1181 Citations
Human T-cell Leukemia Virus type HTLV- is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology especially when compared to the closely related virus HIV A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host Importantly the first in class anti-capsid drug approved lenacapavir can provide long-acting pre-exposure prophylaxis Such a property would provide a means to prevent the transmission of HTLV- but its capsid has not previously ... More
Human T-cell Leukemia Virus type 1 (HTLV-1) is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions. Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology, especially when compared to the closely related virus, HIV. A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host. Importantly, the first in class anti-capsid drug approved, lenacapavir, can provide long-acting pre-exposure prophylaxis. Such a property would provide a means to prevent the transmission of HTLV-1, but its capsid has not previously been considered as a drug target. Here we describe the first high-resolution crystal structures of the HTLV-1 capsid protein, define essential lattice interfaces, and identify a previously unknown ligand-binding pocket. We show that this pocket is essential for virus infectivity, providing a potential target for future anti-capsid drug development. Less
Prophages dormant bacteriophage genomes integrated within the bacterial chromosome play pivotal roles in shaping microbial communities when awakened Our current understanding of prophage activation is largely shaped by a narrow set of traditional DNA-damaging inducers such as mitomycin C and ciprofloxacin which trigger the bacterial SOS response This study employed high-throughput screening of compounds to identify novel prophage inducers using model lambdoid prophage HK We identified multiple new inducers across diverse pharmacological classes including dietary supplements and therapeutics Despite the variety in compounds all acted through SOS-dependent pathways However bleomycin an antineoplastic antibiotic demonstrated broad-spectrum and potent prophage induction exceeding ... More
Prophages, dormant bacteriophage genomes integrated within the bacterial chromosome, play pivotal roles in shaping microbial communities when awakened. Our current understanding of prophage activation is largely shaped by a narrow set of traditional DNA-damaging inducers, such as mitomycin C and ciprofloxacin, which trigger the bacterial SOS response. This study employed high-throughput screening of 3,921 compounds to identify novel prophage inducers using model lambdoid prophage HK97. We identified multiple new inducers across diverse pharmacological classes, including dietary supplements and therapeutics. Despite the variety in compounds, all acted through SOS-dependent pathways. However, bleomycin, an antineoplastic antibiotic, demonstrated broad-spectrum and potent prophage induction exceeding standard inducers, with activity validated across multiple phage-host pairings. These findings expand the repertoire of prophage inducers into commonly ingested xenobiotics and introduce bleomycin as a powerful, cost-effective tool for prophage research.
Around 75% of bacteria carry within them dormant viruses (prophages), which can awaken when the bacterium is stressed, killing the bacterium. Historically, this has been done using DNA-damaging antibiotics, but increasingly, more such signals have been discovered. Here, through a high-throughput screen, we identify phage-waking activity in several commonly consumed compounds, such as the SSRI Prozac, as well as a new DNA-damaging agent that is much more effective in waking phages than the previous gold standard. Less
Around 75% of bacteria carry within them dormant viruses (prophages), which can awaken when the bacterium is stressed, killing the bacterium. Historically, this has been done using DNA-damaging antibiotics, but increasingly, more such signals have been discovered. Here, through a high-throughput screen, we identify phage-waking activity in several commonly consumed compounds, such as the SSRI Prozac, as well as a new DNA-damaging agent that is much more effective in waking phages than the previous gold standard. Less
Photobleaching of fluorescent proteins often limits the acquisition of high-quality images in microscopy StayGold a novel dimeric GFP recently monomerized through sequence engineering addresses this challenge with its high photostability There is now a focus on producing different colored StayGold derivatives to facilitate concurrent tagging of multiple targets The unnatural amino acid -aminotyrosine has previously been shown to redshift superfolder GFP upon incorporation into its chromophore via genetic code expansion Here we apply the same strategy to redshift StayGold through substitution of tyrosine- with -aminotyrosine The resultant red fluorescent protein StayRose shows an excitation wavelength maximum of nm and an ... More
Photobleaching of fluorescent proteins often limits the acquisition of high-quality images in microscopy. StayGold, a novel dimeric GFP recently monomerized through sequence engineering, addresses this challenge with its high photostability. There is now a focus on producing different colored StayGold derivatives to facilitate concurrent tagging of multiple targets. The unnatural amino acid 3-aminotyrosine has previously been shown to redshift superfolder GFP upon incorporation into its chromophore via genetic code expansion. Here, we apply the same strategy to redshift StayGold through substitution of tyrosine-58 with 3-aminotyrosine. The resultant red fluorescent protein, StayRose, shows an excitation wavelength maximum of 530 nm and an emission wavelength maximum of 588 nm. Importantly, the monomeric mStayRose retains the favorable photostability in vivo in Escherichia coli and zebrafish embryos. A high-resolution crystal structure of StayRose confirms the modified structure of the amino chromophore within an unperturbed 3D fold. Although reliant on genetic code expansion, StayRose provides an important step toward developing redshifted StayGold derivatives. Less
Heliorhodopsins HeRs the third rhodopsin family are characterized by inverted membrane topology and confinement to monoderm organisms yet their biological meaning has so far remained a mystery We report the first crystal structure of a eukaryotic HeR supported by structural modeling and comparative analyses across all domains of life A conserved carotenoid-binding site reminiscent of secondary antennae in some microbial rhodopsins is identified and found to be common among HeRs We show that inverted topology allows recruitment of exogenous xanthophylls inaccessible in diderm cells explaining HeRs distinctive orientation and distribution These findings reveal a previously unrecognized light-harvesting mechanism of HeRs ... More
Heliorhodopsins (HeRs), the third rhodopsin family, are characterized by inverted membrane topology and confinement to monoderm organisms, yet their biological meaning has so far remained a mystery. We report the first crystal structure of a eukaryotic HeR, supported by structural modeling and comparative analyses across all domains of life. A conserved carotenoid-binding site, reminiscent of secondary antennae in some microbial rhodopsins, is identified and found to be common among HeRs. We show that inverted topology allows recruitment of exogenous xanthophylls, inaccessible in diderm cells, explaining HeRs’ distinctive orientation and distribution. These findings reveal a previously unrecognized light-harvesting mechanism of HeRs, expand the known repertoire of microbial phototrophy, and suggest evolutionary constraints linking membrane topology to environmental metabolite accessibility. Less
The composition of the primordial genetic material remains uncertain Studies of duplex structure and stability and of nonenzymatic template copying chemistry provide insight into the viability of potentially primordial genetic polymers Recent work suggests that - deoxyribo-purine nucleotides may have been generated together with ribonucleotides on the early Earth Since DNA RNA duplexes are known to be less stable than RNA RNA duplexes we have examined the impact of dA dI and dG substitutions on RNA structure and nonenzymatic template copying We find that single -deoxyribo-purine substitutions reduce RNA duplex stability as expected Crystallographic studies show that such substitutions lead ... More
The composition of the primordial genetic material remains uncertain. Studies of duplex structure and stability, and of nonenzymatic template copying chemistry, provide insight into the viability of potentially primordial genetic polymers. Recent work suggests that 2′- deoxyribo-purine nucleotides may have been generated together with ribonucleotides on the early Earth. Since DNA/RNA duplexes are known to be less stable than RNA/RNA duplexes, we have examined the impact of dA, dI, and dG substitutions on RNA structure and nonenzymatic template copying. We find that single 2′-deoxyribo-purine substitutions reduce RNA duplex stability, as expected. Crystallographic studies show that such substitutions lead to minimal structural changes but point to diminished solvation as a likely reason for duplex destabilization. Kinetic studies show that dI and dG substrates exhibit slightly weaker template binding and slower rates of template-directed primer extension than the corresponding ribo-purine substrates. In contrast, dA substrates exhibit much slower reaction kinetics but higher template affinity than rA substrates. Our results suggest that a mixed RNA/DNA primordial genetic polymer would have suffered from moderately slower rates of template copying, but that this could have been offset by an advantage due to more facile strand separation or exchange. Less
C domains are ubiquitous membrane-binding modules of residues in eukaryotes that are often associated with proteins involved in membrane trafficking and lipid modification The genome of Trichomonas vaginalis the most common non-viral sexually transmitted human pathogen encodes eight genes that contain a N-terminal C module linked to a XYPPX-repeat domain of more than four XYPPX repeats C -XYPPX While the function of the XYPPX-repeat domain remains unknown its multiple association with C domains in T vaginalis suggests it is important The C domain from one of these C -XYPPX-repeat proteins Tv-C - was structurally and physically characterized using X-ray crystallography ... More
C2 domains are ubiquitous membrane-binding modules of ∼130 residues in eukaryotes that are often associated with proteins involved in membrane trafficking and lipid modification. The genome of Trichomonas vaginalis, the most common, non-viral, sexually transmitted human pathogen, encodes eight genes that contain a N-terminal C2 module linked to a XYPPX-repeat domain of more than four XYPPX repeats (C2-XYPPX). While the function of the XYPPX-repeat domain remains unknown, its multiple association with C2 domains in T. vaginalis suggests it is important. The C2 domain from one of these C2-XYPPX-repeat proteins, Tv-C2-1, was structurally and physically characterized using X-ray crystallography and NMR spectroscopy. The crystal structure for Tv-C2-1 shows that this domain shares a fold common to all C2 domains, a compact Greek-key motif composed of eight anti-parallel β-strands in the type-2 topology. An NMR chemical shift perturbation study with Ca2+ showed that Tv-C2-1 bound two Ca2+ atoms primarily via two loops (loop-1 and loop-3) on the predicted calcium binding face of the protein with Kds of 58.0 ± 0.1 μM and 232 ± 6 μM. Estimations of the overall rotational correlation time, τc, in the apo (11.1 ns) and Ca2+-bound (9.2 ns) state suggests the protein becomes more compact upon Ca2+ binding, consistent with a decrease in dynamics in loop-3 and marginally in loop-1 suggested by amide 15N heteronuclear steady-state {1H}-15N NOEs. Showing Tv-C2-1 binds calcium and adopts a compact Greek-key motif structure, two primary features of C2 domains, suggests understanding the function of the XYPPX-repeat domain may be warranted. Less
Spermine a pivotal player in biomolecular condensation and diverse cellular processes has emerged as a focus of investigation in aging neurodegeneration and other diseases Despite its significance the mechanistic details of spermine remain incompletely understood Here we describe the distinct modulation by spermine on Alzheimer s Tau and Parkinson s -synuclein elucidating their condensation behaviors in vitro and in vivo Using biophysical techniques including time-resolved SAXS and NMR we trace electrostatically driven transitions from atomic-scale conformational changes to mesoscopic structures Notably spermine extends lifespan ameliorates movement deficits and restores mitochondrial function in C elegans models expressing Tau and -synuclein Acting ... More
Spermine, a pivotal player in biomolecular condensation and diverse cellular processes, has emerged as a focus of investigation in aging, neurodegeneration, and other diseases. Despite its significance, the mechanistic details of spermine remain incompletely understood. Here, we describe the distinct modulation by spermine on Alzheimer’s Tau and Parkinson’s α-synuclein, elucidating their condensation behaviors in vitro and in vivo. Using biophysical techniques including time-resolved SAXS and NMR, we trace electrostatically driven transitions from atomic-scale conformational changes to mesoscopic structures. Notably, spermine extends lifespan, ameliorates movement deficits, and restores mitochondrial function in C. elegans models expressing Tau and α-synuclein. Acting as a molecular glue, spermine orchestrates in vivo condensation of α-synuclein, influences condensate mobility, and promotes degradation via autophagy, specifically through autophagosome expansion. This study unveils the interplay between spermine, protein condensation, and functional outcomes, advancing our understanding of neurodegenerative diseases and paving the way for therapeutic development. Less
Plasma and cerebrospinal fluid are complementary sources of biomarkers for neurodegenerative diseases The wide dynamic range of protein abundances particularly in plasma hampers detection of low-abundance proteins Depletion of high-abundance proteins and efficient enzymatic digestion can improve proteome coverage but must be carefully optimized for reproducibility throughput and cost-efficiency for use in large-scale clinical proteomic studies We developed a scalable sample preparation workflow for plasma and cerebrospinal fluid CSF that integrates depletion of high-abundance proteins optimized digestion using Lys-C and trypsin and compatibility with both label-free and tandem mass tag TMTpro -based quantification We systematically evaluated protein depletion and enzyme ... More
Plasma and cerebrospinal fluid are complementary sources of biomarkers for
neurodegenerative diseases. The wide dynamic range of protein abundances, particularly in
plasma, hampers detection of low-abundance proteins. Depletion of high-abundance proteins
and efficient enzymatic digestion can improve proteome coverage but must be carefully
optimized for reproducibility, throughput, and cost-efficiency for use in large-scale clinical
proteomic studies.
We developed a scalable sample preparation workflow for plasma and cerebrospinal fluid (CSF)
that integrates depletion of high-abundance proteins, optimized digestion using Lys-C and
trypsin, and compatibility with both label-free and tandem mass tag (TMTpro)-based
quantification. We systematically evaluated protein depletion and enzyme digestion conditions,
and the effect of deoxycholate on digestion, monitoring the number of detectable proteins and
the quantitation precision. Less
neurodegenerative diseases. The wide dynamic range of protein abundances, particularly in
plasma, hampers detection of low-abundance proteins. Depletion of high-abundance proteins
and efficient enzymatic digestion can improve proteome coverage but must be carefully
optimized for reproducibility, throughput, and cost-efficiency for use in large-scale clinical
proteomic studies.
We developed a scalable sample preparation workflow for plasma and cerebrospinal fluid (CSF)
that integrates depletion of high-abundance proteins, optimized digestion using Lys-C and
trypsin, and compatibility with both label-free and tandem mass tag (TMTpro)-based
quantification. We systematically evaluated protein depletion and enzyme digestion conditions,
and the effect of deoxycholate on digestion, monitoring the number of detectable proteins and
the quantitation precision. Less
The Maternal-to-Zygotic transition MZT is a reprograming process encompassing zygotic genome activation ZGA and the clearance of maternally-provided mRNAs While some factors regulating MZT have been identified there are thousands of maternal RNAs whose function has not been ascribed yet Here we have performed a proof-of-principle CRISPR-RfxCas d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT Bckdk mRNA knockdown caused epiboly defects ZGA deregulation H K ac reduction and a partial impairment of miR- processing Phospho-proteomic analysis revealed that Phf Baf a a chromatin remodeling factor is ... More
The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation. Less
High-grade serous ovarian cancer HGSOC is a devastating disease that is frequently detected at an advanced and incurable stage Advances in ultrasensitive mass spectrometry-based spatial proteomics have provided a unique opportunity to uncover early molecular events in tumorigenesis and common dysregulated pathways with high therapeutic potential Here we present a comprehensive proteomic analysis of serous tubal intraepithelial carcinoma STIC the HGSOC precursor lesion covering more than proteins from ultralow input archival tissue We discovered that STICs and concurrent invasive carcinomas were indistinguishable at the global proteome level revealing a similar level of phenotypic and molecular heterogeneity Using cell-type resolved tissue ... More
High-grade serous ovarian cancer (HGSOC) is a devastating disease that is frequently detected at an advanced and incurable stage. Advances in ultrasensitive mass spectrometry-based spatial proteomics have provided a unique opportunity to uncover early molecular events in tumorigenesis and common dysregulated pathways with high therapeutic potential. Here, we present a comprehensive proteomic analysis of serous tubal intraepithelial carcinoma (STIC), the HGSOC precursor lesion, covering more than 10,000 proteins from ultralow input archival tissue. We discovered that STICs and concurrent invasive carcinomas were indistinguishable at the global proteome level, revealing a similar level of phenotypic and molecular heterogeneity. Using cell-type resolved tissue proteomics, we revealed strong cell-of-origin signatures preserved in STICs and invasive tumors and identified early dysregulated pathways of therapeutic relevance. These include proliferation and DNA damage repair signatures, as well as onco-metabolic changes, such as increased cholesterol biosynthesis. Finally, we uncovered substantial remodeling of the co-evolving tumor microenvironment, affecting approximately one-third of the stromal proteome, and derived a common signature associated with progressive immunosuppression and ECM restructuring. In summary, our study highlights the power of spatially resolved quantitative proteomics to dissect the molecular underpinnings of early carcinogenesis and provides a rich proteomic resource for future biomarker and drug target discovery. Less
The increase in multidrug-resistant bacteria underscores an urgent need for additional antibiotics Here we integrate small-molecule high-throughput screening with a deep-learning-based virtual screening approach to uncover new antibacterial compounds We screen million small molecules against a sensitized Escherichia coli strain yielding thousands of hits We use these data to train a deep learning model GNEprop to predict antibacterial activity retrospectively validating robustness with respect to out-of-distribution generalization and activity cliff prediction Virtual screening of over billion synthetically accessible compounds identifies potential candidates of which exhibit antibacterial activity on the same strain illustrating a -fold improved hit rate over the high-throughput ... More
The increase in multidrug-resistant bacteria underscores an urgent need for additional antibiotics. Here, we integrate small-molecule high-throughput screening with a deep-learning-based virtual screening approach to uncover new antibacterial compounds. We screen ~2 million small molecules against a sensitized Escherichia coli strain, yielding thousands of hits. We use these data to train a deep learning model, GNEprop, to predict antibacterial activity, retrospectively validating robustness with respect to out-of-distribution generalization and activity cliff prediction. Virtual screening of over 1.4 billion synthetically accessible compounds identifies potential candidates, of which 82 exhibit antibacterial activity on the same strain, illustrating a 90-fold improved hit rate over the high-throughput screening experiment used for training. Many newly identified compounds exhibit high dissimilarity to known antibiotics, potency beyond the training bacterial strain and selectivity. Biological characterization identifies specific, validated targets, indicating promising avenues for further exploration in antibiotic discovery. Less
The ribosome is a universally conserved and essential protein complex but its biogenesis in mammals is more complex than in single-celled eukaryotes To explore this added complexity we conducted a protein protein interaction screen in human cells This led to the identification of the eumetazoan-specific SPATA SPATA L CINP C ORF LCC complex as a key regulator of ribosome biogenesis Structural analyses using cryo-EM and X-ray crystallography defined the architecture of LCC Functional studies following acute depletion revealed that each component is essential for pre- S maturation Swapping endogenous LCC components with mutant versions pinpointed critical functional interactions and showed ... More
The ribosome is a universally conserved and essential protein complex, but its biogenesis in mammals is more complex than in single-celled eukaryotes. To explore this added complexity, we conducted a protein–protein interaction screen in human cells. This led to the identification of the eumetazoan-specific SPATA5–SPATA5L1–CINP–C1ORF109 (55LCC) complex as a key regulator of ribosome biogenesis. Structural analyses using cryo-EM and X-ray crystallography defined the architecture of 55LCC. Functional studies following acute depletion revealed that each component is essential for pre-60S maturation. Swapping endogenous 55LCC components with mutant versions pinpointed critical functional interactions and showed that SPATA5’s ATPase activity is more important than SPATA5L1’s. Our findings support that SPATA5 evolved from the solitary yeast ATPase Drg1 into the multiprotein 55LCC complex in metazoans. This work provides insights into the complexity of ribosome biogenesis and lays the foundation for deeper exploration of 55LCC’s role in pre-60S maturation. Less
Abstract Background Recombinant human NELL- rhNELL- is a potent osteogenic protein with therapeutic potential in regenerative medicine A stable formulation is essential to prevent aggregation during production filling storage and clinical use Methodology A four-stage rational formulation strategy was used identify intrinsic aggregation risks of rhNELL- screen polysorbate- and cyclodextrin-based formulations to enhance colloidal and conformational stability test lead candidates under agitation freeze thaw pH shifts and elevated temperature Analytical techniques included PEG challenge differential scanning fluorimetry DSF isothermal chemical denaturation ICD and dynamic light scattering DLS Aggregation was assessed via visible particles VP opalescence subvisible particles SVP Micro Flow ... More
Abstract
Background
Recombinant human NELL-1 (rhNELL-1) is a potent osteogenic protein with therapeutic potential in regenerative medicine. A stable formulation is essential to prevent aggregation during production, filling, storage, and clinical use.
Methodology
A four-stage rational formulation strategy was used: (1) identify intrinsic aggregation risks of rhNELL-1; (2) screen polysorbate- and cyclodextrin-based formulations to enhance colloidal and conformational stability; (3–4) test lead candidates under agitation, freeze/thaw, pH shifts, and elevated temperature. Analytical techniques included PEG challenge, differential scanning fluorimetry (DSF), isothermal chemical denaturation (ICD), and dynamic light scattering (DLS). Aggregation was assessed via visible particles (VP), opalescence, subvisible particles (SVP, Micro Flow Imaging), SDS-PAGE, and ultra-high performance size exclusion chromatography (UP-SEC).
Results
rhNELL-1 was prone to self-association via hydrophobic and electrostatic interactions. Polysorbate 20 (PS20) and hydroxypropyl beta cyclodextrin (HPB-LB-BCD) improved protein stability. PS20 markedly reduced VP and SVP formation. While HPB-LB-BCD alone did not further reduce SVP beyond PS20, it enhanced thermal stress resistance. PS20 was more effective under agitation.
Conclusions
Two lead formulations containing potassium phosphate/Tris buffer, sorbitol, PS20, and HPB-LB-BCD demonstrated strong resistance to aggregation under multiple stresses. PS20 mitigated interfacial stress, while HPB-LB-BCD suppressed solution-phase aggregation, especially at high temperatures. This systematic approach offers a framework for stabilizing other aggregation-prone proteins. Less
Background
Recombinant human NELL-1 (rhNELL-1) is a potent osteogenic protein with therapeutic potential in regenerative medicine. A stable formulation is essential to prevent aggregation during production, filling, storage, and clinical use.
Methodology
A four-stage rational formulation strategy was used: (1) identify intrinsic aggregation risks of rhNELL-1; (2) screen polysorbate- and cyclodextrin-based formulations to enhance colloidal and conformational stability; (3–4) test lead candidates under agitation, freeze/thaw, pH shifts, and elevated temperature. Analytical techniques included PEG challenge, differential scanning fluorimetry (DSF), isothermal chemical denaturation (ICD), and dynamic light scattering (DLS). Aggregation was assessed via visible particles (VP), opalescence, subvisible particles (SVP, Micro Flow Imaging), SDS-PAGE, and ultra-high performance size exclusion chromatography (UP-SEC).
Results
rhNELL-1 was prone to self-association via hydrophobic and electrostatic interactions. Polysorbate 20 (PS20) and hydroxypropyl beta cyclodextrin (HPB-LB-BCD) improved protein stability. PS20 markedly reduced VP and SVP formation. While HPB-LB-BCD alone did not further reduce SVP beyond PS20, it enhanced thermal stress resistance. PS20 was more effective under agitation.
Conclusions
Two lead formulations containing potassium phosphate/Tris buffer, sorbitol, PS20, and HPB-LB-BCD demonstrated strong resistance to aggregation under multiple stresses. PS20 mitigated interfacial stress, while HPB-LB-BCD suppressed solution-phase aggregation, especially at high temperatures. This systematic approach offers a framework for stabilizing other aggregation-prone proteins. Less
Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design Nowadays the use of robotic systems for crystal growth and diffraction analysis is widespread and high throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field However the identification of crystals is still largely carried out through manual inspection sometimes involving tens of thousands of images which represents a bottleneck in an otherwise highly automated process Here we describe AXIS an AI-based Crystal Identification System combining the DINOv computer vision model state-of-the-art transfer learning and MARCO the largest crystallization dataset available to date ... More
Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design. Nowadays, the use of robotic systems for crystal growth and diffraction analysis is widespread and high throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field. However, the identification of crystals is still largely carried out through manual inspection, sometimes involving tens of thousands of images, which represents a bottleneck in an otherwise highly automated process. Here we describe AXIS, an AI-based Crystal Identification System combining the DINOv2 computer vision model, state-of-the-art transfer learning and MARCO, the largest crystallization dataset available to date, for automated crystal detection. AXIS can operate both with visible and UV light images and integrates a Lab-In-The-Loop approach combining ML and expert inputs for continuous learning and specialization. AXIS enables automated annotation of large crystallization image datasets with performance and accuracy comparable to that of human experts and the Lab-In-The-Loop approach introduced here enables efficient adaptation to local conditions facilitating widespread application, which has been a major limitation to date. AXIS can help correct human errors in image annotation and removes critical bottlenecks, particularly in the context of extensive crystallization screens or high throughput applications like fragment and ligand screening unlocking the potential for higher levels of automation that are key both in fundamental and translational research. Less
Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase composed of factor Xa fXa and fVa The affinity of fXa for fVa is low with assembly and function dependent on phospholipid PL membranes Some snakes have evolved venom versions of fXa that bind to fVa with high affinity and efficiently activate prothrombin in the absence of PL We created a similar high-affinity PL-independent human prothrombinase with mutations to human fXa M The increase in affinity enabled cryogenic electron microscopy cryo-EM structure determination of M -prothrombinase to a resolution of All protein domains were well resolved ... More
Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase, composed of factor Xa (fXa) and fVa. The affinity of fXa for fVa is low, with assembly and function dependent on phospholipid (PL) membranes. Some snakes have evolved venom versions of fXa that bind to fVa with high affinity and efficiently activate prothrombin in the absence of PL. We created a similar high-affinity, PL-independent human prothrombinase with 17 mutations to human fXa (M17). The increase in affinity enabled cryogenic electron microscopy (cryo-EM) structure determination of M17-prothrombinase to a resolution of 3.3 Å. All protein domains were well resolved in the map, except for the Gla domain of fXa. The main contacts involve the serine protease and EGF2 domains of fXa and the A2 and A3 domains of fVa, resulting in the burying of a total surface area of 4,900 Å2. The map is of sufficient quality to resolve side chain interactions, including several key M17 mutations. To aid in the placement of the loop Cterminal to the A2 domain (a2-loop), we solved a high-resolution crystal structure of fXa in complex with a synthetic a2 peptide. The acidic a2-loop interacts with the basic heparin binding site of fXa, involving a conserved antiparallel -strand interaction. The M17-prothrombinase structure is compatible with data from biochemical and mutagenesis research and provides important new insights into the assembly and function of the prothrombinase complex. Less
Severe acute respiratory syndrome coronavirus SARS-CoV- continues to threaten global health This underpins the need for novel therapeutics against this virus Nonstructural protein Nsp of SARS-CoV- is a multifunctional protein with an essential role in viral replication As such it presents itself as an attractive target for drug discovery Here we describe two crystallographic fragment-screening campaigns against Nsp one using the established F X-Entry Screen and one using a new chemically and structurally diverse fragment library which we call the KIT library Together hits could be identified from screened fragments which constitutes the highest hit rate reported for Nsp to ... More
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten global health. This underpins the need for novel therapeutics against this virus. Nonstructural protein 1 (Nsp1) of SARS-CoV-2 is a multifunctional protein with an essential role in viral replication. As such, it presents itself as an attractive target for drug discovery. Here, we describe two crystallographic fragment-screening campaigns against Nsp1, one using the established F2X-Entry Screen and one using a new, chemically and structurally diverse fragment library, which we call the KIT library. Together, 21 hits could be identified from 192 screened fragments, which constitutes the highest hit rate reported for Nsp1 to date. Many hits bind to a key functional region and interact with residues involved in cellular mRNA cleavage, ribosome binding and viral RNA recognition. Furthermore, most of the identified fragments share a common binding mode, providing promising starting points for further optimization into drug-like compounds that can disrupt the role of Nsp1 in viral replication. Less
Discovery and artificial intelligence-guided mechanistic elucidation of a narrow-spectrum antibiotic
Current clinical antibiotics are largely broad-spectrum agents that can alter the gut microbiome and promote colonization by Enterobacteriaceae which are often drug resistant This includes adherent-invasive Escherichia coli AIEC particularly in patients with inflammatory bowel disease in which dysbiosis creates a niche for this pathogen to colonize There is an urgent and unmet need for novel narrow-spectrum and microbiome-sparing antibiotics Here we screened bioactive small molecules for antibacterial activity against AIEC and discovered enterololin an antibacterial compound with targeted activity against Enterobacteriaceae species Enterololin could overcome intrinsic and acquired resistance mechanisms in clinical isolates when combined with a subinhibitory concentration ... More
Current clinical antibiotics are largely broad-spectrum agents that can alter the gut microbiome and promote colonization by Enterobacteriaceae, which are often drug resistant. This includes adherent-invasive Escherichia coli (AIEC), particularly in patients with inflammatory bowel disease, in which dysbiosis creates a niche for this pathogen to colonize. There is an urgent and unmet need for novel narrow-spectrum and microbiome-sparing antibiotics. Here we screened 10,747 bioactive small molecules for antibacterial activity against AIEC and discovered enterololin, an antibacterial compound with targeted activity against Enterobacteriaceae species. Enterololin could overcome intrinsic and acquired resistance mechanisms in clinical isolates when combined with a subinhibitory concentration of SPR741, a polymyxin B analogue used here to increase outer membrane permeability in Gram-negative bacteria. Molecular substructure- and deep learning-guided mechanism-of-action investigations revealed that enterololin perturbs lipoprotein trafficking through a mechanism involving the LolCDE complex, laboratory-evolved resistant mutants predominantly mapped to lolC and lolE, with an in vitro frequency of resistance of ~10-8 to 10-7. Enterololin showed low mammalian cytotoxicity (HEK293 half-maximal inhibitory concentration ~100 µg ml-1) and suppressed AIEC infection in mouse models when administered in combination with SPR741, while largely preserving the overall microbiome composition. This study highlights the utility of deep learning methods for predicting molecular interactions and identifies a promising Enterobacteriaceae-specific antibacterial candidate for further development. Less
Antibody-drug conjugates ADCs are a promising therapeutic modality that enables the delivery of cytotoxic drugs to the target cells that express the corresponding antigen However the purification of ADCs while ensuring product safety homogeneity and stability is a challenging task due to their complex and fragile structure Size exclusion chromatography SEC the conventional method for ADC purification is time-consuming as it requires multiple column washes and equilibration steps Moreover subsequent formulation of ADCs typically using dead-end filtration DEF further complicates the production workflow We compared SEC DEF with the Pulse a miniaturized and automated tangential flow filtration system for purification ... More
Antibody-drug conjugates (ADCs) are a promising therapeutic modality that enables the delivery of cytotoxic drugs to the target cells that express the corresponding antigen. However, the purification of ADCs while ensuring product safety, homogeneity, and stability is a challenging task due to their complex and fragile structure. Size exclusion chromatography (SEC), the conventional method for ADC purification, is time-consuming as it requires multiple column washes and equilibration steps. Moreover, subsequent formulation of ADCs, typically using dead-end filtration (DEF), further complicates the production workflow. We compared SEC+DEF with the µPulse®, a miniaturized and automated tangential flow filtration system, for purification and formulation of ADCs. Quality analysis revealed that both approaches were equally gentle as comparable drug-to-antibody ratios (DARs) and monomer purities were observed in the purified samples. Most importantly, both methods exhibited equivalent cleanup efficiency with a 99.8% reduction in free linker-drug concentration. The endotoxin loads comprised 0.11 EU mg-1 for the µPulse and 0.07 EU mg-1 for SEC+DEF, ensuring validation of the safe application of purified ADCs in living systems. However, the µPulse performed purification and formulation of ADCs simultaneously as compared to SEC+DEF, which required multiple manual interventions. Our results indicate that the µPulse is a gentle, single-step, and walk-away approach for the purification of ADCs. Less
Ferritins are a widespread family of proteins involved in iron homeostasis While classic ferritins consist of four -helices and form -meric nanocages related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity Here by analyzing the available genomic data we identify a family of double ferritin-like proteins DFLPs composed of two four-helical domains which arose by duplication of a ferritin fold protein We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens TmDFLP and CaDFLP and show that they form homodimers and bind heme We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase ... More
Ferritins are a widespread family of proteins involved in iron homeostasis. While classic ferritins consist of four α-helices and form 24-meric nanocages, related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity. Here, by analyzing the available genomic data, we identify a family of double ferritin-like proteins (DFLPs) composed of two four-helical domains, which arose by duplication of a ferritin fold protein. We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens, TmDFLP and CaDFLP, and show that they form homodimers and bind heme. We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase activity. Furthermore, we show that some DFLPs, including TmDFLP and CaDFLP, are highly likely to be targeted into encapsulin shells. Our work expands the range of known iron metabolism systems and highlights the power of genome mining for discovery of new proteins. Less
Flap endonuclease FEN is a long-standing target of interest in the DNA damage response DDR field due to its therapeutic potential in BRCA mutant cancers To-date there have only been a handful of FEN inhibitors reported in the literature most of which display modest selectivity and or weak cellular activity As such there is a need for more advanced pharmacological tools to probe the biology of FEN Here we report the discovery of MSC the first potent selective and orally bioavailable FEN inhibitor We describe our metal-chelating fragment screening approach and structure-based optimization to identify MSC using structural insights to ... More
Flap endonuclease 1 (FEN1) is a long-standing target of interest in the DNA damage response (DDR) field due to its therapeutic potential in BRCA mutant cancers. To-date there have only been a handful of FEN1 inhibitors reported in the literature, most of which display modest selectivity and/or weak cellular activity. As such, there is a need for more advanced pharmacological tools to probe the biology of FEN1. Here, we report the discovery of MSC778, the first potent, selective, and orally bioavailable FEN1 inhibitor. We describe our metal-chelating fragment screening approach and structure-based optimization to identify MSC778, using structural insights to drive design. Consistent with FEN1 inhibition, MSC778 selectively kills BRCA2-deficient cells and potentiates the activity of PARPi niraparib in vivo to induce tumor stasis in a BRCA2 KO DLD-1 mouse xenograft. Furthermore, we illustrate how development of this approach has the potential for addressing nucleases as a target class. Less
Efficient drug discovery relies on workflows that integrate structural insights with rapid and cost-effective exploration of chemical space Here we present a data-driven fragment-based lead discovery approach to target Neuronal Calcium Sensor NCS- protein-protein interactions PPIs This study represents a complete implementation of a single high-value design-make-test-analyze cycle that directly yields compounds with micromolar affinity with the potential to modulate NCS- interactions with key targets including the G-protein chaperone Ric- A and the dopamine D and cannabinoid CB receptors X-ray crystallographic fragment screening CFS revealed diverse interaction patterns within the NCS- hydrophobic crevice Algorithmically guided fragment evolution and automated synthesis ... More
Efficient drug discovery relies on workflows that integrate structural insights with rapid and cost-effective exploration of chemical space. Here, we present a data-driven fragment-based lead discovery approach to target Neuronal Calcium Sensor 1 (NCS-1) protein-protein interactions (PPIs). This study represents a complete implementation of a single high-value design-make-test-analyze cycle that directly yields compounds with micromolar affinity with the potential to modulate NCS-1 interactions with key targets, including the G-protein chaperone Ric-8A and the dopamine D2 and cannabinoid CB1 receptors. X-ray crystallographic fragment screening (CFS) revealed diverse interaction patterns within the NCS-1 hydrophobic crevice. Algorithmically guided fragment evolution and automated synthesis enabled the rapid generation of over 250 derivatives, with biophysical validation using LC-MS and Grating-coupled interferometry. Structural analyses highlighted key pharmacophores, with selected compounds exhibiting favorable drug-like properties and potential blood-brain barrier penetration, making them promising candidates for neurodegenerative and neurodevelopmental disorders. Our results demonstrate the feasibility of accelerated hit-to-lead development at synchrotrons, demonstrating a robust, scalable platform for PPI-targeting drug discovery. The generated chemically diverse scaffolds provide a strong foundation for future therapeutic optimization. Less
The enzymatic degradation of polyethylene terephthalate PET offers a sustainable solution for PET recycling Over the past two decades more than PETases have been characterized primarily exhibiting similar sequences and structures Here we report new PET-degrading hydrolases including HaloPETase from the marine Halopseudomonas lineage thereby extending the narrow sequence space by novel features at the active site The crystal structure of HaloPETase was determined to a resolution of revealing a unique active site architecture and a lack of the canonical -stacking clamp found in PETases so far Further variations in active site composition and loop structures were observed Additionally we ... More
The enzymatic degradation of polyethylene terephthalate (PET) offers a sustainable solution for PET recycling. Over the past two decades, more than 100 PETases have been characterized, primarily exhibiting similar sequences and structures. Here, we report new PET-degrading α/β hydrolases, including HaloPETase1 from the marine Halopseudomonas lineage, thereby extending the narrow sequence space by novel features at the active site. The crystal structure of HaloPETase1 was determined to a resolution of 1.16 Å, revealing a unique active site architecture and a lack of the canonical π-stacking clamp found in PETases so far. Further, variations in active site composition and loop structures were observed. Additionally, we found five more enzymes from the same lineage, two of which have a high similarity to type IIa bacterial PETases, while the other three resemble HaloPETase1. All these enzymes exhibited high salt tolerance ranging from 2.5 to 5 M NaCl leading to higher total product releases upon PET degradation at 40 or 50 °C. Based on these findings, we propose an extension of the existing PETase classification system to include type III PETases. Less
Poly-ADP-ribosylation at sites of DNA damage catalyzed by PARP enzymes activates the DNA damage response chromatin remodeling and DNA repair The modification is reversed by two enzymes in humans PARG which efficiently hydrolyzes the poly-ADP-ribose chains and ARH which is the key enzyme for removing the last proximal mono-ADP-ribose from serine residues While inhibitor development has largely focused on PARPs and PARG no potent and selective inhibitors for ARH are currently available We optimized a FRET-based competition assay for ARH and carried out high-throughput screening of small-molecule inhibitors One hit compound with a potency of M was discovered and through ... More
Poly-ADP-ribosylation at sites of DNA damage, catalyzed by PARP enzymes, activates the DNA damage response, chromatin remodeling, and DNA repair. The modification is reversed by two enzymes in humans: PARG, which efficiently hydrolyzes the poly-ADP-ribose chains, and ARH3, which is the key enzyme for removing the last proximal mono-ADP-ribose from serine residues. While inhibitor development has largely focused on PARPs and PARG, no potent and selective inhibitors for ARH3 are currently available. We optimized a FRET-based competition assay for ARH3 and carried out high-throughput screening of small-molecule inhibitors. One hit compound, 1, with a potency of 22 μM was discovered, and through structure–activity relationship studies and synthesis, we improved its potency 10-fold to 2 μM (compound 27, MDOLL-0286). We demonstrate that the compound inhibits ARH3’s poly-ADP-ribose hydrolytic activity on cellular substrates. Intriguingly, it does not effectively inhibit the hydrolysis of mono-ADP-ribosylation from natural protein substrates. This is despite the fact that the cocrystal structure of compound 1 bound to ARH3 reveals its overlap with the enzyme’s ADP-ribose binding site, agreeing with the competition in the FRET assay. The first experimental ARH3 inhibitor complex provides a valuable starting point for developing more potent chemical probes to study DNA damage response mechanisms in the future. Less
Liquid dispensing technologies that enhance experimental efficiency and deliver high accuracy across multiple volume ranges are critically important in high-throughput experimentation HTE workflows for reaction exploration and optimization The Mantis liquid handler stands out as a compact highly modular system featuring a wide array of input options and near plug-and-play functionality with carousels all while employing positive displacement dispensing technology for precise liquid delivery that is largely independent of liquid properties However the system was originally designed to handle aqueous-based biological media at low volumes L dispense limiting its applications in nonaqueous chemistry Herein we report a hardware development effort ... More
Liquid dispensing technologies that enhance experimental efficiency and deliver high accuracy across multiple volume
ranges are critically important in high-throughput experimentation (HTE) workflows for reaction exploration and optimization. The
Mantis liquid handler stands out as a compact, highly modular system featuring a wide array of input options and near plug-and-play
functionality with carousels, all while employing positive displacement dispensing technology for precise liquid delivery that is largely
independent of liquid properties. However, the system was originally designed to handle aqueous-based biological media at low
volumes (1−5 μL/dispense), limiting its applications in nonaqueous chemistry. Herein, we report a hardware development effort
from a cross-industry collaboration aimed at enhancing Mantis’ capabilities to handle organic solvents and chemical reagents, even at
larger deliverable volumes (up to 50 μL/dispense). Various chemistry examples are provided to demonstrate the implementation of
newly developed chip designs, which allow the acquisition of accurate, reproducible, and robust data, enabling more efficient
workflows for new reaction discovery, reaction optimization, and data set production. Most importantly, by implementing the Mantis
liquid handler from the outset, an increase of 77% in HTE execution efficiency relative to a manual workflow incorporating
traditional liquid handling operations was achieved in an amide coupling demonstration. Less
ranges are critically important in high-throughput experimentation (HTE) workflows for reaction exploration and optimization. The
Mantis liquid handler stands out as a compact, highly modular system featuring a wide array of input options and near plug-and-play
functionality with carousels, all while employing positive displacement dispensing technology for precise liquid delivery that is largely
independent of liquid properties. However, the system was originally designed to handle aqueous-based biological media at low
volumes (1−5 μL/dispense), limiting its applications in nonaqueous chemistry. Herein, we report a hardware development effort
from a cross-industry collaboration aimed at enhancing Mantis’ capabilities to handle organic solvents and chemical reagents, even at
larger deliverable volumes (up to 50 μL/dispense). Various chemistry examples are provided to demonstrate the implementation of
newly developed chip designs, which allow the acquisition of accurate, reproducible, and robust data, enabling more efficient
workflows for new reaction discovery, reaction optimization, and data set production. Most importantly, by implementing the Mantis
liquid handler from the outset, an increase of 77% in HTE execution efficiency relative to a manual workflow incorporating
traditional liquid handling operations was achieved in an amide coupling demonstration. Less
An enzyme activity-based workflow for the identification and characterization of covalent inhibitors
The field of covalent drug development has advanced rapidly offering promising therapeutic potential due to the ability of these drugs to form slowly reversible or irreversible bonds with target proteins resulting in prolonged pharmacodynamic effects This distinctive mechanism of action has sparked resurging interest in covalent inhibitors across various disease areas including oncology neurological disorders and infectious diseases However characterization of covalent inhibitors poses unique challenges highlighting the need for simplified and robust assay methods This protocol describes an enzyme activity-based workflow designed to identify and characterize covalent inhibitors efficiently By streamlining the evaluation process this approach enhances the reliability ... More
The field of covalent drug development has advanced rapidly, offering promising therapeutic potential due to the ability of these drugs to form slowly reversible or irreversible bonds with target proteins, resulting in prolonged pharmacodynamic effects. This distinctive mechanism of action has sparked resurging interest in covalent inhibitors across various disease areas, including oncology, neurological disorders, and infectious diseases. However, characterization of covalent inhibitors poses unique challenges, highlighting the need for simplified and robust assay methods. This protocol describes an enzyme activity-based workflow designed to identify and characterize covalent inhibitors efficiently. By streamlining the evaluation process, this approach enhances the reliability and reproducibility of covalent inhibitor assessment, ultimately accelerating the discovery and optimization of novel covalent therapeutics. Less
In more and more drug discovery projects crystallographic fragment screening CFS is employed as an early screening method Here we demonstrate that choosing the right crystal form has a profound influence on the hit rates and hence success and speed of downstream lead generation Two CFS campaigns with the same fragment library and an almost identical experimental setup were carried out against the two crystal forms of the SARS-CoV- main protease While both crystal forms exhibit similar diffraction properties the observed hit rates in the two campaigns were vastly different For the monoclinic crystals a hit rate of was determined ... More
In more and more drug discovery projects, crystallographic fragment screening (CFS) is employed as an early screening method. Here, we demonstrate that choosing the right crystal form has a profound influence on the hit rates and hence success and speed of downstream lead generation. Two CFS campaigns with the same fragment library and an almost identical experimental setup were carried out against the two crystal forms of the SARS-CoV-2 main protease.While both crystal forms exhibit similar diffraction properties, the observed hit rates in the two campaigns were vastly different. For the monoclinic crystals a hit rate of 3% was determined, while a hit rate of 16% was observed for the orthorhombic crystals. These findings align with the more open molecular packing in the orthorhombic crystals where the solvent channels leading to the active sites are about twice larger than in the monoclinic crystal form. Our results highlight the critical importance of the crystal system in a crystallographic fragment-screening campaign and identify this parameter as one of the most important ones to be optimized during preparation of a campaign. Less
Glutarimide analogs such as thalidomide redirect the E ubiquitin ligase CRL CRBN to induce degradation of certain zinc finger ZF proteins Although the core structural motif recognized by CRBN has been characterized it does not fully explain substrate specificity To explore the role of residues adjacent to this core motif we constructed a comprehensive ZF reporter library of reporters derived from human ZF proteins and conducted a library-on-library screen with glutarimide analogs to identify compounds that collectively degrade ZF reporters Cryo-electron microscopy and crystal structures of ZFs in complex with CRBN revealed the importance of interactions beyond the core ZF ... More
Glutarimide analogs, such as thalidomide, redirect the E3 ubiquitin ligase CRL4CRBN to induce degradation of certain zinc finger (ZF) proteins. Although the core structural motif recognized by CRBN has been characterized, it does not fully explain substrate specificity. To explore the role of residues adjacent to this core motif, we constructed a comprehensive ZF reporter library of 9,097 reporters derived from 1,655 human ZF proteins and conducted a library-on-library screen with 29 glutarimide analogs to identify compounds that collectively degrade 38 ZF reporters. Cryo-electron microscopy and crystal structures of ZFs in complex with CRBN revealed the importance of interactions beyond the core ZF degron. We used systematic mutagenesis of ZFs and CRBN to identify modes of neosubstrate recruitment requiring distinct amino acids. Finally, we found subtle chemical variations in glutarimide analogs that alter target scope and selectivity, thus providing a roadmap for their rational design. Less
Deep learning has revolutionized soluble protein design yet de novo transmembrane TM protein engineering remains hindered by scarce structural data complex membrane-specific interactions and conformational dynamics We developed TMDiffusion TMDF a joint all-heavy-atom sequence structure diffusion model trained to capture the full interaction diversity of natural TM proteins including weak and polar contact networks TMDF designs diverse TM architectures associating domains inhibitors and conformational switches in a single step achieving experimental success A crystal structure of designed proteins matches predictions with atomic accuracy Leveraging TMDF we built synthetic single-pass receptors whose de novo TM domains toggle between conformations enabling precise ... More
Deep learning has revolutionized soluble protein design, yet de novo transmembrane (TM) protein engineering remains hindered by scarce structural data, complex membrane-specific interactions and conformational dynamics. We developed TMDiffusion (TMDF), a joint all-heavy-atom sequence–structure diffusion model trained to capture the full interaction diversity of natural TM proteins, including weak and polar contact networks. TMDF designs diverse TM architectures—associating domains, inhibitors, and conformational switches—in a single step, achieving >70% experimental success. A crystal structure of designed proteins matches predictions with atomic accuracy. Leveraging TMDF, we built synthetic single-pass receptors whose de novo TM domains toggle between conformations, enabling precise control of signalling outputs consistent with predicted equilibria. These results show that membrane-adapted DL models can accurately encode and program TM association energetics and conformations. TMDF establishes a general framework for bottom-up design of TM proteins with programmable functions, advancing both mechanistic studies of membrane proteins and development of next-generation therapeutics. Less
Transcription factors TFs regulate gene expression by engaging chromatin remodeling complexes yet the structural principles governing these critical interactions remain poorly defined Here we uncover the molecular mechanism by which lineage-specific pioneer transcription factor PU encoded by SPI directly engages the BAF mSWI SNF chromatin remodeling complex First using a variety of genomic approaches we establish that BAF collaborates with PU to regulate transcription in AML cells Then using a combination of biochemistry and biophysics mass spectrometry-based protein footprinting and crystallography we map the PU -BAF A interface to a disordered region of PU that adopts a helical conformation upon ... More
Transcription factors (TFs) regulate gene expression by engaging chromatin remodeling complexes, yet the structural principles governing these critical interactions remain poorly defined. Here, we uncover the molecular mechanism by which lineage-specific pioneer transcription factor PU.1 (encoded by SPI1) directly engages the BAF (mSWI/SNF) chromatin remodeling complex. First, using a variety of genomic approaches, we establish that BAF collaborates with PU.1 to regulate transcription in AML cells. Then, using a combination of biochemistry and biophysics, mass spectrometry-based protein footprinting, and crystallography, we map the PU.1-BAF60A interface to a disordered region of PU.1 that adopts a helical conformation upon binding to the YEATS-like domain of BAF60A. Disruption of this functionally critical interface via knockdown abrogates the ability of PU.1 to rescue cell viability. Finally, we conducted a high-throughput screen that yielded small molecules which selectively bind BAF60A and disrupt PU.1 binding. Co-crystal structures reveal distinct compound binding modes that converge on a critical PU.1-BAF60A interaction hotspot. These findings define, for the first time, the structural interface between a pioneer transcription factor and the BAF complex and establish a platform that enables targeting transcription factor-chromatin remodeling complex interactions in cancer. Less
Dual-specificity mitogen-activated protein kinase MAPK phosphatases MKPs directly dephosphorylate and inactivate the MAPKs Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored Here we sought to define the molecular mechanism of MKP regulation through an allosteric site within its catalytic domain We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y is required to maintain the structural integrity of the allosteric pocket Along with molecular dynamics simulations these data provide insight into how changes in ... More
Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (MKPs) directly dephosphorylate and inactivate the MAPKs. Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established, a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored. Here, we sought to define the molecular mechanism of MKP5 regulation through an allosteric site within its catalytic domain. We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y435 is required to maintain the structural integrity of the allosteric pocket. Along with molecular dynamics simulations, these data provide insight into how changes in the allosteric pocket propagate conformational flexibility in the surrounding loops to reorganize catalytically crucial residues in the active site. Furthermore, Y435 contributes to the interaction with p38 MAPK and JNK, thereby promoting dephosphorylation. Collectively, these results highlight the role of Y435 in the allosteric site as a novel mode of MKP5 regulation by p38 MAPK and JNK Less
Transport and Golgi Organization Homolog TANGO protein deficiency disorder TDD is a rare autosomal recessive disorder characterized by multi-systemic abnormalities and significant phenotypic variability including neurodevelopmental delay seizures intermittent ataxia hypothyroidism rhabdomyolysis life-threatening metabolic derangements and cardiac arrhythmias Mutations in TANGO result in mitochondrial dysfunction abnormal lipid homeostasis with cardiolipin deficiency and impaired Golgi-ER trafficking in TANGO patient-derived cells Despite the wide recognition of the clinical manifestations of TDD and numerous molecular studies the precise function of TANGO and the pathophysiology of TDD remain poorly understood A computationally derived three-dimensional structure model suggested that TANGO adopts an -fold similar to ... More
Transport and Golgi Organization 2 Homolog (TANGO2) protein deficiency disorder (TDD) is a rare autosomal recessive disorder characterized by multi-systemic abnormalities and significant phenotypic variability including neurodevelopmental delay, seizures, intermittent ataxia, hypothyroidism, rhabdomyolysis, life-threatening metabolic derangements, and cardiac arrhythmias. Mutations in TANGO2 result in mitochondrial dysfunction, abnormal lipid homeostasis with cardiolipin deficiency, and impaired Golgi-ER trafficking in TANGO2 patient-derived cells. Despite the wide recognition of the clinical manifestations of TDD and numerous molecular studies, the precise function of TANGO2 and the pathophysiology of TDD remain poorly understood. A computationally derived three-dimensional structure model suggested that TANGO2 adopts an αββα-fold, similar to the N-terminal nucleophile aminohydrolase (Ntn) superfamily of proteins, but the experimentally verified structure has not been available thus far. Here, we present the first crystal structure of the recombinant human TANGO2, determined at 1.70 Å resolution. The X-ray structure data confirmed its predicted tertiary fold with similarity to the Ntn-hydrolase family of proteins, and the comparative analysis of the active site architecture, including residues involved in catalysis and putative ligand binding site, suggests a potential hydrolase function. Additional examination of the common mutation sites found in TDD patients provides insight regarding their potential effect on protein structure integrity. Less
This protocol describes the crystallization of Enterovirus EV- A protease mutant C A containing the VP - A junction in the active site The crystals form within - hours using a crystallization screen composed of M NaCl and ethanol The crystal structure was determined using X-ray diffraction resulting in hexagonal prism crystals in space group P with unit cell dimensions of and an average resolution of The protein was expressed using the plasmid Enterovirus Coxsackievirus A A protease
This protocol describes the crystallization of Enterovirus EV- A protease mutant C A containing the VP - A junction in the active site The crystals form within - hours using a crystallization screen composed of M NaCl and ethanol The crystal structure was determined using X-ray diffraction resulting in hexagonal prism crystals in space group P with unit cell dimensions of and an average resolution of The protein was expressed using the plasmid Enterovirus Coxsackievirus A A protease
Genetic studies have identified thousands of individual disease-associated non-coding alleles but identification of the causal alleles and their functions remain critical bottlenecks Even though CRISPR-Cas editing has enabled targeted modification of DNA inefficient editing leads to heterogeneous outcomes across individual cells limiting the ability to detect functional consequences of disease alleles To overcome these challenges we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits assays the transcriptome and measures cell surface protein expression We apply this approach to investigate the effects of gene disruption deletions in regulatory regions and non-coding single nucleotide polymorphisms We identify ... More
Genetic studies have identified thousands of individual disease-associated non-coding alleles, but identification of the causal alleles and their functions remain critical bottlenecks. Even though CRISPR-Cas editing has enabled targeted modification of DNA, inefficient editing leads to heterogeneous outcomes across individual cells, limiting the ability to detect functional consequences of disease alleles. To overcome these challenges, we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome, and measures cell surface protein expression. We apply this approach to investigate the effects of gene disruption, deletions in regulatory regions, and non-coding single nucleotide polymorphisms. We identify the specific effects of individual SNPs, including the state-specific effects of an IL2RA autoimmune variant in primary human T cells. Multimodal functional genomic single cell assays including DNA sequencing bridge a crucial gap in our understanding of complex human diseases by directly identifying causal variation in primary human cells. Less
Electroporation-mediated gene delivery is a cornerstone of synthetic biology offering several advantages over other methods higher efficiencies broader applicability and simpler sample preparation Yet electroporation protocols are often challenging to integrate into highly multiplexed workflows owing to limitations in their scalability and tunability These challenges ultimately increase the time and cost per transformation As a result rapidly screening genetic libraries exploring combinatorial designs or optimizing electroporation parameters requires extensive iterations consuming large quantities of expensive custom-made DNA and cell lines or primary cells To address these limitations we have developed a High-Throughput Microfluidic Electroporation HTME platform that includes a -well ... More
Electroporation-mediated gene delivery is a cornerstone of synthetic biology, offering several advantages over other methods: higher efficiencies, broader applicability, and simpler sample preparation. Yet, electroporation protocols are often challenging to integrate into highly multiplexed workflows, owing to limitations in their scalability and tunability. These challenges ultimately increase the time and cost per transformation. As a result, rapidly screening genetic libraries, exploring combinatorial designs, or optimizing electroporation parameters requires extensive iterations, consuming large quantities of expensive custom-made DNA and cell lines or primary cells. To address these limitations, we have developed a High-Throughput Microfluidic Electroporation (HTME) platform that includes a 384-well electroporation plate (E-Plate) and control electronics capable of rapidly electroporating all wells in under a minute with individual control of each well. Fabricated using scalable and cost-effective printed-circuit-board (PCB) technology, the E-Plate significantly reduces consumable costs and reagent consumption by operating on nano to microliter volumes. Furthermore, individually addressable wells facilitate rapid exploration of large sets of experimental conditions to optimize electroporation for different cell types and plasmid concentrations/types. Use of the standard 384-well footprint makes the platform easily integrable into automated workflows, thereby enabling end-to-end automation. We demonstrate transformation of E. coli with pUC19 to validate the HTME’s core functionality, achieving at least a single colony forming unit in more than 99% of wells and confirming the platform’s ability to rapidly perform hundreds of electroporations with customizable conditions. This work highlights the HTME’s potential to significantly accelerate synthetic biology Design-Build-Test-Learn (DBTL) cycles by mitigating the transformation/transfection bottleneck. Less
This review highlights the development and evolution of three macromolecular crystallography MX beamlines at the Swiss Light Source SLS over the past two decades We discuss key advancements in X-ray optics detectors goniometers sample changers and MX methodology emphasizing their impact on high-throughput and high-resolution structural biology Our contributions are presented within the broader context of global efforts in synchrotron-based MX Looking ahead we explore the future experiments enabled by SLS and new opportunities at SwissFEL to enhance experimental capabilities and drive scientific discoveries
Fungal cell walls composed of polysaccharides and proteins play critical roles in adaptation cell division and protection against environmental stress Their polyglucan components are continuously remodeled by various types of glycosyl hydrolases GHs and transferases GTs In Saccharomyces cerevisiae and other ascomycetes enzymes of the Dfg subfamily which belong as GTs to the GH family cleave an linkage between glucosamine and mannose to facilitate covalent linkage of GPI-anchored proteins to the cell wall s polyglucans In contrast the functions of other fungal GH subfamilies are not understood We characterized CtGH from the sordariomycete Chaetomium thermophilum a member of the Fungi ... More
Fungal cell walls, composed of polysaccharides and proteins, play critical roles in adaptation, cell division, and protection against environmental stress. Their polyglucan components are continuously remodeled by various types of glycosyl hydrolases (GHs) and transferases (GTs). In Saccharomyces cerevisiae and other ascomycetes, enzymes of the Dfg5 subfamily, which belong as GTs to the GH76 family, cleave an α1,4 linkage between glucosamine and mannose to facilitate covalent linkage of GPI-anchored proteins to the cell wall’s polyglucans. In contrast, the functions of other fungal GH76 subfamilies are not understood. We characterized CtGH76 from the sordariomycete Chaetomium thermophilum, a member of the Fungi/Bacteria-mixed GH76 subfamily, revealing conserved structural features and functional divergence within the GH76 family. Notably, our structural characterization by X-ray crystallography combined with glycan fragment screening indicated that CtGH76 can recognize GPI-anchors like members of the Dfg5 subfamily but shows a broader promiscuity toward other glycans with central α1,6-mannobiose motifs due to the presence of an elongated glycan binding canyon. These findings provide new insights into GH76 enzyme diversity and fungal cell wall maturation. Less
Malaria remains a global health threat exacerbated by emerging resistance to antimalarial therapies and insecticides climate-driven outbreaks and limited chemoprotective options Here we report the characterization of RUPB- the first orally bioavailable inhibitor of Plasmodium falciparum cGMP-dependent protein kinase PfPKG RUPB- prevents infection by P falciparum and P cynomolgi sporozoites including the formation of hypnozoites by the latter A single oral dose blocks liver infection by P berghei sporozoites in vivo demonstrating efficacy consistent with further development as a once-weekly prophylaxis based on pharmacokinetic modeling The compound retains activity against field isolates resistant to chloroquine mefloquine cycloguanil sulfadoxine and pyrimethamine ... More
Malaria remains a global health threat exacerbated by emerging resistance to antimalarial therapies and insecticides, climate-driven outbreaks, and limited chemoprotective options. Here, we report the characterization of RUPB-61, the first orally bioavailable inhibitor of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG). RUPB-61 prevents infection by P. falciparum and P. cynomolgi sporozoites, including the formation of hypnozoites by the latter. A single oral dose blocks liver infection by P. berghei sporozoites in vivo, demonstrating efficacy consistent with further development as a once-weekly prophylaxis based on pharmacokinetic modeling. The compound retains activity against field isolates resistant to chloroquine, mefloquine, cycloguanil, sulfadoxine and pyrimethamine, suggesting low likelihood of cross-resistance to existing antimalarials. Structural studies and free energy-based modeling guided compound design and prospectively validated the predictive accuracy of an in silico model of PfPKG interactions with this chemotype. While selectivity profiling identified off-target activity against human kinases, structural modeling provides a clear path for optimization. These results establish PfPKG inhibitors as promising candidates for chemoprotection and support further preclinical development of the RUPB-61 chemotype. Less
Malaria poses an enormous threat to human health With ever-increasing resistance to currently deployed antimalarials new targets and starting point compounds with novel mechanisms of action need to be identified Here we explore the antimalarial activity of the Streptomyces sp natural product -O-sulfamoyl- -chloroadenosine dealanylascamycin DACM and compare it with the synthetic adenosine monophosphate AMP mimic -O-sulfamoyladenosine AMS These nucleoside sulfamates exhibit potent inhibition of P falciparum growth with an efficacy comparable to that of the current front-line antimalarial dihydroartemisinin Exposure of P falciparum to DACM leads to inhibition of protein translation driven by eIF phosphorylation We show that DACM ... More
Malaria poses an enormous threat to human health. With ever-increasing resistance to currently deployed antimalarials, new targets and starting point compounds with novel mechanisms of action need to be identified. Here, we explore the antimalarial activity of the Streptomyces sp natural product, 5ʹ-O-sulfamoyl-2-chloroadenosine (dealanylascamycin, DACM) and compare it with the synthetic adenosine monophosphate (AMP) mimic, 5-O-sulfamoyladenosine (AMS). These nucleoside sulfamates exhibit potent inhibition of P. falciparum growth with an efficacy comparable to that of the current front-line antimalarial dihydroartemisinin. Exposure of P. falciparum to DACM leads to inhibition of protein translation, driven by eIF2α phosphorylation. We show that DACM targets multiple amino acyl tRNA synthetase (aaRS) targets, including the cytoplasmic aspartyl tRNA synthetase (AspRS). The mechanism involves hijacking of the reaction product, leading to the formation of a tightly bound inhibitory amino acid-sulfamate conjugate. We show that recombinant P. falciparum and P. vivax AspRS are susceptible to hijacking by DACM and AMS, generating Asp-DACM and Asp-AMS adducts that stabilize these proteins. By contrast, human AspRS appears less susceptible to hijacking. X-ray crystallography reveals that apo P. vivax AspRS exhibits a stabilized flipping loop over the active site that is poised to bind substrates. By contrast, human AspRS exhibits disorder in an extended region around the flexible flipping loop as well as in a loop in motif II. These structural differences may underpin the decreased susceptibility of human AspRS to reaction-hijacking by DACM and AMS. Our work reveals Plasmodium AspRS as a promising antimalarial target and highlights structural features that underpin differences in the susceptibility of aaRSs to reaction hijacking inhibition. Less
Glaucoma is a neurodegenerative condition involving optic nerve damage and retinal ganglion cells death Animal studies suggested that the pathway linking these events can be mediated by mobile zinc secreted into the intraretinal space and exerting cytotoxic effects Whether this mechanism is relevant for human glaucoma and what are the targets of extracellular zinc is unknown We report that increased zinc content in the aqueous humor and retina is indeed a characteristic of glaucomatous neuropathy and excess extracellular zinc may be recognized by the key retinal neurotrophic factor PEDF Biophysical and X-ray crystallographic studies show that PEDF coordinates zinc ions ... More
Glaucoma is a neurodegenerative condition involving optic nerve damage and retinal ganglion cells death. Animal studies suggested that the pathway linking these events can be mediated by mobile zinc secreted into the intraretinal space and exerting cytotoxic effects. Whether this mechanism is relevant for human glaucoma and what are the targets of extracellular zinc is unknown. We report that increased zinc content in the aqueous humor and retina is indeed a characteristic of glaucomatous neuropathy, and excess extracellular zinc may be recognized by the key retinal neurotrophic factor PEDF. Biophysical and X-ray crystallographic studies show that PEDF coordinates zinc ions in five types of intermolecular high-affinity sites, leading to a decrease in negative surface charge and reversible oligomerization of the protein, thereby masking the target recognition sites responsible for its neurotrophic and antiangiogenic activities and collagen binding. Notably, PEDF secretion is enhanced in both glaucoma and retinal cell models in response to zinc stress; however, zinc binding negatively affects axogenic, differentiative and prosurvival functions of PEDF by suppressing its ability to activate receptor PEDF-R/PNPLA2. We suggest that glaucomatous neurodegeneration is associated with direct inhibition of PEDF signaling by extracellular zinc, making their complex a promising target for neuroprotective therapy. Less
Glycyl tRNA synthetases GlyRSs are prospective drug targets for combating Mycobacterium tuberculosis Mtb and cancer in humans These synthetases are of the -subtype with the ortholog in humans being dual targeted to the cytosol and mitochondria Whereas the human enzyme has been structurally characterized previously in several liganded states no structures of MtbGlyRS have thus far been reported Here we describe our recent work with MtbGlyRS and the closely-related Mycobacterium thermoresitibile GlyRS MtrGlyRS which progressed through all phases of the structural genomics pipeline for the purpose of facilitating structure-based drug discovery MtbGlyRS was expressed in Mycobacterium smegmatis and MtrGlyRS in ... More
Glycyl tRNA synthetases (GlyRSs) are prospective drug targets for combating Mycobacterium tuberculosis (Mtb) and cancer in humans. These synthetases are of the α2-subtype, with the ortholog in humans being dual targeted to the cytosol and mitochondria. Whereas the human enzyme has been structurally characterized previously in several liganded states, no structures of MtbGlyRS have thus far been reported. Here, we describe our recent work with MtbGlyRS and the closely-related Mycobacterium thermoresitibile GlyRS (MtrGlyRS), which progressed through all phases of the structural genomics pipeline, for the purpose of facilitating structure-based drug discovery. MtbGlyRS was expressed in Mycobacterium smegmatis and MtrGlyRS in Escherichia coli. Crystal structures are described for complexes of the two enzymes with adenosine monophosphate (AMP) and glycyl-sulfamoyl-adenylate (glycyl-AMS) at resolutions of 1.65/2.90 and 2.25/1.95 Å, respectively, and for MtrGlyRS in its apo state at 2.85 Å. Despite crystallizing in the dimeric state characteristic of many class II synthetases, the two enzymes elute predominantly as monomers during size exclusion chromatography. Strikingly, significant portions of the dimer interface and active site are unstructured in the MtrGlyRS apoenzyme crystal. AMP orders two tRNA recognition loops and a section of the insertion domain, and glycyl-AMS further stabilizes the structure, including the closure of a lid motif. Both the active and anticodon binding sites display structural differences with the human GlyRS and thus the collection of crystal structures should be useful for guiding drug development efforts targeting the various characterized structural states. Less
Millions of platelet units are needed each year to manage thrombocytopenia and other conditions linked to excessive bleeding These life-saving treatments still depend entirely on donated platelets despite the numerous shortcomings associated with them such as limited shelf life supply shortages unpredictable functionality potential for infection as well as immune-incompatibility issues These challenges could be overcome with universal donor platelets generated from human induced pluripotent stem cell hiPSC -derived megakaryocytes MKs We recently developed expandable hiPSC-derived megakaryocytic cell lines imMKCLs as a potentially unlimited source for platelet production imMKCL-derived platelets are functional and have already been tested in patients In ... More
Millions of platelet units are needed each year to manage thrombocytopenia and other conditions linked to excessive bleeding. These life-saving treatments still depend entirely on donated platelets, despite the numerous shortcomings associated with them, such as limited shelf life, supply shortages, unpredictable functionality, potential for infection, as well as immune-incompatibility issues. These challenges could be overcome with universal donor platelets generated from human induced pluripotent stem cell (hiPSC)-derived megakaryocytes (MKs). We recently developed expandable hiPSC-derived megakaryocytic cell lines (imMKCLs) as a potentially unlimited source for platelet production. imMKCL-derived platelets are functional and have already been tested in patients. In this study, we demonstrate through single-cell time-course imaging that imMKCL maturation is heterogeneous and asynchronous, with only a few imMKCLs generating platelets at any given time under static culture conditions. Using a chemical screen, we identify microtubule (MT) destabilizing agents, including vincristine (VCR), as promising hits, with a larger proportion of VCR-exposed imMKCLs developing proplatelet extensions and more platelets being produced per imMKCL. VCR use reduces the MT content of imMKCLs and results in the production of platelets with a diminished peripheral MT ring structure. Nevertheless, these platelets are functional, as evidenced by their normal response to agonists, their ability to attach to and spread on fibrinogen-coated surfaces, and their capacity to restore hemostasis in vivo. Interestingly, we also observed a negative correlation between the MT content of imMKCLs and platelet yields when we compared imMKCLs differentiated under static conditions (MThigh, low yield) to our turbulence-optimized VerMES™ bioreactor (MTlow, high yield). Taken together, our findings highlight the importance of MT dynamics in megakaryocyte biology, provide a possible explanation for the still poorly understood link between vinca alkaloid in vivo use and thrombocytosis, and bring us closer to realizing the clinical potential of affordable, off-the-shelf hiPSC-derived platelets. Less
Efficient drug discovery relies on workflows that integrate structural insights with rapid and cost-effective exploration of chemical space Here we present a data-driven fragment-based lead discovery approach to target Neuronal Calcium Sensor NCS- protein-protein interactions PPIs This study represents the first implementation of a complete design-make-test-analyze cycle leading to the identification of micromolar affinity compounds with the potential to modulate NCS- interactions with key targets including the G-protein chaperone Ric- A and the dopamine D and cannabinoid CB receptors Through X-ray crystallographic fragment screening CFS diverse interaction patterns within the NCS- hydrophobic crevice were revealed Algorithmically guided fragment evolution and ... More
Efficient drug discovery relies on workflows that integrate structural insights with rapid and cost-effective exploration of chemical space. Here, we present a data-driven fragment-based lead discovery approach to target Neuronal Calcium Sensor 1 (NCS-1) protein-protein interactions (PPIs). This study represents the first implementation of a complete design-make-test-analyze cycle leading to the identification of micromolar affinity compounds with the potential to modulate NCS-1 interactions with key targets, including the G-protein chaperone Ric-8A and the dopamine D2 and cannabinoid CB1 receptors. Through X-ray crystallographic fragment screening (CFS), diverse interaction patterns within the NCS-1 hydrophobic crevice were revealed. Algorithmically guided fragment evolution and automated synthesis enabled the rapid generation of over 400 derivatives, with biophysical validation using LC-MS and waveRAPID technology. Structural analyses highlighted key pharmacophores, with selected compounds exhibiting favorable drug-like properties and potential blood-brain barrier penetration, making them promising candidates for neurodegenerative and neurodevelopmental disorders. Our results demonstrate the feasibility of accelerated hit-to-lead development at synchrotrons, demonstrating a robust, scalable platform for PPI-targeting drug discovery. The generated chemically diverse scaffolds provide a strong foundation for future therapeutic optimization. Less
Room-temperature RT X-ray diffraction experiments enable us to investigate protein dynamics efficiently probe fragment binding and perform time-resolved crystallography experiments The Versatile Macromolecular Crystallography in-situ VMXi beamline at Diamond Light Source DLS in the United Kingdom specializes in the collection of RT X-ray diffraction data in situ directly from crystallization trays without any manipulation of protein crystals improving crystal integrity for fragile crystals While many X-ray sources are now equipped to grow crystals on site for in-situ experiments to date there has been no comprehensive analysis that we are aware of on the effect of shipping crystals on plates at ... More
Room-temperature (RT) X-ray diffraction experiments enable us to investigate protein dynamics, efficiently probe fragment binding, and perform time-resolved crystallography experiments. The Versatile Macromolecular Crystallography in-situ (VMXi) beamline at Diamond Light Source (DLS) in the United Kingdom specializes in the collection of RT X-ray diffraction data in situ directly from crystallization trays without any manipulation of protein crystals, improving crystal integrity for fragile crystals. While many X-ray sources are now equipped to grow crystals on site for in-situ experiments, to date there has been no comprehensive analysis that we are aware of on the effect of shipping crystals on plates at ambient temperature for RT data collection, while the equivalent methodology for cryo-cooled crystals is well established. Here we examine the impact of shipping on crystals grown on MiTeGen In Situ-1 plates at the University of Buffalo Hauptman Woodward Research Institute (UB-HWI) in Buffalo, NY, United States transatlantic to DLS in Didcot, United Kingdom. We utilized the Stanford Synchrotron Radiation Lightsource (SSRL) Blue Box Thermal Shipper (Blue Box), which can maintain temperature for up to 168 hours, to ship crystallization plates at room temperature from UB-HWI to DLS. We hypothesized that long-distance shipping might compromise data quality through mechanical stress or temperature fluctuations. Instead, we found that room-temperature data collected at VMXi showed no significant differences for crystals set up at UB-HWI and shipped relative to crystals set up on site in the UK. High-resolution structures were successfully determined for all proteins in the study, demonstrating that long-distance shipment of crystals at non-cryogenic temperatures is feasible without compromising diffraction quality. This study provides a proof-of-concept workflow for expanding access to room-temperature crystallography worldwide, enabling more researchers to leverage cutting-edge techniques without needing to grow crystals on site. Less
ADP-ribosylation is an enzymatic process where an ADP-ribose moiety is transferred from NAD to an acceptor molecule While ADP-ribosylation is well-established as a post-translational modification of proteins rifamycin antibiotics are its only known small-molecule targets ADP-ribosylation of rifampicin was first identified in Mycolicibacterium smegmatis whose Arr enzyme transfers the ADP-ribose moiety to the -hydroxy group of rifampicin preventing its interaction with the bacterial RNA polymerase thereby inactivating the antibiotic Arr homologues are widely spread among bacterial species and present in several pathogenic species often associated with mobile genetic elements Inhibition of Arr enzymes offers a promising strategy to overcome ADP-ribosylation ... More
ADP-ribosylation is an enzymatic process where an ADP-ribose moiety is transferred from NAD+ to an acceptor molecule. While ADP-ribosylation is well-established as a post-translational modification of proteins, rifamycin antibiotics are its only known small-molecule targets. ADP-ribosylation of rifampicin was first identified in Mycolicibacterium smegmatis, whose Arr enzyme transfers the ADP-ribose moiety to the 23-hydroxy group of rifampicin preventing its interaction with the bacterial RNA polymerase thereby inactivating the antibiotic. Arr homologues are widely spread among bacterial species and present in several pathogenic species often associated with mobile genetic elements. Inhibition of Arr enzymes offers a promising strategy to overcome ADP-ribosylation mediated rifamycin resistance. We developed a high-throughput activity assay, which was applied to screen an in-house library of human ADP-ribosyltransferase-targeted compounds. We identified 15 inhibitors with IC50 values below 5 µM against four Arr enzymes from M. smegmatis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Mycobacteroides abscessus. The observed overall selectivity of the hit compounds over the other homologues indicated structural differences between the proteins. We crystallized M. smegmatis and P. aeruginosa Arr enzymes, the former in complex with its most potent hit compound with an IC50 value of 1.3 µM. We observed structural differences in the NAD+ binding pockets of the two Arr homologues explaining the selectivity. Although the Arr inhibitors did not sensitize M. smegmatis to rifampicin in a growth inhibition assay, the structural information and the collection of inhibitors provide a foundation for rational modifications and further development of the compounds. Less
In this work we present the high-resolution structure of human aconitate decarboxylase hACOD in its true apo form active site empty as well in complex with the inhibitor citraconate These two new structures show the architecture of the active site and the structure-activity relationships of citraconate inhibition Careful analysis of the structures indicates probable dynamics required for substrate inhibitor binding and catalysis These observations were further explored using molecular dynamic simulations which show a clear open-close mechanism of hACOD between the A and A loops the lid- and helical-domain respectively As part of the biochemical characterization of the protein we ... More
In this work, we present the high-resolution structure of human aconitate decarboxylase 1 (hACOD1) in its true apo form (active site empty) as well in complex with the inhibitor citraconate. These two new structures show the architecture of the active site and the structure-activity relationships of citraconate inhibition. Careful analysis of the structures indicates probable dynamics required for substrate/inhibitor binding and catalysis. These observations were further explored using molecular dynamic simulations, which show a clear open-close mechanism of hACOD1 between the A1 and A2 loops, the lid- and helical-domain respectively. As part of the biochemical characterization of the protein, we also developed an alternative kinetic assay which measures the rate of catalysis of hACOD1 by direct observation of the conversion of cis-aconitate to itaconate by NMR spectroscopy. The work herein offers a foundation for structure- and dynamic-driven design of novel hACOD1 inhibitors as novel chemotherapeutics. Less
Microbial production of target molecules has advanced significantly in recent years driven by innovations in enzyme engineering DNA synthesis and genomic editing However to access the massive potential of microbial production a vast parametric space remains to be investigated to optimize these biobased processes for a robust bioeconomy Here we review the current state of the art some key challenges and possible solutions We see a critical role of automation high-throughput technologies self-driving and cloud labs and data management to enable Artificial Intelligence Machine Learning and mechanistic models to overcome the design space challenges and accelerate the development of novel ... More
Microbial production of target molecules has advanced significantly in recent years driven by innovations in enzyme engineering, DNA synthesis, and genomic editing. However, to access the massive potential of microbial production, a vast parametric space remains to be investigated to optimize these biobased processes for a robust bioeconomy. Here, we review the current state of the art, some key challenges and possible solutions. We see a critical role of automation, high-throughput technologies, self-driving and cloud labs, and data management to enable Artificial Intelligence/Machine Learning and mechanistic models to overcome the design space challenges and accelerate the development of novel bio-based solutions. Accurate models will expedite the development and scale-up of engineered microbes for a range of final products from many starting materials. Less
Flavin-binding proteins flavoproteins are widespread in nature revealing versatile oxidation-reduction reactions and photochemistry Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics as well as fluorescent markers and photogenerators of reactive oxygen species Despite extensiev efforts all currently used LOV-derived proteins have similar absorption spectra with maxima around - and - nm Here we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein LSSFbFP which can be obtainedin vivo and in vitro with absorption maxima at - and - nm Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ... More
Flavin-binding proteins (flavoproteins) are widespread in nature, revealing versatile oxidation-reduction reactions and photochemistry. Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics, as well as fluorescent markers and photogenerators of reactive oxygen species. Despite extensiev efforts, all currently used LOV-derived proteins have similar absorption spectra with maxima around 275, 35-0375, and 450-485 nm. Here, we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein, LSSFbFP, which can be obtainedin vivo and in vitro, with absorption maxima at 340-350 and 395-405 nm. Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ~500 nm. We sho that the protein binds lumichrome as the chromophore and use low temperature and time-resolved spectroscopy, X-ray crystallography and modeling to prove that the apparent Stokes shift of LSSFbFP occurs due to excited state proton phenomena observed in flavoproteni s and pave the way for engineering of new flavin-based molecular instruments. Less
West Nile virus NS B-NS innactive fusion protease was crystallized using vapor diffusion in Morpheus screen conditions at pH Hexagonal rod-shaped crystals grew to m in length after days at C The crystals belonged to space group P and diffracted to resolution at Diamond Light Source beamline I The structure has been deposited as PDB ID CO In this version we added the Addgene id of the plasmid used for the protein expresssion and purification
Crystal-based structural methods including X-ray crystallography are frequently utilized for the determination of high-resolution structures of biomolecules All crystal-based diffraction methods first require the preparation of biomolecular crystals and careful sample preparation for crystallization experiments can increase the frequency of success In this article strategies to optimize factors that can impact crystallization are presented from which buffers and reducing agents are most favorable to which crystallization techniques could be used