888 Citations
This review highlights the development and evolution of three macromolecular crystallography MX beamlines at the Swiss Light Source SLS over the past two decades We discuss key advancements in X-ray optics detectors goniometers sample changers and MX methodology emphasizing their impact on high-throughput and high-resolution structural biology Our contributions are presented within the broader context of global efforts in synchrotron-based MX Looking ahead we explore the future experiments enabled by SLS and new opportunities at SwissFEL to enhance experimental capabilities and drive scientific discoveries
Fungal cell walls composed of polysaccharides and proteins play critical roles in adaptation cell division and protection against environmental stress Their polyglucan components are continuously remodeled by various types of glycosyl hydrolases GHs and transferases GTs In Saccharomyces cerevisiae and other ascomycetes enzymes of the Dfg subfamily which belong as GTs to the GH family cleave an linkage between glucosamine and mannose to facilitate covalent linkage of GPI-anchored proteins to the cell wall s polyglucans In contrast the functions of other fungal GH subfamilies are not understood We characterized CtGH from the sordariomycete Chaetomium thermophilum a member of the Fungi ... More
Fungal cell walls, composed of polysaccharides and proteins, play critical roles in adaptation, cell division, and protection against environmental stress. Their polyglucan components are continuously remodeled by various types of glycosyl hydrolases (GHs) and transferases (GTs). In Saccharomyces cerevisiae and other ascomycetes, enzymes of the Dfg5 subfamily, which belong as GTs to the GH76 family, cleave an α1,4 linkage between glucosamine and mannose to facilitate covalent linkage of GPI-anchored proteins to the cell wall’s polyglucans. In contrast, the functions of other fungal GH76 subfamilies are not understood. We characterized CtGH76 from the sordariomycete Chaetomium thermophilum, a member of the Fungi/Bacteria-mixed GH76 subfamily, revealing conserved structural features and functional divergence within the GH76 family. Notably, our structural characterization by X-ray crystallography combined with glycan fragment screening indicated that CtGH76 can recognize GPI-anchors like members of the Dfg5 subfamily but shows a broader promiscuity toward other glycans with central α1,6-mannobiose motifs due to the presence of an elongated glycan binding canyon. These findings provide new insights into GH76 enzyme diversity and fungal cell wall maturation. Less
Malaria remains a global health threat exacerbated by emerging resistance to antimalarial therapies and insecticides climate-driven outbreaks and limited chemoprotective options Here we report the characterization of RUPB- the first orally bioavailable inhibitor of Plasmodium falciparum cGMP-dependent protein kinase PfPKG RUPB- prevents infection by P falciparum and P cynomolgi sporozoites including the formation of hypnozoites by the latter A single oral dose blocks liver infection by P berghei sporozoites in vivo demonstrating efficacy consistent with further development as a once-weekly prophylaxis based on pharmacokinetic modeling The compound retains activity against field isolates resistant to chloroquine mefloquine cycloguanil sulfadoxine and pyrimethamine ... More
Malaria remains a global health threat exacerbated by emerging resistance to antimalarial therapies and insecticides, climate-driven outbreaks, and limited chemoprotective options. Here, we report the characterization of RUPB-61, the first orally bioavailable inhibitor of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG). RUPB-61 prevents infection by P. falciparum and P. cynomolgi sporozoites, including the formation of hypnozoites by the latter. A single oral dose blocks liver infection by P. berghei sporozoites in vivo, demonstrating efficacy consistent with further development as a once-weekly prophylaxis based on pharmacokinetic modeling. The compound retains activity against field isolates resistant to chloroquine, mefloquine, cycloguanil, sulfadoxine and pyrimethamine, suggesting low likelihood of cross-resistance to existing antimalarials. Structural studies and free energy-based modeling guided compound design and prospectively validated the predictive accuracy of an in silico model of PfPKG interactions with this chemotype. While selectivity profiling identified off-target activity against human kinases, structural modeling provides a clear path for optimization. These results establish PfPKG inhibitors as promising candidates for chemoprotection and support further preclinical development of the RUPB-61 chemotype. Less
Malaria poses an enormous threat to human health With ever-increasing resistance to currently deployed antimalarials new targets and starting point compounds with novel mechanisms of action need to be identified Here we explore the antimalarial activity of the Streptomyces sp natural product -O-sulfamoyl- -chloroadenosine dealanylascamycin DACM and compare it with the synthetic adenosine monophosphate AMP mimic -O-sulfamoyladenosine AMS These nucleoside sulfamates exhibit potent inhibition of P falciparum growth with an efficacy comparable to that of the current front-line antimalarial dihydroartemisinin Exposure of P falciparum to DACM leads to inhibition of protein translation driven by eIF phosphorylation We show that DACM ... More
Malaria poses an enormous threat to human health. With ever-increasing resistance to currently deployed antimalarials, new targets and starting point compounds with novel mechanisms of action need to be identified. Here, we explore the antimalarial activity of the Streptomyces sp natural product, 5ʹ-O-sulfamoyl-2-chloroadenosine (dealanylascamycin, DACM) and compare it with the synthetic adenosine monophosphate (AMP) mimic, 5-O-sulfamoyladenosine (AMS). These nucleoside sulfamates exhibit potent inhibition of P. falciparum growth with an efficacy comparable to that of the current front-line antimalarial dihydroartemisinin. Exposure of P. falciparum to DACM leads to inhibition of protein translation, driven by eIF2α phosphorylation. We show that DACM targets multiple amino acyl tRNA synthetase (aaRS) targets, including the cytoplasmic aspartyl tRNA synthetase (AspRS). The mechanism involves hijacking of the reaction product, leading to the formation of a tightly bound inhibitory amino acid-sulfamate conjugate. We show that recombinant P. falciparum and P. vivax AspRS are susceptible to hijacking by DACM and AMS, generating Asp-DACM and Asp-AMS adducts that stabilize these proteins. By contrast, human AspRS appears less susceptible to hijacking. X-ray crystallography reveals that apo P. vivax AspRS exhibits a stabilized flipping loop over the active site that is poised to bind substrates. By contrast, human AspRS exhibits disorder in an extended region around the flexible flipping loop as well as in a loop in motif II. These structural differences may underpin the decreased susceptibility of human AspRS to reaction-hijacking by DACM and AMS. Our work reveals Plasmodium AspRS as a promising antimalarial target and highlights structural features that underpin differences in the susceptibility of aaRSs to reaction hijacking inhibition. Less
Glaucoma is a neurodegenerative condition involving optic nerve damage and retinal ganglion cells death Animal studies suggested that the pathway linking these events can be mediated by mobile zinc secreted into the intraretinal space and exerting cytotoxic effects Whether this mechanism is relevant for human glaucoma and what are the targets of extracellular zinc is unknown We report that increased zinc content in the aqueous humor and retina is indeed a characteristic of glaucomatous neuropathy and excess extracellular zinc may be recognized by the key retinal neurotrophic factor PEDF Biophysical and X-ray crystallographic studies show that PEDF coordinates zinc ions ... More
Glaucoma is a neurodegenerative condition involving optic nerve damage and retinal ganglion cells death. Animal studies suggested that the pathway linking these events can be mediated by mobile zinc secreted into the intraretinal space and exerting cytotoxic effects. Whether this mechanism is relevant for human glaucoma and what are the targets of extracellular zinc is unknown. We report that increased zinc content in the aqueous humor and retina is indeed a characteristic of glaucomatous neuropathy, and excess extracellular zinc may be recognized by the key retinal neurotrophic factor PEDF. Biophysical and X-ray crystallographic studies show that PEDF coordinates zinc ions in five types of intermolecular high-affinity sites, leading to a decrease in negative surface charge and reversible oligomerization of the protein, thereby masking the target recognition sites responsible for its neurotrophic and antiangiogenic activities and collagen binding. Notably, PEDF secretion is enhanced in both glaucoma and retinal cell models in response to zinc stress; however, zinc binding negatively affects axogenic, differentiative and prosurvival functions of PEDF by suppressing its ability to activate receptor PEDF-R/PNPLA2. We suggest that glaucomatous neurodegeneration is associated with direct inhibition of PEDF signaling by extracellular zinc, making their complex a promising target for neuroprotective therapy. Less
Glycyl tRNA synthetases GlyRSs are prospective drug targets for combating Mycobacterium tuberculosis Mtb and cancer in humans These synthetases are of the -subtype with the ortholog in humans being dual targeted to the cytosol and mitochondria Whereas the human enzyme has been structurally characterized previously in several liganded states no structures of MtbGlyRS have thus far been reported Here we describe our recent work with MtbGlyRS and the closely-related Mycobacterium thermoresitibile GlyRS MtrGlyRS which progressed through all phases of the structural genomics pipeline for the purpose of facilitating structure-based drug discovery MtbGlyRS was expressed in Mycobacterium smegmatis and MtrGlyRS in ... More
Glycyl tRNA synthetases (GlyRSs) are prospective drug targets for combating Mycobacterium tuberculosis (Mtb) and cancer in humans. These synthetases are of the α2-subtype, with the ortholog in humans being dual targeted to the cytosol and mitochondria. Whereas the human enzyme has been structurally characterized previously in several liganded states, no structures of MtbGlyRS have thus far been reported. Here, we describe our recent work with MtbGlyRS and the closely-related Mycobacterium thermoresitibile GlyRS (MtrGlyRS), which progressed through all phases of the structural genomics pipeline, for the purpose of facilitating structure-based drug discovery. MtbGlyRS was expressed in Mycobacterium smegmatis and MtrGlyRS in Escherichia coli. Crystal structures are described for complexes of the two enzymes with adenosine monophosphate (AMP) and glycyl-sulfamoyl-adenylate (glycyl-AMS) at resolutions of 1.65/2.90 and 2.25/1.95 Å, respectively, and for MtrGlyRS in its apo state at 2.85 Å. Despite crystallizing in the dimeric state characteristic of many class II synthetases, the two enzymes elute predominantly as monomers during size exclusion chromatography. Strikingly, significant portions of the dimer interface and active site are unstructured in the MtrGlyRS apoenzyme crystal. AMP orders two tRNA recognition loops and a section of the insertion domain, and glycyl-AMS further stabilizes the structure, including the closure of a lid motif. Both the active and anticodon binding sites display structural differences with the human GlyRS and thus the collection of crystal structures should be useful for guiding drug development efforts targeting the various characterized structural states. Less
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
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
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
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
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
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
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
The human heterogeneous nuclear ribonucleoprotein hnRNP A is a prototypical RNA-binding protein essential in regulating a wide range of post-transcriptional events in cells As a multifunctional protein with a key role in RNA metabolism deregulation of its functions has been linked to neurodegenerative diseases tumour aggressiveness and chemoresistance which has fuelled efforts to develop novel therapeutics that modulates its RNA binding activities Here using a combination of Molecular Dynamics MD simulations and graph neural network pockets predictions we showed that hnRNPA N-terminal RNA binding domain UP contains several cryptic pockets capable of binding small molecules To identify chemical entities for ... More
The human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a prototypical RNA-binding protein essential in regulating a wide range of post-transcriptional events in cells. As a multifunctional protein with a key role in RNA metabolism, deregulation of its functions has been linked to neurodegenerative diseases, tumour aggressiveness and chemoresistance, which has fuelled efforts to develop novel therapeutics that modulates its RNA binding activities. Here, using a combination of Molecular Dynamics (MD) simulations and graph neural network pockets predictions, we showed that hnRNPA1 N-terminal RNA binding domain (UP1) contains several cryptic pockets capable of binding small molecules. To identify chemical entities for development of potent drug candidates and experimentally validate identified druggable hotspots, we carried out a large fragment screening on UP1 protein crystals. Our screen identified 36 hits which extensively samples UP1 functional regions involved in RNA recognition and binding, as well as mapping hotspots onto novel protein interaction surfaces. We observed a wide range of ligand-induced conformational variation, by stabilisation of dynamic protein regions. Our high-resolution structures, the first of an hnRNP in complex with a fragment or small molecule, provides rapid routes for the rational development of a range of different inhibitors and chemical tools for studying molecular mechanisms of hnRNPA1 mediated splicing regulation. Less
Introduction The MHC-class-I-related molecule MR presents small metabolites of microbial and self-origin to T cells bearing semi-invariant or variant T cell receptors One such T cell receptor MC G was previously shown to confer broad MR -restricted reactivity to tumor cells but not normal cells sparking interest in the development of non-MHC-restricted immunotherapy approaches Methods Results Here we provide cellular biophysical and crystallographic evidence that the MC G TCR does not have pan-cancer specificity but is restricted to a rare allomorph of MR bearing the R H mutation Discussion Our results underscore the importance of in-depth characterization of MR -reactive ... More
Introduction: The MHC-class-I-related molecule MR1 presents small metabolites of microbial and self-origin to T cells bearing semi-invariant or variant T cell receptors. One such T cell receptor, MC.7.G5, was previously shown to confer broad MR1-restricted reactivity to tumor cells but not normal cells, sparking interest in the development of non-MHC-restricted immunotherapy approaches.
Methods/Results: Here we provide cellular, biophysical, and crystallographic evidence that the MC.7.G5 TCR does not have pan-cancer specificity but is restricted to a rare allomorph of MR1, bearing the R9H mutation.
Discussion: Our results underscore the importance of in-depth characterization of MR1-reactive TCRs against targets expressing the full repertoire of MR1 allomorphs. Less
Methods/Results: Here we provide cellular, biophysical, and crystallographic evidence that the MC.7.G5 TCR does not have pan-cancer specificity but is restricted to a rare allomorph of MR1, bearing the R9H mutation.
Discussion: Our results underscore the importance of in-depth characterization of MR1-reactive TCRs against targets expressing the full repertoire of MR1 allomorphs. Less
Human lens fiber membrane intrinsic protein MP is the second most abundant membrane protein of the human eye lens Despite decades of effort its structure and function remained elusive Here we determined the MicroED structure of full-length human MP in lipidic-cubic phase to a resolution of MP forms tetramers each of which contain transmembrane -helices that are packed against one another forming a helical bundle Both the N- and C-termini of MP are cytoplasmic We found that each MP tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion These interactions were mediated by the extracellular loops of ... More
Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we determined the MicroED structure of full-length human MP20 in lipidic-cubic phase to a resolution of 3.5 Å. MP20 forms tetramers each of which contain 4 transmembrane α-helices that are packed against one another forming a helical bundle. Both the N- and C-termini of MP20 are cytoplasmic. We found that each MP20 tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion. These interactions were mediated by the extracellular loops of the protein. The dimensions of the MP20 adhesive junctions are consistent with the 11 nm thin lens junctions. Investigation of MP20 localization in human lenses indicated that in young fiber cells MP20 was stored intracellularly in vesicles and upon fiber cell maturation MP20 inserted into the plasma membrane and restricted the extracellular space. Together these results suggest that MP20 forms lens thin junctions in vivo confirming its role as a structural protein in the human eye lens, essential for its optical transparency. Less
Fatty acid esters of hydroxy fatty acids FAHFAs are a newly discovered lipid class known for their potential anti-inflammatory and insulin-sensitizing properties A sustainable and efficient synthesis route is essential to realize the potential of FAHFAs and enable cost-effective large-scale production Enzymatic synthesis favored for its scalability and environmental impact is the preferred approach Candida Moesziomyces antarctica lipase A CalA previously known for its thermostability and limited ability to catalyze FAHFA esterification was investigated along with its orthologues for their ability to produce a variety of FAHFAs We developed a systematic workflow to identify uncharacterized enzymes for FAHFA synthesis from ... More
Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered lipid class known for their potential anti-inflammatory and insulin-sensitizing properties. A sustainable and efficient synthesis route is essential to realize the potential of FAHFAs and enable cost-effective, large-scale production. Enzymatic synthesis, favored for its scalability and environmental impact, is the preferred approach. Candida (Moesziomyces) antarctica lipase A (CalA), previously known for its thermostability and limited ability to catalyze FAHFA esterification, was investigated along with its orthologues for their ability to produce a variety of FAHFAs. We developed a systematic workflow to identify uncharacterized enzymes for FAHFA synthesis from natural sources, using an automation-compatible method, leading to the discovery of several novel lipases capable of synthesizing diverse FAHFAs. Among these lipases, two newly discovered enzymes, CL20 and CL23, demonstrated superior performance in FAHFA biosynthesis, achieving faster and higher yields than the benchmark enzyme, CalA. Our work advances methodologies and processes critical for industrial FAHFA production and provides a foundation for sustainable commercial-scale synthesis via synthetic enzymology. Less
Candida albicans is a critical priority fungal pathogen causing invasive fungal infections with high mortality rates in immunocompromised patients The increasing fungal infection rate and resistance of fungal pathogens to existing antifungal treatments have emphasized the need for the development of novel antifungal medicine The ergosterol biosynthesis pathway has been a successful target for antifungal compounds but many enzymatic steps remain unexplored C-sterol methyltransferase C-SMT catalyzes a critical fungal-specific step in ergosterol biosynthesis When C-SMT is disrupted fungal pathogens are sensitized to temperature various inhibitors and antifungals and a loss of virulence can be observed In this study five C-SMT ... More
Candida albicans is a critical priority fungal pathogen causing invasive fungal infections with high mortality rates in immunocompromised patients. The increasing fungal infection rate and resistance of fungal pathogens to existing antifungal treatments have emphasized the need for the development of novel antifungal medicine. The ergosterol biosynthesis pathway has been a successful target for antifungal compounds, but many enzymatic steps remain unexplored. 24C-sterol methyltransferase (24C-SMT) catalyzes a critical fungal-specific step in ergosterol biosynthesis. When 24C-SMT is disrupted, fungal pathogens are sensitized to temperature, various inhibitors, and antifungals, and a loss of virulence can be observed. In this study, five 24C-SMT variants with different lengths of N-termini were heterologously produced in Escherichia coli and three were purified to near-homogeneity with immobilized metal-affinity and size-exclusion chromatography. N-terminally truncated C. albicans 24C-SMT was utilized for crystallization trials due to its increased stability and higher purity compared to the full-length protein. 24C-SMT crystals were obtained in the presence of Sadenosyl-homocysteine, but diffracted to low resolution. Therefore, we established a starting point for 24C-SMT crystallization by providing an optimized protocol for heterologous 24C-SMT production, purification and initial crystallization conditions, which could be used for further downstream crystallographic studies. Less
Crystallisation and stereochemical stability are pivotal factors in pharmaceutical development particularly for complex beyond Rule of bRo compounds In this study we explore the intricate interplay between atropisomerism and crystallisation using two model bRo compounds namely ACBI and BI both violating three of four Lipinski s rules One of the tool compounds exhibits Class atropisomeric behaviour and the other devoid of it A diverse array of crystallisation methods including solution-phase crystallisation cocrystallisation and salt formation was applied revealing the critical role of atropisomerism induced stereochemistry in polymorphism and nucleation outcomes In-silico torsion profile calculations and NMR studies were employed to ... More
Crystallisation and stereochemical stability are pivotal factors in pharmaceutical development, particularly for complex beyond Rule of 5 (bRo5) compounds. In this study, we explore the intricate interplay between atropisomerism and crystallisation using two model bRo5 compounds, namely ACBI1 and BI201335, both violating three of four Lipinski’s rules. One of the tool compounds exhibits Class 2 atropisomeric behaviour and the other devoid of it. A diverse array of crystallisation methods—including solution-phase crystallisation, cocrystallisation, and salt formation—was applied, revealing the critical role of atropisomerism induced stereochemistry in polymorphism and nucleation outcomes. In-silico torsion profile calculations and NMR studies were employed to elucidate the rotational energy barriers and confirm the presence or absence of atropisomerism. This comprehensive analysis highlights the significance of understanding stereochemical phenomena like atropisomerism in designing and developing bRo5 compounds. By integrating advanced analytical techniques and crystallisation strategies, this work provides novel insights into tailoring pharmaceutical properties for nextgeneration therapeutics. Less
The C carbon concentrating mechanism relies on specialized enzymes that have evolved unique expression patterns and biochemical properties distinct to their ancestral housekeeping forms In maize and sorghum the evolution of C -NADP-malic enzyme C -NADP-ME involved gene duplication and neofunctionalization leading to the emergence of two plastidic isoforms C -NADP-ME and nonC -NADP-ME each with distinct kinetic and structural features While C -NADP-ME functions primarily as a tetramer nonC -NADP-ME exists in an equilibrium between dimeric and tetrameric forms favoring the dimer in solution This study shows which evolutionary changes in amino acid sequences influence the structure and function ... More
The C4 carbon concentrating mechanism relies on specialized enzymes that have evolved unique expression patterns and biochemical properties distinct to their ancestral housekeeping forms. In maize and sorghum, the evolution of C4-NADP-malic enzyme (C4-NADP-ME) involved gene duplication and neofunctionalization, leading to the emergence of two plastidic isoforms: C4-NADP-ME and nonC4-NADP-ME, each with distinct kinetic and structural features. While C4-NADP-ME functions primarily as a tetramer, nonC4-NADP-ME exists in an equilibrium between dimeric and tetrameric forms, favoring the dimer in solution. This study shows which evolutionary changes in amino acid sequences influence the structure and function of these isoforms. By integrating X-ray crystallography, cryo-electron microscopy, computational molecular modeling and targeted biochemical analysis of mutant and truncated protein variants, we identify crucial roles for the N- and C-terminal regions and specific amino acid residues in governing isoform oligomerization. Our results reveal that the N-terminal region is essential for stabilizing the dimeric form of nonC4-NADP-ME, whereas specific adaptive substitutions and interactions with the C-terminal region enhance the stability of the tetrameric state characteristic of the C4-adapted isoform. We propose that differences in the N-terminal domain between the C4 and nonC4 isoforms reflect distinct selective pressures, which have driven their evolutionary divergence to fulfill specialized cellular functions. Less
Acetyl CoA synthetases ACS have emerged as drug targets for the treatment of cancer metabolic diseases as well as fungal and parasitic infections Although a variety of small molecule ACS inhibitors have been discovered the systematic optimization of these molecules has been slowed by a lack of structural information regarding their mechanism of inhibition Through a chemical genetic-based synthetic lethal screen of the human fungal pathogen Cryptococcus neoformans we identified an isoxazole-based ACS inhibitor with antifungal activity and exquisite selectivity for the C neoformans Acs relative to human ACSS as well as other fungal ACSs Xray crystallographic characterization of the ... More
Acetyl CoA synthetases (ACS) have emerged as drug targets for the treatment of cancer, metabolic diseases as well as fungal and parasitic infections. Although a variety of small molecule ACS inhibitors have been discovered, the systematic optimization of these molecules has been slowed by a lack of structural information regarding their mechanism of inhibition. Through a chemical genetic-based, synthetic lethal screen of the human fungal pathogen Cryptococcus neoformans, we identified an isoxazole-based ACS inhibitor with antifungal activity and exquisite selectivity for the C. neoformans Acs1 relative to human ACSS2 as well as other fungal ACSs. Xray crystallographic characterization of the isoxazole-CnAcs1 complex revealed that the isoxazole functions as an acetyl CoA mimic and occupies both the acetyl- and CoA-binding sites of CnAcs1. Consistent with this novel mode of inhibition, the isoxazoles display uncompetitive inhibition kinetics that are similar to antimalarial ACS inhibitors also proposed to target the CoA binding site. Consequently, these data provide structural and mechanistic insights into the remarkable selectivity of Acetyl CoA pocket-targeting ACS inhibitors. In addition, these data provide strong proof-of-principle that targeting fungal and parasitic ACSs for the development of novel anti-infectives can be achieved with high selectivity and, thereby, low host toxicity. Less
Many viral proteins form biomolecular condensates via liquid-liquid phase separation LLPS to support viral replication and evade host antiviral responses and thus they are potential targets for designing antivirals In the case of nonenveloped positive-sense RNA viruses forming such condensates for viral replication is unclear and less understood Human noroviruses HuNoVs are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide Here we show that the RNA-dependent RNA polymerase RdRp of pandemic GII HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication ... More
Many viral proteins form biomolecular condensates via liquid-liquid phase separation (LLPS) to support viral replication and evade host antiviral responses, and thus, they are potential targets for designing antivirals. In the case of nonenveloped positive-sense RNA viruses, forming such condensates for viral replication is unclear and less understood. Human noroviruses (HuNoVs) are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide. Here, we show that the RNA-dependent RNA polymerase (RdRp) of pandemic GII.4 HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication. We show that such condensates are formed in HuNoV-infected human intestinal enteroid cultures and are the sites for genome replication. Our studies demonstrate the formation of phase-separated condensates as replication factories in a positive-sense RNA virus, which plausibly is an effective mechanism to dynamically isolate RdRp replicating the genomic RNA from interfering with the ribosomal translation of the same RNA. Less
Crimean-Congo hemorrhagic fever virus CCHFV is a tickborne virus that can cause severe disease in humans with case fatality rates of Although structures of CCHFV glycoproteins GP and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies the structure of glycoprotein Gn and its interactions with GP and Gc have remained elusive Here we use structure-guided protein engineering to produce a stabilized GP -Gn-Gc heterotrimeric glycoprotein complex GP -GnH-DS-Gc A cryo-electron microscopy cryo-EM structure of this complex provides the molecular basis for GP s association on the viral surface reveals the structure of Gn ... More
Crimean-Congo hemorrhagic fever virus (CCHFV) is a tickborne virus that can cause severe disease in humans with case fatality rates of 10%–40%. Although structures of CCHFV glycoproteins GP38 and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies, the structure of glycoprotein Gn and its interactions with GP38 and Gc have remained elusive. Here, we use structure-guided protein engineering to produce a stabilized GP38-Gn-Gc heterotrimeric glycoprotein complex (GP38-GnH-DS-Gc). A cryo-electron microscopy (cryo-EM) structure of this complex provides the molecular basis for GP38’s association on the viral surface, reveals the structure of Gn, and demonstrates that GP38-Gn restrains the Gc fusion loops in the prefusion conformation, facilitated by an N-linked glycan attached to Gn. Immunization with GP38-GnH-DS-Gc conferred 40% protection against lethal IbAr10200 challenge in mice. These data define the architecture of a GP38-Gn-Gc protomer and provide a template for structure-guided vaccine antigen development. Less
The transcription factor BCL A is a critical regulator of the switch from fetal hemoglobin HbF to adult hemoglobin HbA during development BCL A binds at a cognate recognition site TGACCA in the -globin gene promoter and represses its expression DNA-binding is mediated by a triple zinc finger domain designated ZnF Here we report comprehensive investigation of ZnF leveraging X-ray crystallography and NMR to determine the structures in both the presence and absence of DNA We delve into the dynamics and mode of interaction with DNA Moreover we discovered that the last zinc finger of BCL A ZnF plays a ... More
The transcription factor BCL11A is a critical regulator of the switch from fetal hemoglobin (HbF: α 2 γ 2 ) to adult hemoglobin (HbA: α 2 β 2 ) during development. BCL11A binds at a cognate recognition site (TGACCA) in the γ-globin gene promoter and represses its expression. DNA-binding is mediated by a triple zinc finger domain, designated ZnF456. Here, we report comprehensive investigation of ZnF456, leveraging X-ray crystallography and NMR to determine the structures in both the presence and absence of DNA. We delve into the dynamics and mode of interaction with DNA. Moreover, we discovered that the last zinc finger of BCL11A (ZnF6) plays a special role in DNA binding and γ-globin gene repression. Our findings help account for some rare γ-globin gene promoter mutations that perturb BCL11A binding and lead to increased HbF in adults (hereditary persistence of fetal hemoglobin). Comprehending the DNA binding mechanism of BCL11A opens avenues for the strategic, structure-based design of novel therapeutics targeting sickle cell disease and β-thalassemia. Less
GPR is an orphan G protein coupled receptor with high constitutive activity found in D -type dopamine receptor expressing medium spiny neurons of the striatopallidal pathway which is aberrantly hyperactivated in Parkinson s disease Here we solved crystal structures of GPR without the addition of a ligand a pseudo-apo state and in complex with two inverse agonists including CVN which improved motor symptoms in patients with Parkinson s disease in clinical trials In addition we obtained a cryo electron microscopy structure of the signaling complex between GPR and its cognate Gs heterotrimer The pseudo-apo structure revealed a strong density in ... More
GPR6 is an orphan G protein–coupled receptor with high constitutive activity found in D2-type dopamine receptor–expressing medium spiny neurons of the striatopallidal pathway, which is aberrantly hyperactivated in Parkinson’s disease. Here, we solved crystal structures of GPR6 without the addition of a ligand (a pseudo-apo state) and in complex with two inverse agonists, including CVN424, which improved motor symptoms in patients with Parkinson’s disease in clinical trials. In addition, we obtained a cryo–electron microscopy structure of the signaling complex between GPR6 and its cognate Gs heterotrimer. The pseudo-apo structure revealed a strong density in the orthosteric pocket of GPR6 corresponding to a lipid-like endogenous ligand. A combination of site-directed mutagenesis, native mass spectrometry, and computer modeling suggested potential mechanisms for high constitutive activity and inverse agonism in GPR6 and identified a series of lipids and ions bound to the receptor. The structures and results obtained in this study could guide the rational design of drugs that modulate GPR6 signaling. Less
Heliorhodopsins HeRs constitute a novel and distinct group of microbial rhodopsins characterized by the inverted position of C- and N- termini relative to conventional Type I rhodopsins The production of HeRs for structural and functional investigations has proven challenging as evidenced by the structural elucidation of only two proteins and the functional characterization of a few others to date Notably no eukaryotic HeRs have been reported thus far In this study we report the first expression of three eukaryotic HeRs in the LEXSY expression system from marine and freshwater algae and a free-living marine unicellular eukaryote We spectroscopically characterized these ... More
Heliorhodopsins (HeRs) constitute a novel and distinct group of microbial rhodopsins, characterized by the inverted position of C- and N- termini relative to conventional Type I rhodopsins. The production of HeRs for structural and functional investigations has proven challenging, as evidenced by the structural elucidation of only two proteins and the functional characterization of a few others to date. Notably, no eukaryotic HeRs have been reported thus far. In this study, we report the first expression of three eukaryotic HeRs in the LEXSY expression system: from marine and freshwater algae and a free-living marine unicellular eukaryote. We spectroscopically characterized these HeRs, demonstrating that they were expressed in the functional states. Finally, we report their successful crystallization, thus paving the way for their further structural and functional studies Less
Phosphopentomutases catalyze the isomerization of ribose -phosphate and ribose -phosphate Thermococcus kodakarensis a hyperthermophilic archaeon harbors a novel enzyme PPMTk that exhibits high homology with phosphohexomutases but has no significant phosphohexomutase activity Instead PPMTk catalyzes the interconversion of ribose -phosphate and ribose -phosphate Here we report biophysical analysis crystallization and three-dimensional structure determination of PPMTk by X-ray diffraction at resolution The solved structure revealed a novel catalytic motif unique to PPMTk which makes this enzyme distinct from the homologous counterparts We postulate that this novel catalytic motif may enable PPMTk to isomerize phosphopentose instead of phosphohexose To the best of ... More
Phosphopentomutases catalyze the isomerization of ribose 1-phosphate and ribose 5-phosphate. Thermococcus kodakarensis, a hyperthermophilic archaeon, harbors a novel enzyme (PPMTk) that exhibits high homology with phosphohexomutases but has no significant phosphohexomutase activity. Instead, PPMTk catalyzes the interconversion of ribose 1-phosphate and ribose 5-phosphate. Here, we report biophysical analysis, crystallization, and three-dimensional structure determination of PPMTk by X-ray diffraction at 2.39 Å resolution. The solved structure revealed a novel catalytic motif, unique to PPMTk, which makes this enzyme distinct from the homologous counterparts. We postulate that this novel catalytic motif may enable PPMTk to isomerize phosphopentose instead of phosphohexose. To the best of our knowledge, this is the first biophysical and structural analysis of a phosphopentomutase from hyperthermophilic archaea. Less
The tripartite ATP-independent periplasmic TRAP transporters enable Vibrio cholerae and Haemophilus influenzae to acquire sialic acid aiding their colonization of human hosts This process depends on SiaP a substrate-binding protein SBP that captures and delivers sialic acid to the transporter We identified nanobodies that bind specifically to the SiaP proteins from H influenzae HiSiaP and V cholerae VcSiaP Two nanobodies inhibited sialic acid binding Detailed structural and biophysical studies of one nanobody-SBP complex revealed an allosteric inhibition mechanism preventing ligand binding and releasing pre-bound sialic acid A hydrophobic surface pocket of the SBP is crucial for the allosteric mechanism and ... More
The tripartite ATP-independent periplasmic (TRAP) transporters enable Vibrio cholerae and Haemophilus influenzae to acquire sialic acid, aiding their colonization of human hosts. This process depends on SiaP, a substrate-binding protein (SBP) that captures and delivers sialic acid to the transporter. We identified 11 nanobodies that bind specifically to the SiaP proteins from H. influenzae (HiSiaP) and V. cholerae (VcSiaP). Two nanobodies inhibited sialic acid binding. Detailed structural and biophysical studies of one nanobody-SBP complex revealed an allosteric inhibition mechanism, preventing ligand binding and releasing pre-bound sialic acid. A hydrophobic surface pocket of the SBP is crucial for the allosteric mechanism and for the conformational rearrangement that occurs upon binding of sialic acid to the SBP. Our findings provide new clues regarding the mechanism of TRAP transporters, as well as potential starting points for novel drug design approaches to starve these human pathogens of important host-derived molecules. Less
The non-polymorphic HLA-E molecule offers opportunities for new universal immunotherapeutic approaches to chronic infectious diseases Chronic Hepatitis B virus HBV infection is driven in part by T cell dysfunction due to elevated levels of the HBV envelope Env protein hepatitis B surface antigen HBsAg Here we report the characterization of three genotypic variants of an HLA-E-binding HBsAg peptide Env - identified through bioinformatic predictions and verified by biochemical and cellular assays Using a soluble affinity-enhanced T cell receptor TCR a b -anti-CD bispecific molecule to probe HLA-E presentation of the Env - peptides we demonstrate that only the most stable ... More
The non-polymorphic HLA-E molecule offers opportunities for new universal immunotherapeutic approaches to chronic infectious diseases. Chronic Hepatitis B virus (HBV) infection is driven in part by T cell dysfunction due to elevated levels of the HBV envelope (Env) protein hepatitis B surface antigen (HBsAg). Here we report the characterization of three genotypic variants of an HLA-E-binding HBsAg peptide, Env371-379, identified through bioinformatic predictions and verified by biochemical and cellular assays. Using a soluble affinity-enhanced T cell receptor (TCR) (a09b08)-anti-CD3 bispecific molecule to probe HLA-E presentation of the Env371-379 peptides, we demonstrate that only the most stable Env371-379 variant, L6I, elicits functional responses to a09b08-anti-CD3-redirected polyclonal T cells co-cultured with targets expressing endogenous HBsAg. Furthermore, HLA-E-Env371-379 L6I-specific CD8+ T cells are detectable in HBV-naïve donors and people with chronic HBV after in vitro priming. In conclusion, we provide evidence for HLA-E-mediated HBV Env peptide presentation, and highlight the effect of viral mutations on the stability and targetability of pHLA-E molecules. Less
Specificity of a T cell receptor TCR is determined by the combination of its interactions to the peptide and human leukocyte antigen HLA TCR-based therapeutic molecules have to date targeted a single peptide in the context of a single HLA allele Some peptides are presented on multiple HLA alleles and by engineering TCRs for specific recognition of more than one allele there is potential to expand the targetable patient population Here as a proof of concept we studied two TCRs S and S binding to the PRAME peptide antigen ELFSYLIEK presented by HLA alleles HLA-A and HLA-A By structure-guided affinity ... More
Specificity of a T cell receptor (TCR) is determined by the combination of its interactions to the peptide and human leukocyte antigen (HLA). TCR-based therapeutic molecules have to date targeted a single peptide in the context of a single HLA allele. Some peptides are presented on multiple HLA alleles, and by engineering TCRs for specific recognition of more than one allele, there is potential to expand the targetable patient population. Here, as a proof of concept, we studied two TCRs, S2 and S8, binding to the PRAME peptide antigen (ELFSYLIEK) presented by HLA alleles HLA-A*03:01 and HLA-A*11:01. By structure-guided affinity maturation targeting a specific residue on the HLA surface, we show that the affinity of the TCR can be modulated for different alleles. Using a combination of affinity maturation and functional T cell assay, we demonstrate that an engineered TCR can target the same peptide on two different HLA alleles with similar affinity and potency. This work highlights the importance of engineering alloselectivity for designing TCR based therapeutics suitable for differing global populations. Less
Eukaryotic innate immune systems use pattern recognition receptors to sense infection by detecting pathogen-associated molecular patterns which then triggers an immune response Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators known as bacteriophages Although different immunity proteins can recognize different phage-encoded triggers individual bacterial immunity proteins have been found to sense only a single trigger during infection suggesting a one-to-one relationship between bacterial pattern recognition receptors and their ligands Here we demonstrate that the antiphage defence protein CapRelSJ in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using ... More
Eukaryotic innate immune systems use pattern recognition receptors to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators, known as bacteriophages1,2,3,4,5,6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have been found to sense only a single trigger during infection, suggesting a one-to-one relationship between bacterial pattern recognition receptors and their ligands7,8,9,10,11. Here we demonstrate that the antiphage defence protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the notable versatility of an immune sensory domain, which may be a common property of antiphage defence systems that enables them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbour both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phages can fully evade CapRelSJ46 defence only when both triggers are mutated. Our work shows how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow the detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defence systems and complex coevolutionary relationships between them and their phage-encoded triggers. Less
Leishmania a protozoan parasite is responsible for significant morbidity and mortality worldwide manifesting as cutaneous mucocutaneous and visceral leishmaniasis These diseases pose a substantial burden especially in impoverished regions with limited access to effective medical treatments Current therapies are toxic have low efficacy and face growing resistance Understanding the metabolic pathways of Leishmania particularly those differing from its host can unveil potential therapeutic targets In this study we investigated the acetyl-CoA synthetase ACS enzyme from Leishmania infantum LiAcs which unlike many organisms also exhibits acetoacetyl-CoA synthetase KBC activity This dual functionality is unique among ANL superfamily enzymes and crucial for ... More
Leishmania, a protozoan parasite, is responsible for significant morbidity and mortality worldwide, manifesting as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases pose a substantial burden, especially in impoverished regions with limited access to effective medical treatments. Current therapies are toxic, have low efficacy, and face growing resistance. Understanding the metabolic pathways of Leishmania, particularly those differing from its host, can unveil potential therapeutic targets. In this study, we investigated the acetyl-CoA synthetase (ACS) enzyme from Leishmania infantum (LiAcs1), which, unlike many organisms, also exhibits acetoacetyl-CoA synthetase (KBC) activity. This dual functionality is unique among ANL superfamily enzymes and crucial for the parasite's reliance on leucine catabolism, energy production and sterol biosynthesis. Our biochemical characterization of LiAcs1 revealed its ability to utilize both acetate and acetoacetate substrates. Additionally, LiAcs1 displayed a distinct CoA substrate inhibition pattern, partially alleviated by acetoacetate. Structural analysis provided insights into the substrate binding flexibility of LiAcs1, highlighting a more promiscuous substrate pocket compared to other ACS or KBC-specific enzymes. Substrate mimetics elucidated its ability to accommodate both small and large AMP-ester derivatives, contributing to its dual ACS/KBC functionality. These findings not only advance our understanding of Leishmania metabolism but also present LiAcs1 as a promising drug target. The dual functionality of LiAcs1 underscores the potential for developing selective inhibitors that could disrupt critical metabolic pathways across Leishmania spp. as it appears this enzyme is highly conserved across this genus. This paves the way for developing novel effective treatments against this devastating disease. Less