement is limited by its nonspecific regioselectivity in producing a side product along with CBGA. Herein, we demonstrated a detailed and extensive computational structure-guided approach in identifying target residues of mutation for engineering NphB for optimal CBGA production. Our comprehensive computational workflow has led to the discovery of several highly regiospecific variants that produce CBGA exclusively, with the best-performing V49W/Y288P variant having a 13.6-fold yield improvement, outperforming all previous work on NphB enzyme engineering. We subsequently investigated the effects of these mutations by X-ray crystallographic studies of the mutant variants and performed molecular dynamics simulations to uncover an interplay of a H-bonding network and an optimal ligand orientation that favors the CBGA production over the side product. Collectively, this study not only recapitulates the utility of computational tools in informing and accelerating experimental design but also contributes to a better understanding of molecular mechanisms that govern enzyme regioselectivity and readily aids in cannabinoid synthetic biology production for future research into maximizing their therapeutic potential. Less