1209 Citations
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
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 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
Physical activity improves health yet the molecular mechanisms remain partially understood This study presents a high-resolution time-resolved atlas profiling proteins across plasma saliva and urine from healthy adults post-acute exercise Exercise regulated over proteins revealing distinct fluid-specific temporal dynamics By integrating fluid-specific exercise signatures with tissue and disease atlases we delineated the contribution of tissues and associations to various diseases Network analysis across body fluids elucidated coordinated remodeling in the extracellular matrix and immune activation orchestrating exercise-induced networks Many exercise-responsive plasma proteins were robust across age sex and exercise modalities indicating a conserved systemic signature Integration with genetic data established ... More
Physical activity improves health, yet the molecular mechanisms remain partially understood.
This study presents a high-resolution, time-resolved atlas profiling 10,127 proteins across
plasma, saliva, and urine from healthy adults post-acute exercise. Exercise regulated over
3,000 proteins, revealing distinct, fluid-specific temporal dynamics. By integrating fluid-specific
exercise signatures with tissue and disease atlases, we delineated the contribution of tissues
and associations to various diseases. Network analysis across body fluids elucidated
coordinated remodeling in the extracellular matrix and immune activation orchestrating
exercise-induced networks. Many exercise-responsive plasma proteins were robust across
age, sex, and exercise modalities, indicating a conserved systemic signature. Integration with
genetic data established exercise-regulated proteins as modulators of metabolic traits and
identified over 200 targeted by approved drugs, highlighting their impact on disease-relevant
pathways. This comprehensive atlas, available as an open-access resource
https://cbmr.ku.dk/research/research-groups/deshmukh-group/shiny-apps/, advances our
molecular insight into exercise adaptations and enables exerkine discovery, biomarker
development, and pharmacological exercise-mimetic strategies. Less
This study presents a high-resolution, time-resolved atlas profiling 10,127 proteins across
plasma, saliva, and urine from healthy adults post-acute exercise. Exercise regulated over
3,000 proteins, revealing distinct, fluid-specific temporal dynamics. By integrating fluid-specific
exercise signatures with tissue and disease atlases, we delineated the contribution of tissues
and associations to various diseases. Network analysis across body fluids elucidated
coordinated remodeling in the extracellular matrix and immune activation orchestrating
exercise-induced networks. Many exercise-responsive plasma proteins were robust across
age, sex, and exercise modalities, indicating a conserved systemic signature. Integration with
genetic data established exercise-regulated proteins as modulators of metabolic traits and
identified over 200 targeted by approved drugs, highlighting their impact on disease-relevant
pathways. This comprehensive atlas, available as an open-access resource
https://cbmr.ku.dk/research/research-groups/deshmukh-group/shiny-apps/, advances our
molecular insight into exercise adaptations and enables exerkine discovery, biomarker
development, and pharmacological exercise-mimetic strategies. Less
Mobile phones contaminated with pathogenic microorganisms have the potential to act as trojan horses The microbial signatures present on their surfaces most probably vary across different geographical regions As a result mobile phones belonging to international conference attendees may serve as a model for global microbial dissemination posing potential risks to public health and biosecurity This study aimed to profile the microbes present on mobile phones belonging to delegates attending an international scientific conference through use of metagenomic shotgun DNA sequencing Results A total of microbial hits were accumulated across mobile phones inclusive of bacteria viruses fungi and protozoa Of ... More
Mobile phones, contaminated with pathogenic microorganisms, have
the potential to act as “trojan horses”. The microbial signatures present on their surfaces most probably vary across different geographical regions. As a result, mobile phones belonging to
international conference attendees may serve as a model for global microbial dissemination,
posing potential risks to public health and biosecurity. This study aimed to profile the microbes present on mobile phones belonging to delegates
attending an international scientific conference through use of metagenomic shotgun DNA
sequencing. Results: A total of 2204 microbial hits were accumulated across 20 mobile phones inclusive of
882 bacteria, 1229 viruses, 88 fungi and 5 protozoa. Of particular concern was the identification of 65 distinct antibiotic resistance genes and 86 virulence genes. Plant, animal and human
pathogens, including ESKAPE and HACEK bacteria were found on mobile phones Less
the potential to act as “trojan horses”. The microbial signatures present on their surfaces most probably vary across different geographical regions. As a result, mobile phones belonging to
international conference attendees may serve as a model for global microbial dissemination,
posing potential risks to public health and biosecurity. This study aimed to profile the microbes present on mobile phones belonging to delegates
attending an international scientific conference through use of metagenomic shotgun DNA
sequencing. Results: A total of 2204 microbial hits were accumulated across 20 mobile phones inclusive of
882 bacteria, 1229 viruses, 88 fungi and 5 protozoa. Of particular concern was the identification of 65 distinct antibiotic resistance genes and 86 virulence genes. Plant, animal and human
pathogens, including ESKAPE and HACEK bacteria were found on mobile phones Less
In the human commensal Gram-positive bacterial pathogen Streptococcus pneumoniae the essential extracellular cell-division-associated peptidoglycan PG hydrolase PcsB interacts directly with the cytoplasmic-membrane-bound complex between FtsE and FtsX PcsB contains a cysteine hishdine-dependent amidohydrolase pephdase CHAP domain responsible for PG hydrolysis as well as a coiled-coil domain required for interaction with FtsEX ATP hydrolysis of FtsE in the cytoplasm drives conformational changes in FtsX in the cytoplasmic membrane which ultimately regulates the PG hydrolase on the outside of the cell In this work we show using in vitro and in vivo approaches that the CHAP domain of PcsB predominately functions as ... More
In the human commensal Gram-positive bacterial pathogen Streptococcus pneumoniae, the essential extracellular cell-division-associated peptidoglycan (PG) hydrolase PcsB interacts directly with the cytoplasmic-membrane-bound complex between FtsE and FtsX (1–3). PcsB contains a cysteine, hishdine-dependent amidohydrolase/pephdase (CHAP) domain responsible for PG hydrolysis, as well as a coiled-coil domain required for interaction with FtsEX (1,4). ATP hydrolysis of FtsE in the cytoplasm drives conformational changes in FtsX in the cytoplasmic membrane, which ultimately regulates the PG hydrolase on the outside of the cell (5). In this work we show using in vitro and in vivo approaches, that the CHAP domain of PcsB predominately functions as an iso-D-Glutaminyl-Lysyl D,L-endopeptidase, with particular substrate specificity for Lys-containing, amidated PG, cleaving between the second and third amino acids of the peptidoglycan stem peptide. The catalytic activity of PcsB is regulated and activated by conformation changes of the coiled-coil region of PcsB and in part by a short helical region immediately adjacent to the CHAP domain to guard against PcsB hydrolytic activation outside of its cell division specific functional requirement. This work supports a model for the overall biological activity of the FtsEX-PcsB complex, in which ATP hydrolysis by FtsE in the cytoplasm, drives conformational changes in FtsX and PcsB resulting in the liberation of the hydrolytic CHAP domain of PcsB from its regulatory helix to allow PG stem peptide cleavage that splits the septal disk and marks a region of the peptidoglycan sacculus for subsequent cell division remodelling. Less
Nonenzymatic RNA copying is thought to have been responsible for the replication of genetic information during the origin of life However chemical copying with the canonical nucleotides A U G and C strongly favors the incorporation of G and C and disfavors the incorporation of A and especially U because of the stronger G C vs A U base pair and the weaker stacking interactions of U Recent advances in prebiotic chemistry suggest that the -thiopyrimidines were precursors to the canonical pyrimidines raising the possibility that they may have played an important early role in RNA copying chemistry Furthermore -thiouridine ... More
Nonenzymatic RNA copying is thought to have been responsible for the replication of genetic information during the origin of life. However, chemical copying with the canonical nucleotides (A, U, G, and C) strongly favors the incorporation of G and C and disfavors the incorporation of A and especially U, because of the stronger G:C vs. A:U base pair, and the weaker stacking interactions of U. Recent advances in prebiotic chemistry suggest that the 2-thiopyrimidines were precursors to the canonical pyrimidines, raising the possibility that they may have played an important early role in RNA copying chemistry. Furthermore, 2-thiouridine (s2U) and inosine (I) form by deamination of 2-thiocytidine (s2C) and A respectively. We used thermodynamic and crystallographic analyses to compare the I:s2C and A:s2U base pairs. We find that the I:s2C base pair is isomorphic and isoenergetic with the A:s2U base pair. The I:s2C base pair is weaker than a canonical G:C base pair, while the A:s2U base pair is stronger than the canonical A:U base pair, so that a genetic alphabet consisting of s2U, s2C, I and A generates RNA duplexes with uniform base pairing energies. Consistent with these results, kinetic analysis of nonenzymatic template-directed primer extension reactions reveals that s2C and s2U substrates bind similarly to I and A in the template, and vice versa. Our work supports the plausibility of a potentially primordial genetic alphabet consisting of s2U, s2C, I and A, and offers a potential solution to the long-standing problem of biased nucleotide incorporation during nonenzymatic template copying. Less
Collagen prolyl -hydroxylase C-P H catalyzes the -hydroxylation of Y-prolines of the XYG-repeat of procollagen C-P Hs are tetrameric enzymes The -subunit provides the N-terminal dimerization domain the middle peptide-substrate binding PSB domain and the C-terminal catalytic CAT domain There are three isoforms of the -subunit complexed with a -subunit that is protein disulfide isomerase forming C-P H I-III The PSB domain of the -subunit binds proline-rich peptides but its function with respect to the prolyl hydroxylation mechanism is unknown An extended mode of binding of proline-rich peptides PPII polyproline type-II conformation to the PSB-I domain has previously been reported ... More
Collagen prolyl 4-hydroxylase (C-P4H) catalyzes the 4-hydroxylation of Y-prolines of the XYG-repeat of procollagen. C-P4Hs are tetrameric α2β2 enzymes. The α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate–binding (PSB) domain, and the C-terminal catalytic (CAT) domain. There are three isoforms of the α-subunit, complexed with a β-subunit that is protein disulfide isomerase, forming C-P4H I-III. The PSB domain of the α-subunit binds proline-rich peptides, but its function with respect to the prolyl hydroxylation mechanism is unknown. An extended mode of binding of proline-rich peptides (PPII, polyproline type-II, conformation) to the PSB-I domain has previously been reported for the PPG-PPG-PPG and P9 peptides. Crystal structures now show that peptides with the motif PxGP (PPG-PRG-PPG, PPG-PAG-PPG) (where x, at Y-position 5, is not a proline) bind to the PSB-I domain differently, more deeply, in the peptide-binding groove. The latter mode of binding has previously been reported for structures of the PSB-II domain complexed with these PxGP-peptides. In addition, it is shown here by crystallographic binding studies that the POG-PAG-POG peptide (with 4-hydroxyprolines at Y-positions 2 and 8) also adopts the PxGP mode of binding to PSB-I as well as to PSB-II. Calorimetric binding studies show that the affinities of these peptides are lower for PSB-I than for PSB-II, with, respectively, KD values of about 70 μM for PSB-I and 20 μM for PSB-II. The importance of these results for understanding the reaction mechanism of C-P4H, in particular concerning the function of the PSB domain, is discussed. Less
Immunomodulatory imide drugs IMiDs degrade specific C H zinc finger degrons in transcription factors making them effective against certain cancers SALL a cancer driver contains seven C H zinc fingers in four clusters including an IMiD degron in zinc finger cluster two ZFC Surprisingly IMiDs do not inhibit growth of SALL expressing cancer cells To overcome this limit we focused on a non-IMiD degron SALL zinc finger cluster four ZFC By combining AlphaFold and the ZFC -DNA crystal structure we identified a potential ZFC drug pocket Utilizing an in silico docking algorithm and cell viability assays we screened chemical libraries ... More
Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in four clusters, including an IMiD degron in zinc finger cluster two (ZFC2). Surprisingly, IMiDs do not inhibit growth of SALL4 expressing cancer cells. To overcome this limit, we focused on a non-IMiD degron, SALL4 zinc finger cluster four (ZFC4). By combining AlphaFold and the ZFC4-DNA crystal structure, we identified a potential ZFC4 drug pocket. Utilizing an in silico docking algorithm and cell viability assays, we screened chemical libraries and discovered SH6, which selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 led to significant 62% tumor growth inhibition of SALL4+ xenografts in vivo and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors in cancer. Less
The COVID- pandemic has highlighted the need to identify novel therapeutic interventions and strategies for pandemic preparedness Other than Severe Acute Respiratory Syndrome Coronavirus SARS-CoV- there are several human coronaviruses that are of pandemic concern these include SARS-CoV and Middle Eastern Respiratory Syndrome MERS-CoV MERS-CoV is a zoonotic virus that was first discovered in The disease has spread rapidly with large outbreaks as recent as and Currently there is no therapeutic intervention for MERS-CoV with of reported cases resulting in human death Like-wise to SARS-CoV- MERS-CoV produces a main protease Mpro which is essential for viral replication and therefore an ... More
The COVID-19 pandemic has highlighted the need to identify novel therapeutic interventions and strategies for pandemic preparedness. Other than Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there are several human coronaviruses that are of pandemic concern, these include SARS-CoV and Middle Eastern Respiratory Syndrome (MERS-CoV). MERS-CoV is a zoonotic virus that was first discovered in 2012. The disease has spread rapidly with large outbreaks as recent as 2015 and 2018. Currently there is no therapeutic intervention for MERS-CoV with 35% of reported cases resulting in human death. Like-wise to SARS-CoV-2, MERS-CoV produces a main protease (Mpro) which is essential for viral replication and therefore an attractive target to inhibit the virus. In this new version we added the protein purification protocol and Addgene id, together with the solvent test and compound soaking conditions. Less
Bacteriorhodopsin is a seven-helical light-driven proton pump and a model membrane protein Here we report engineering of soluble analogues of bacteriorhodopsin NeuroBRs which bind retinal and photocycle under illumination We also report the crystallographic structure of NeuroBR A determined at anisotropic resolution reaching that reveals a conserved chromophore binding pocket and tertiary structure Our results highlight the power of modern protein engineering approaches and pave the way towards wider development of molecular tools derived from membrane proteins
The Fe II - and -oxoglutarate -OG -dependent dioxygenase FOGDD enzyme superfamily catalyzes the oxidation of RNA DNA and peptide substrates to install chemical modifications that regulate the diverse functions of these biomolecules For decades it has been appreciated that many FOGDDs require ascorbate Vitamin C as a cofactor for efficient catalysis but ascorbate requirements across different FOGDD enzymes its effects on the catalysis of different substrates and how it engages the FOGDD active site remain poorly understood Here we use RNA demethylases FTO and AlkBH as model FOGDD enzymes and show that their ascorbate requirements for efficient demethylation reactions ... More
The Fe(II)- and 2-oxoglutarate(2-OG)-dependent dioxygenase (FOGDD) enzyme superfamily catalyzes the oxidation of RNA, DNA, and peptide substrates to install chemical modifications that regulate the diverse functions of these biomolecules. For decades, it has been appreciated that many FOGDDs require ascorbate (Vitamin C) as a cofactor for efficient catalysis, but ascorbate requirements across different FOGDD enzymes, its effects on the catalysis of different substrates, and how it engages the FOGDD active site, remain poorly understood. Here, we use RNA demethylases FTO and AlkBH5 as model FOGDD enzymes and show that their ascorbate requirements for efficient demethylation reactions are dramatically different. Furthermore, FTO was found to have strikingly different ascorbate requirements for its own demethylation reactions with different methylated RNA substrates. Our enzymology experiments suggest FTO and AlkBH5, and likely FOGDDs generally, can have widely different ascorbate dependencies based on the balance between an enzyme’s intrinsic ability to decarboxylate co-substrate 2-oxoglutarate and the kinetics of its substrate oxidation reaction. Finally, we determined a crystal structure of FTO in complex with ascorbate, which for the first time shows how ascorbate selectively engages the FOGDD active site. Together, our biochemical and structural data show that ascorbate can selectively tune the RNA demethylation reactions of FTO and AlkBH5, and that more globally, differences in ascorbate concentrations in different environments or diseases may regulate the activity and specificity of diverse FOGDD-catalyzed oxidation reactions in the cell. Less
Solid-state nuclear magnetic resonance ssNMR is a powerful technique for studying membrane protein structure and dynamics Ideally measurements are performed with the protein in a lipid bilayer However homogenous reconstitution of functional protein into intact bilayers at sufficiently high concentrations is often difficult to achieve In this work we investigate the suitability of the lipid cubic phase LCP which incorporates a lipid bilayer as an alternative medium for ssNMR of integral membrane peptides and proteins The cubic mesophase has long been used to generate membrane protein crystals for use in X-ray crystallographic structure determination by the so-called in meso method ... More
Solid-state nuclear magnetic resonance (ssNMR) is a powerful technique for studying membrane protein structure and dynamics. Ideally, measurements are performed with the protein in a lipid bilayer. However, homogenous reconstitution of functional protein into intact bilayers at sufficiently high concentrations is often difficult to achieve. In this work, we investigate the suitability of the lipid cubic phase (LCP), which incorporates a lipid bilayer, as an alternative medium for ssNMR of integral membrane peptides and proteins. The cubic mesophase has long been used to generate membrane protein crystals for use in X-ray crystallographic structure determination by the so-called in meso method and for protein functional and biophysical characterization. Preparing and handling protein-laden LCP is straightforward. LCP may therefore provide a valuable alternative to native membranes and other membrane mimetics for ssNMR. We tested this idea by conducting standard magic-angle spinning ssNMR experiments on LCP into which gramicidin, a ∼4-kDa transmembrane peptide, or bacterial lipoprotein signal peptidase II (LspA), a ∼20-kDa integral membrane enzyme, had been reconstituted. We report one- and two-dimensional ssNMR spectra for both gramicidin and LspA and the parameters for optimizing spectral quality. The high protein-carrying capacity of the cubic phase facilitated 13C ssNMR at natural abundance. Lowering temperature and raising magic-angle spinning frequency enabled significant improvements in spectral quality. One-dimensional 13C and 15N spectra were collected for LspA. Two-dimensional ssNMR experiments provided information on LspA dynamics and its interaction with the water and lipid components of the cubic phase. Solution NMR measurements carried out in parallel yielded information on the effect of the antibiotic, globomycin, on LspA structure and dynamics. Less
The Elettra synchrotron radiation facility located in Trieste Italy is a third-generation storage ring operating in top-up mode at both and GeV The facility currently hosts one beamline fully dedicated to macromolecular crystallography XRD XRD is based on a superconducting wiggler and it has been open to users since On-site and remote access for data collection as well as monitoring tools and automatic data analysis pipelines are available to its users In addition since Elettra has operated a general-purpose diffraction beamline XRD offering the macromolecular community a wide spectrum extending to long wavelengths for phasing and ion identification Ancillary facilities ... More
The Elettra synchrotron radiation facility, located in Trieste, Italy, is a third-generation storage ring, operating in top-up mode at both 2.0 and 2.4 GeV. The facility currently hosts one beamline fully dedicated to macromolecular crystallography, XRD2. XRD2 is based on a superconducting wiggler, and it has been open to users since 2018. On-site and remote access for data collection, as well as monitoring tools and automatic data analysis pipelines are available to its users. In addition, since 1994 Elettra has operated a general-purpose diffraction beamline, XRD1, offering the macromolecular community a wide spectrum extending to long wavelengths for phasing and ion identification. Ancillary facilities support the beamlines, providing sample preparation and a high-throughput crystallization platform for the user community. A new CryoEM facility is being established on campus and jointly operated by the Consiglio Nazionale della Ricerche – Istituto Officina dei Materiali (CNR–IOM) and Elettra, providing further opportunities to the Elettra user community. This review outlines the current capabilities and anticipated developments for macromolecular crystallography at Elettra to accompany the upcoming upgrade to Elettra 2.0, featuring a six-bend enhanced achromat lattice. The new source is expected to deliver a high-brilliance beam, enabling the macromolecular crystallography community to better address the emerging and future scientific challenges. Less
Bacteria face a constant existential threat in the form of infection by viruses along with other forms of mobile genetic elements such as bacteriophage and transposable elements To survive bacteria and other prokaryotes have evolved various immune systems to evade these would-be invaders One such immune system is the CRISPR-Cas system an adaptive immune system able to record the genetic signature of invading viruses in order to recognize and destroy them should they be encountered again in the future In this thesis I present data that sheds light on the mechanism of one particular subtype of CRISPR-Cas systems the type ... More
Bacteria face a constant existential threat in the form of infection by viruses along with other forms of mobile genetic elements, such as bacteriophage and transposable elements. To survive, bacteria and other prokaryotes have evolved various immune systems to evade these would-be invaders. One such immune system is the CRISPR-Cas system, an adaptive immune system able to record the genetic signature of invading viruses in order to recognize and destroy them should they be encountered again in the future. In this thesis I present data that sheds light on the mechanism of one particular subtype of CRISPR-Cas systems: the type IV-A1 system from Pseudomonas aeruginosa. I also report on some of the newly identified tools used by viruses and plasmids to evade this system, called anti-CRISPRs.
The type IV-A1 system is unique in that unlike most CRISPR-Cas systems, it doesn’t appear to destroy or degrade the genome of invading viruses. Instead, it relies on an additional helicase protein called CasDinG to repress the expression of any genes near its target. I report data which explains the genetic signatures necessary to activate type IV-A CRISPR system, and I also explore the significance of a particular domain of the CasDinG helicase.
This thesis also identifies the first-ever reported anti-CRISPRs against the type IV-A system, along with hypothesized mechanisms by which they repress immunity. Less
The type IV-A1 system is unique in that unlike most CRISPR-Cas systems, it doesn’t appear to destroy or degrade the genome of invading viruses. Instead, it relies on an additional helicase protein called CasDinG to repress the expression of any genes near its target. I report data which explains the genetic signatures necessary to activate type IV-A CRISPR system, and I also explore the significance of a particular domain of the CasDinG helicase.
This thesis also identifies the first-ever reported anti-CRISPRs against the type IV-A system, along with hypothesized mechanisms by which they repress immunity. Less
DNA replication is tightly regulated to ensure genomic stability and prevent several diseases including cancers Eukaryotes and archaea partly achieve this regulation by strictly controlling the activation of hexameric minichromosome maintenance MCM helicase rings that unwind DNA during its replication In eukaryotes MCM activation critically relies on the sequential recruitment of the essential factors Cdc and a tetrameric GINS complex at the onset of the S-phase to generate a larger CMG complex We present the crystal structure of the tetrameric GINS complex from the archaeal organism Saccharolobus solfataricus Sso to reveal a core structure that is highly similar to the ... More
DNA replication is tightly regulated to ensure genomic stability and prevent several diseases, including cancers. Eukaryotes and archaea partly achieve this regulation by strictly controlling the activation of hexameric minichromosome maintenance (MCM) helicase rings that unwind DNA during its replication. In eukaryotes, MCM activation critically relies on the sequential recruitment of the essential factors Cdc45 and a tetrameric GINS complex at the onset of the S-phase to generate a larger CMG complex. We present the crystal structure of the tetrameric GINS complex from the archaeal organism Saccharolobus solfataricus (Sso) to reveal a core structure that is highly similar to the previously determined GINS core structures of other eukaryotes and archaea. Using molecular modeling, we illustrate that a subdomain of SsoGINS would need to move to accommodate known interactions of the archaeal GINS complex and to generate a SsoCMG complex analogous to that of eukaryotes. Less
T cell receptors TCRs recognize specific peptides presented by human leukocyte antigens HLAs on the surface of antigen-presenting cells and are involved in fighting pathogens and cancer surveillance Canonical docking orientation of TCRs to their target peptide-HLAs pHLAs is essential for T cell activation with reverse binding TCRs lacking functionality TCR binding geometry and molecular interaction footprint with pHLAs are typically obtained by determining the crystal structure Here we describe the use of a cross-linking tandem mass spectrometry XL-MS MS method to decipher the binding orientation of several TCRs to their target pHLAs Cross-linking sites were localized to specific residues ... More
T cell receptors (TCRs) recognize specific peptides presented by human leukocyte antigens (HLAs) on the surface of antigen-presenting cells and are involved in fighting pathogens and cancer surveillance. Canonical docking orientation of TCRs to their target peptide-HLAs (pHLAs) is essential for T cell activation, with reverse binding TCRs lacking functionality. TCR binding geometry and molecular interaction footprint with pHLAs are typically obtained by determining the crystal structure. Here, we describe the use of a cross-linking tandem mass spectrometry (XL-MS/MS) method to decipher the binding orientation of several TCRs to their target pHLAs. Cross-linking sites were localized to specific residues and their molecular interactions showed differentiation between TCRs binding in canonical or reverse orientations. Structural prediction and crystal structure determination of two TCR-pHLA complexes validated these findings. The XL-MS/MS method described herein offers a faster and simpler approach for elucidating TCR-pHLA binding orientation and interactions. Less
Since the Macromolecular Crystallography MX group at Helmholtz-Zentrum Berlin HZB has been operating three MX beamlines at the BESSY II storage ring in Berlin These beamlines were established to support the emerging structural genomics initiatives founded in Germany Europe and overseas around the turn of the century Over the past two decades these beamlines have been continuously developed to enable state-of-the-art diffraction experiments and to provide supporting facilities such as a sample preparation laboratory a spectroscopy laboratory a Biosafety Level laboratory and all necessary computing resources for the MX and chemical crystallography user community Currently more than independent research groups ... More
Since 2003, the Macromolecular Crystallography (MX) group at Helmholtz-Zentrum Berlin (HZB) has been operating three MX beamlines at the BESSY II storage ring in Berlin. These beamlines were established to support the emerging structural genomics initiatives founded in Germany, Europe, and overseas around the turn of the century. Over the past two decades, these beamlines have been continuously developed to enable state-of-the-art diffraction experiments and to provide supporting facilities such as a sample preparation laboratory, a spectroscopy laboratory, a Biosafety Level 1 laboratory and all necessary computing resources for the MX and chemical crystallography user community. Currently, more than 100 independent research groups from the greater Berlin area, Germany, and Europe utilize these beamlines. Over time, more than 4500 Protein Data Bank depositions have been accrued based on data collected at the beamlines. This paper presents historical aspects of the beamlines, their current status including their research output, and future directions. Less
Personalised cell therapies utilising T cell receptors TCRs show tremendous clinical promise though TCR synthesis and validation techniques lag far behind current TCR repertoire sequencing capacity To address this gap we developed makeTCR a modular TCR cloning system that enables rapid single-step fidelity assembly of human or murine TCR sequences into diverse expression vectors We provide pre-cloned modules for and TCRs as well as many native and engineered constant regions We show how implementing cell-free manufacturing both facilitates the propagation of precloned modules and allows testable TCR material to be synthesised in hours enabling patient-derived TCRs to be prototyped prior ... More
Personalised cell therapies utilising T cell receptors (TCRs) show tremendous clinical promise, though TCR synthesis and validation techniques lag far behind current TCR repertoire sequencing capacity. To address this gap we developed makeTCR: a modular TCR cloning system that enables rapid, single-step, 100% fidelity assembly of human or murine TCR sequences into diverse expression vectors. We provide pre-cloned modules for αβ and γδ TCRs, as well as many native and engineered constant regions. We show how implementing cell-free manufacturing both facilitates the propagation of precloned modules, and allows testable TCR material to be synthesised in 24 hours, enabling patient-derived TCRs to be prototyped prior to use in personalised cell therapies. makeTCR scales to making thousands of TCRs, at high fidelity and at substantially reduced cost. makeTCR is facilitated by a free, open-source, extensible, graphical platform to simplify, standardise, and accelerate TCR functionality testing for personalised medicine and beyond. Less
The development of effective broad-spectrum antivirals forms an important part of preparing for future pandemics A current cause for concern is the emerging pathogen Enterovirus D EV-D which primarily spreads through respiratory routes While it mostly causes mild to severe respiratory illness in severe cases it can lead to acute flaccid myelitis The C protease of EV-D is a potential target for antiviral drug development due to its essential role in the viral life cycle and high sequence conservation This protocol was used to grow EV-D C crystals that were subjected to high-throughput fragment screening crystallography PDB group deposition G ... More
The development of effective broad-spectrum antivirals forms an important part of preparing for future pandemics. A current cause for concern is the emerging pathogen Enterovirus D68 (EV-D68), which primarily spreads through respiratory routes. While it mostly causes mild to severe respiratory illness, in severe cases it can lead to acute flaccid myelitis. The 3C protease of EV-D68 is a potential target for antiviral drug development due to its essential role in the viral life cycle and high sequence conservation. This protocol was used to grow EV-D68 3C crystals that were subjected to high-throughput fragment screening crystallography (PDB group deposition G_10002271). In this new version, we have added the protocols for protein expression and purification, soaking conditions, and fragment screening information, as well as the affiliation with the ASAP Discovery Consortium. Less
Picornaviridae coxsackievirus A is the causative agent of paediatric hand-foot-and-mouth disease and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak The A protease of the virus is responsible for self-cleavage from the poly protein allowing for correct folding and assembly of capsid proteins in the final stages of viral replication Inhibition deranges capsid folding and assembly preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity This protocol was used to grow coxsackievirus A crystals PDB POA that were used in high-throughput crystallographic ... More
Picornaviridae coxsackievirus A16 is the causative agent of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of the virus is responsible for self-cleavage from the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. Inhibition deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. This protocol was used to grow coxsackievirus A16 crystals (PDB 8POA) that were used in high-throughput crystallographic fragment screening, and follow up compounds on the target. In this new version we added: the group deposition code; details about the fragment screen and solvent tolerance; also the protein production protocol. Less
Zika virus ZIKV NS protease with its NS B cofactor is essential for the cleavage of Zika polyprotein precursor into individual structural and non-structural proteins and is therefore an attractive drug target We optimized a robust crystal system of co-expressed NS protease with its NS B cofactor The crystals appeared within hours and diffracted to in average The NS B-NS structure is in closed conformation and has been deposited to PDB PDB code PN In this version we added the addgene id and the protein production protocol
Bacterial proton pumps proteorhodopsins PRs are a major group of light-driven membrane proteins found in marine bacteria They are functionally and structurally distinct from archaeal and eukaryotic proton pumps To elucidate the proton transfer mechanism by PRs and understand the differences to nonbacterial pumps on a molecular level high-resolution structures of PRs functional states are needed In this work we have determined atomic-resolution structures of MAR a PR from marine actinobacteria in various functional states notably the challenging late O intermediate state These data and information from recent atomic-resolution structures on an archaeal outward proton pump bacteriorhodopsin and bacterial inward ... More
Bacterial proton pumps, proteorhodopsins (PRs), are a major group of light-driven membrane proteins found in marine bacteria. They are functionally and structurally distinct from archaeal and eukaryotic proton pumps. To elucidate the proton transfer mechanism by PRs and understand the differences to nonbacterial pumps on a molecular level, high-resolution structures of PRs’ functional states are needed. In this work, we have determined atomic-resolution structures of MAR, a PR from marine actinobacteria, in various functional states, notably the challenging late O intermediate state. These data and information from recent atomic-resolution structures on an archaeal outward proton pump bacteriorhodopsin and bacterial inward proton pump xenorhodopsin allow for deducing key universal elements for light-driven proton pumping. First, long hydrogen-bonded chains characterize proton pathways. Second, short hydrogen bonds allow proton storage and inhibit their backflow. Last, the retinal Schiff base is the active proton donor and acceptor to and from hydrogen-bonded chains. Less
Reinvigoration of tumor-reactive T-cells using co-stimulatory bispecific antibodies bsAbs targeting CD or CD is emerging as a promising therapeutic strategy Conditional tumor-specific recruitment can offer a necessary layer of control and specificity We developed pH-selective CD xVISTA bsAbs to act specifically within the acidic tumor microenvironment TME aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome CRS risk CD agonism by CD xVISTA bsAbs relies on pH-selective engagement of VISTA a protein robustly expressed on myeloid cells highly prevalent in most solid tumors This modality avoids engagement of tumor-associated antigens TAAs with the ... More
Reinvigoration of tumor-reactive T-cells using co-stimulatory bispecific antibodies (bsAbs) targeting CD28 or CD137 is emerging as a promising therapeutic strategy. Conditional, tumor-specific recruitment can offer a necessary layer of control and specificity. We developed pH-selective CD28xVISTA bsAbs to act specifically within the acidic tumor microenvironment (TME), aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome (CRS) risk. CD28 agonism by CD28xVISTA bsAbs relies on pH-selective engagement of VISTA, a protein robustly expressed on myeloid cells highly prevalent in most solid tumors. This modality avoids engagement of tumor-associated antigens (TAAs) with the potential to provide highly tumor specific activity with minimal on-target/off-tumor side effects.
We report the identification of a lead candidate with pH-dependent simultaneous engagement of both targets, and VISTA-dependent CD28 signaling in a reporter cell line. CD28xVISTA avidly bound VISTA-positive cells, and co-stimulation was shown in vitro by its ability to activate and expand T-cells and enhance T-cell mediated cancer cell killing in co-cultures of human PBMCs and cancer cells in the presence of a TAA-targeted anti-CD3 T-cell engager. Interestingly, our findings support both signaling in cis (between T-cell and cell displaying peptide-MHC complex) and in trans with stimulation occurring through CD28 clustering outside of the immune synapse. Our lead candidate displayed efficient tumor growth inhibition of human VISTA-expressing MC38 cells in a humanized CD28 syngeneic mouse model in combination with PD-1 blockade. Importantly, our CD28xVISTA bsAb showed no signs of superagonistic properties in several in vitro assays geared towards revealing induction of CRS. Our data supports clinical development in combination with anti-PD-1 or any TAA-targeted anti-CD3 T-cell engagers developed for solid tumors. Less
We report the identification of a lead candidate with pH-dependent simultaneous engagement of both targets, and VISTA-dependent CD28 signaling in a reporter cell line. CD28xVISTA avidly bound VISTA-positive cells, and co-stimulation was shown in vitro by its ability to activate and expand T-cells and enhance T-cell mediated cancer cell killing in co-cultures of human PBMCs and cancer cells in the presence of a TAA-targeted anti-CD3 T-cell engager. Interestingly, our findings support both signaling in cis (between T-cell and cell displaying peptide-MHC complex) and in trans with stimulation occurring through CD28 clustering outside of the immune synapse. Our lead candidate displayed efficient tumor growth inhibition of human VISTA-expressing MC38 cells in a humanized CD28 syngeneic mouse model in combination with PD-1 blockade. Importantly, our CD28xVISTA bsAb showed no signs of superagonistic properties in several in vitro assays geared towards revealing induction of CRS. Our data supports clinical development in combination with anti-PD-1 or any TAA-targeted anti-CD3 T-cell engagers developed for solid tumors. Less
NEMO is an essential component in the activation of the canonical NF B pathway and exerts its function by recruiting the I B kinases IKK to the IKK complex Inhibition of the NEMO IKKs interaction is an attractive therapeutic paradigm for diseases related to NF B mis-regulation but a difficult endeavor because of the extensive protein-protein interface Here we report the design and characterization of novel engineered constructs of the IKK-binding domain of NEMO programmed to render this difficult protein domain amenable to NMR and X-ray characterization while preserving the biological function ZipNEMO binds IKK with nanomolar affinity is amenable ... More
NEMO is an essential component in the activation of the canonical NFκ B pathway and exerts its function by recruiting the I κ B kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NFκ B mis-regulation, but a difficult endeavor because of the extensive protein-protein interface. Here we report the design and characterization of novel engineered constructs of the IKK-binding domain of NEMO, programmed to render this difficult protein domain amenable to NMR and X-ray characterization, while preserving the biological function. ZipNEMO binds IKK β with nanomolar affinity, is amenable to heteronuclear NMR techniques and structure determination by X-ray crystallography. We show that NMR spectra of zipNEMO allow to detect inhibitor binding in solution and resonance assignment. The X-ray structure of zipNEMO highlights a novel ligand binding motif and the adaptability of the binding pocket and inspired the design of new peptide inhibitors. Less