425 Citations
The ribosome is a universally conserved and essential protein complex but its biogenesis in mammals is more complex than in single-celled eukaryotes To explore this added complexity we conducted a protein protein interaction screen in human cells This led to the identification of the eumetazoan-specific SPATA SPATA L CINP C ORF LCC complex as a key regulator of ribosome biogenesis Structural analyses using cryo-EM and X-ray crystallography defined the architecture of LCC Functional studies following acute depletion revealed that each component is essential for pre- S maturation Swapping endogenous LCC components with mutant versions pinpointed critical functional interactions and showed ... More
The ribosome is a universally conserved and essential protein complex, but its biogenesis in mammals is more complex than in single-celled eukaryotes. To explore this added complexity, we conducted a protein–protein interaction screen in human cells. This led to the identification of the eumetazoan-specific SPATA5–SPATA5L1–CINP–C1ORF109 (55LCC) complex as a key regulator of ribosome biogenesis. Structural analyses using cryo-EM and X-ray crystallography defined the architecture of 55LCC. Functional studies following acute depletion revealed that each component is essential for pre-60S maturation. Swapping endogenous 55LCC components with mutant versions pinpointed critical functional interactions and showed that SPATA5’s ATPase activity is more important than SPATA5L1’s. Our findings support that SPATA5 evolved from the solitary yeast ATPase Drg1 into the multiprotein 55LCC complex in metazoans. This work provides insights into the complexity of ribosome biogenesis and lays the foundation for deeper exploration of 55LCC’s role in pre-60S maturation. Less
Gram-negative bacteria pose a threat to global healthcare mainly because their outer membrane OM provides an intrinsic barrier to many antimicrobials Key to this barrier function is the asymmetric structure of the OM with phospholipids constituting the inner leaflet and lipopolysaccharides the outer leaflet Although the mechanism of phospholipid transport between the inner membrane IM and OM remains poorly understood recent studies implicate TamB YhdP and YdbH as functionally redundant proteins mediating this process in Escherichia coli Accordingly collective loss of these three paralogs is lethal and any one of them is sufficient for growth YdbH is anchored to the ... More
Gram-negative bacteria pose a threat to global healthcare mainly because their outer membrane (OM) provides an intrinsic barrier to many antimicrobials. Key to this barrier function is the asymmetric structure of the OM, with phospholipids constituting the inner leaflet and lipopolysaccharides the outer leaflet. Although the mechanism of phospholipid transport between the inner membrane (IM) and OM remains poorly understood, recent studies implicate TamB, YhdP, and YdbH as functionally redundant proteins mediating this process in Escherichia coli. Accordingly, collective loss of these three paralogs is lethal and any one of them is sufficient for growth. YdbH is anchored to the IM and its periplasmic repeating β-sheet groove domain interacts with the OM lipoprotein YnbE via β-strand augmentation to form an intermembrane bridge. Additionally, YnbE multimerizes, and the periplasmic protein YdbL is proposed to modulate YnbE multimerization to facilitate its stacking on the C-terminus of YdbH. Here, we demonstrate that excess YdbL specifically inhibits the function of the YdbH-YnbE complex since overexpression of ydbL causes lethality in the ΔyhdP ΔtamB double mutant but the presence of both ydbH and ynbE in trans abrogates this lethality. We resolve high-resolution structural data for YdbL and ascertain its interaction site with the YnbE C-terminal α-helix, with residues mediating this interface highly conserved and critical for YdbL function. Finally, we show that YdbL is protected from degradation by the protease DegP when complexed with YnbE. Overall, our data supports a model in which YdbL ensures proper YdbH-YnbE intermembrane bridge formation by directly interacting with YnbE. Less
Mycobacterium tuberculosis Mtb encodes a huge repertoire of toxin antitoxin TA systems many of which remain uncharacterized Here we report the crystal structures of the VapC toxin and VapBC TA complex at a resolution of and respectively We show that VapC adopts a unique open dimeric conformation and inhibits mycobacterial growth by cleaving tRNA at the variable or anticodon loop region Structure reveals that VapB adopts a distinct structural architecture and binds VapC with a stoichiometry respectively Interestingly binding of ssDNA activates VapB peptidase domain resulting in auto-cleavage of VapB N-terminal domain which is critical for VapBC complex formation and ... More
Mycobacterium tuberculosis (Mtb) encodes a huge repertoire of toxin–antitoxin (TA) systems, many of which remain uncharacterized. Here, we report the crystal structures of the VapC52 toxin and VapBC52 TA complex at a resolution of 2.6 and 3.2 Å, respectively. We show that VapC52 adopts a unique open dimeric conformation and inhibits mycobacterial growth by cleaving tRNA at the variable or anticodon loop region. Structure reveals that VapB52 adopts a distinct structural architecture and binds VapC52 with a 1:2 stoichiometry, respectively. Interestingly, binding of ssDNA activates VapB52 peptidase domain, resulting in auto-cleavage of VapB52 N-terminal domain which is critical for VapBC complex formation and neutralization. In addition to VapB52, co-expression of several other non-cognate VapB antitoxins abrogates the growth inhibition associated with VapC52 overexpression in Mycobacterium smegmatis (Msm) suggesting crosstalk among VapBC TA systems. Further, we demonstrate that the vapBC52 locus is dispensable for in vitro growth but essential for Mtb intracellular growth in macrophages and guinea pigs. Notably, VapC52 also cleaves mycobacteriophage D29 encoded tRNAs and confers resistance to phage infection in Msm. Taken together, we show that VapBC52 adopts a unique structural architecture, plays role in pathogenesis, and is possibly involved in mycobacterial antiphage defense mechanisms. Less
EV-A has been responsible for recent severe HFMD outbreaks We report structures for potently neutralizing human anti EV-A monoclonal antibody Fabs alone and complexed with virus Most recognize the native antigenic state with epitopes that span interfaces together covering of the capsid surface The majority of bind the canyon while the others cluster around the icosahedral two- and threefold axes Blocking SCARB receptor binding likely contributes to neutralization for all and a subset induces empty particles A predominant gene family IGHV - does not dictate a common binding pose Long CDR-H loops are frequently key to binding especially at the ... More
EV-A71 has been responsible for recent severe HFMD outbreaks. We report structures for 12 potently neutralizing human anti–EV-A71 monoclonal antibody Fabs, alone and complexed with virus. Most recognize the native antigenic state with epitopes that span interfaces, together covering 85% of the capsid surface. The majority (8 of 12) bind the canyon, while the others cluster around the icosahedral two- and threefold axes. Blocking SCARB2 receptor binding likely contributes to neutralization for all, and a subset induces empty particles. A predominant gene family (IGHV4-39) does not dictate a common binding pose. Long CDR-H3 loops are frequently key to binding, especially at the canyon, suggesting that antigenicity data based on antibodies with shorter CDR3s (e.g., murine) may be misleading. This dataset reveals neutralization mechanisms for recently circulating EV-A71 genotypes, which will inform immunotherapies. We demonstrate synergy in vitro between canyon binding and both two- and threefold binding antibodies to increase neutralization potency. Less
Ribosome stalling caused by polyproline PPs motifs is common Their translation is enhanced by accessory proteins such as YebC in bacteria whose homolog TRANSLATIONAL ACTIVATOR OF CYTOCHROME C OXIDASE TACO aids the translation of mitochondria-encoded proteins The prevalence of PP motifs across plastid-encoded genes and their impact on the translation of photosynthesis-relevant proteins remains unexplored Equally a translation-enhancer of PP motifs equivalent to TACO for plastid ribosomes has not been reported Here we show that plastid genomes encode proteins with a minimum of one PP motif on average half of which are conserved in their cyanobacterial homologs and that the ... More
Ribosome stalling caused by polyproline (PPs) motifs is common. Their translation is enhanced by accessory proteins such as YebC in bacteria, whose homolog, TRANSLATIONAL ACTIVATOR OF CYTOCHROME C OXIDASE 1 (TACO1), aids the translation of mitochondria-encoded proteins. The prevalence of PP motifs across plastid-encoded genes and their impact on the translation of photosynthesis-relevant proteins remains unexplored. Equally, a translation-enhancer of PP motifs equivalent to TACO1 for plastid ribosomes has not been reported. Here, we show that plastid genomes encode 24 proteins with a minimum of one PP motif on average, half of which are conserved in their cyanobacterial homologs, and that the vast majority of eukaryotes, including plants, encode a single TACO1 that we demonstrate to be dually targeted to mitochondria and plastids of Marchantia polymorpha. We resolved the MpTACO1 structure at 2.34 Å by X-ray crystallography and the flexibility by small-angle X-ray scattering. Through modelling, we demonstrate that MpTACO1 can fit into the peptidyl transfer centre of plant chlororibosomes in a similar manner as human TACO1 in the mitoribosome. The identification and structure determination of the first plastid-targeted YebC/TACO1 allows us to sketch a unified model for the function and evolution of this ancient family of ribosomal accessory proteins, underscoring their indispensable role in the translation of bioenergetic membrane proteins reaching back almost 4 billion years. Less
Linking of fragments in neighboring binding sites is one of the optimization strategies in fragment-based drug discovery where additive or even more substantial bioactivity improvements can be realized However such efforts present a considerable challenge when one fragment binds covalently to the target protein as small modifications can influence the correct positioning of the covalent warhead toward the targeted nucleophilic residue Here we present a case study of fragment linking that yielded single-digit micromolar covalent inhibitors of the SARS-CoV- main protease starting from fragments that were inactive in the biochemical assay Using structural information from a recent high-throughput crystallographic fragment ... More
Linking of fragments in neighboring binding sites is one of the optimization strategies in fragment-based drug discovery, where additive or even more substantial bioactivity improvements can be realized. However, such efforts present a considerable challenge when one fragment binds covalently to the target protein, as small modifications can influence the correct positioning of the covalent warhead toward the targeted nucleophilic residue. Here, we present a case study of fragment linking that yielded single-digit micromolar, covalent inhibitors of the SARS-CoV-2 main protease, starting from fragments that were inactive in the biochemical assay. Using structural information from a recent, high-throughput crystallographic fragment screen, we show that the success of fragment linking in the design of targeted covalent inhibitors is heavily impacted by several factors, including the warhead type, the labeling chemistry, and even subtle changes in the designed linker. Notably, we observe that induced fit effects might override the original fragment orientations in the linked molecule, highlighting the need for reliable structure verification, especially in consecutive rounds of fragment elaboration. Less
To establish infection phytopathogens deploy effectors to compromise host defences and facilitate invasive growth As part of this the battle for control of symplastic connectivity via plasmodesmata is a key determinant of infection outcomes yet little is known about how fungal effectors directly exploit these channels and in turn how hosts defend them Here we have identified ChEC as a plasmodesmal-targeting cell-to-cell mobile effector from the anthracnose fungus Colletotrichum higginsianum ChEC binds the plasmodesmal protein HEAVY METAL-ASSOCIATED HMA ISOPRENYLATED PLANT PROTEIN HIPP from Arabidopsis via a tetrahedral metal ion coordination site with either of its HMA domains Constitutive in planta ... More
To establish infection, phytopathogens deploy effectors to compromise host defences and facilitate invasive growth. As part of this, the battle for control of symplastic connectivity via plasmodesmata is a key determinant of infection outcomes, yet little is known about how fungal effectors directly exploit these channels, and in turn, how hosts defend them. Here, we have identified ChEC108 as a plasmodesmal-targeting, cell-to-cell mobile effector from the anthracnose fungus, Colletotrichum higginsianum. ChEC108 binds the plasmodesmal protein HEAVY METAL-ASSOCIATED (HMA) ISOPRENYLATED PLANT PROTEIN 6 (HIPP6) from Arabidopsis via a tetrahedral metal ion coordination site with either of its HMA domains. Constitutive in planta expression of ChEC108 induces plasmodesmal closure and the upregulation of defence-associated genes in a manner dependent on its capacity to bind HIPP6. Further, HIPP6 binding impairs cell-to-cell mobility of ChEC108. Alongside the finding that loss of ChEC108 favoured C. higginsianum infection, this suggests ChEC108-HIPP6 interaction at plasmodesmata positively regulates defence. Less
Background and objectives The PWWP domain of lens epithelium-derived growth factor p LEDGF p mediates chromatin engagement through recognition of histone H lysine di- and trimethylation H K me and nucleosomal DNA LEDGF p plays a role in multiple human diseases In particular its interaction with HIV- integrase enables viral genome integration However the LEDGF PWWP domain remains difficult to target with small molecules as it lacks optimally shaped binding pockets Here we report the generation of high-affinity nanobodies Nbs to investigate the structure and function of this domain Methods Camelids were immunized with recombinant LEDGF PWWP domain and immune ... More
Background and objectives: The PWWP domain of lens epithelium-derived growth factor p75 (LEDGF/p75) mediates chromatin engagement through recognition of histone H3 lysine 36 di- and trimethylation (H3K36me2/3) and nucleosomal DNA. LEDGF/p75 plays a role in multiple human diseases. In particular, its interaction with HIV-1 integrase enables viral genome integration. However, the LEDGF PWWP domain remains difficult to target with small molecules as it lacks optimally shaped binding pockets. Here we report the generation of high-affinity nanobodies (Nbs) to investigate the structure and function of this domain. Methods: Camelids were immunized with recombinant LEDGF PWWP domain, and immune phage display libraries were screened for affinity. Selected Nbs were recombinantly expressed in E. coli and purified. Their interaction with the PWWP domain of LEDGF and its close homolog HRP-2 was characterized using size-exclusion chromatography and surface plasmon resonance. Structural characterization of the Nbs was performed by X-ray crystallography. Functional effects on chromatin engagement were evaluated using the AlphaScreen assay. Results: Nine sequence-distinct Nbs were identified, seven of which were confirmed to bind the LEDGF PWWP domain with nanomolar affinities. Five Nbs also bound the HRP-2 domain, consistent with conserved functional surfaces, while two showed reduced affinity. Crystal structures of two Nbs (NbC03 and NbH10) confirmed canonical immunoglobulin folds, while the latter additionally revealed a domain-swapped dimer. Moreover, NbH10 dose-dependently inhibited the interaction between full-length LEDGF/p75 and H3K36me3-modified nucleosomes in vitro. Conclusions: This work establishes a validated panel of Nbs targeting the LEDGF PWWP domain and demonstrates their ability to functionally disrupt the LEDGF-chromatin interaction. These Nbs serve as valuable tools towards functional studies and structure-based drug design. Less
Visualizing and manipulating proteins in live cells is crucial for studying complex biological processes Self-labelling protein SLP tags such as HaloTag and SNAP-tag are widely used for protein labelling and new systems are needed to expand multiplexing capabilities and broaden the scope of applications Here we present BromoCatch a small kDa bromodomain BD -based SLP platform engineered with a nucleophilic cysteine for covalent ligand engagement A structure-based designed library of electrophilic ligands was screened against two cysteine-containing mutants using differential scanning fluorimetry and intact protein mass spectrometry to assess covalent complex formation We identified a para-acrylamide bumped derivative MR and ... More
Visualizing and manipulating proteins in live cells is crucial for studying complex biological processes. Self-labelling protein (SLP) tags such as HaloTag and SNAP-tag are widely used for protein labelling, and new systems are needed to expand multiplexing capabilities and broaden the scope of applications. Here we present BromoCatch, a small ~13 kDa bromodomain (BD)-based SLP platform, engineered with a nucleophilic cysteine for covalent ligand engagement. A structure-based designed library of electrophilic ligands was screened against two cysteine-containing mutants using differential scanning fluorimetry and intact protein mass spectrometry to assess covalent complex formation. We identified a para-acrylamide bumped derivative MR116 and the Brd4-BD2 double mutant L387A,E438C as the optimal protein-ligand pair, and reveal the binding mode through an X-ray co-crystal structure solved to 1.3 Å resolution. BromoCatch demonstrated potent and irreversible cellular target engagement in NanoBRET and residence-time assays. Its versatility was demonstrated through the design of a biotinylated conjugate, PROTAC-based degraders, and fluorescent full-on and “switch-on” probes for ex-cellulo and live-cell imaging, including side-by-side comparison and orthogonality with HaloTag. Together, these results establish BromoCatch as a robust, modular, and orthogonal SLP tool with broad potential for multiplexed labelling and targeted protein manipulation. Less
Conversion of prothrombin to thrombin occurs in the final step of the blood coagulation cascade and depends on association of the serine protease factor f Xa and the cofactor fVa on activated cell surfaces to form the prothrombinase complex Prothrombinase cleaves prothrombin at two sites in a processive manner -times faster than fXa on its own How fVa confers rapid and processive cleavage of prothrombin is an enzymatical mystery with profound consequence We created a variant of fXa that binds to fVa with high affinity in the absence of phospholipids that preserves the activity of wild-type prothrombinase and recently reported ... More
Conversion of prothrombin to thrombin occurs in the final step of the blood coagulation cascade and depends on association of the serine protease, factor (f) Xa, and the cofactor fVa on activated cell surfaces to form the prothrombinase complex. Prothrombinase cleaves prothrombin at two sites in a processive manner ∼500,000-times faster than fXa on its own. How fVa confers rapid and processive cleavage of prothrombin is an enzymatical mystery with profound consequence. We created a variant of fXa that binds to fVa with high affinity in the absence of phospholipids that preserves the activity of wild-type prothrombinase, and recently reported on the cryo-EM structure of the complex. It revealed an extensive interface between the two proteins, including a critical interaction between the first acidic region C-terminal to the A2 domain of fVa (the N-terminal portion of the a2-loop) with the heparin binding site of fXa. Here we present the cryo-EM structures of prothrombinase bound to prothrombin and the intermediate meizothrombin, both to 3.1 Å resolution. The prothrombin complex revealed a surprising interaction between the second acidic region of the a2-loop with exosite I of prothrombin, accounting for 70% of the total buried surface area. Cleavage at Arg320 triggers the zymogen-to-protease conformational change in meizothrombin which alters all domain-domain and fVa interactions, and results in the presentation of the second cleavage site (Arg271) for processing. Together, these structures reveal a remarkable enzymatic mechanism that depends on the active participation of the substrate itself, and introduce the new paradigm of substrate allostery. Less
The evolution of transcription factor TF DNA-binding specificity is a major driver of gene regulatory innovation Unlike most TFs which diversify through gene duplication and neofunctionalization the plant-specific LEAFY LFY TF evolved novel binding specificities without extensive duplication Here we combine experimental structural determination and biochemical assays to reveal how LFY s dimerization and DNA-binding preferences shifted during the water-to-land transition We present crystal structures of the LFY DNA-binding domain DBD from the hornwort Nothoceros aenigmaticus and the alga Interfilum paradoxum bound to DNA demonstrating two distinct dimerization mechanisms one mediated by direct protein-protein interactions and another driven by DNA-mediated ... More
The evolution of transcription factor (TF) DNA-binding specificity is a major driver of gene regulatory innovation. Unlike most TFs, which diversify through gene duplication and neofunctionalization, the plant-specific LEAFY (LFY) TF evolved novel binding specificities without extensive duplication. Here, we combine experimental structural determination and biochemical assays to reveal how LFY’s dimerization and DNA-binding preferences shifted during the water-to-land transition. We present crystal structures of the LFY DNA-binding domain (DBD) from the hornwort Nothoceros aenigmaticus and the alga Interfilum paradoxum bound to DNA, demonstrating two distinct dimerization mechanisms: one mediated by direct protein-protein interactions and another driven by DNA-mediated cooperativity. In the ancestral state, LFY likely bound DNA as a dimer through DNA-mediated cooperativity, with protein-protein dimerization emerging later, enforcing new DNA-binding preferences. Our findings support a revised evolutionary scenario for LFY, highlighting the dynamic interplay between protein-DNA and protein-protein interactions as key drivers of TF binding specificity. This work deepens our understanding of how structural adaptations in TFs underpin evolutionary transitions in gene regulation. Less
AXIS: a Lab-in-the-Loop machine learning approach for automated detection of macromolecular crystals
Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design Nowadays the use of robotic systems for crystal growth and diffraction analysis is widespread and high-throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field However the identification of crystals is still largely carried out through manual inspection sometimes involving tens of thousands of images which represents a bottleneck in an otherwise highly automated process Here we describe AXIS an AI-based Crystal Identification System combining the DINOv computer vision model state-of-the-art transfer learning and MARCO the largest crystallization dataset available to date for ... More
Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design. Nowadays, the use of robotic systems for crystal growth and diffraction analysis is widespread and high-throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field. However, the identification of crystals is still largely carried out through manual inspection, sometimes involving tens of thousands of images, which represents a bottleneck in an otherwise highly automated process. Here we describe AXIS, an AI-based Crystal Identification System combining the DINOv2 computer vision model, state-of-the-art transfer learning and MARCO, the largest crystallization dataset available to date, for automated crystal detection. AXIS can operate with both visible and UV light images and integrates a Lab-in-the-Loop approach combining ML and expert inputs for iterative learning and specialization. AXIS enables automated annotation of large crystallization image datasets with performance and accuracy comparable to that of human experts, and the Lab-in-the-Loop approach introduced here enables efficient adaptation to local conditions, facilitating widespread application, which has been a major limitation to date. AXIS can help to correct human errors in image annotation and removes critical bottlenecks, particularly in the context of extensive crystallization screens or high-throughput applications like fragment and ligand screening, unlocking the potential for higher levels of automation that are key in both fundamental and translational research. Less
The advancement of single-crystal structural analysis has emerged as a pivotal technology surpassing spectroscopic methods in revealing the intricate structural details of organic small molecules including crystal packing and stereochemical configurations It plays a critical role across scientific domains such as chemistry biology agronomy and medicine Traditional single-crystal X-ray diffraction SCXRD has always been restricted by its stringent requirements on the physical state size and quality of crystals This review discusses the arsenal of equipment and theoretical techniques for obtaining single-crystal structures including SCXRD PXRD CSP and more recently Micro-ED It further explores the significant crystal growth techniques based on ... More
The advancement of single-crystal structural analysis has emerged as a pivotal technology surpassing spectroscopic methods in revealing the intricate structural details of organic small molecules, including crystal packing and stereochemical configurations. It plays a critical role across scientific domains such as chemistry, biology, agronomy, and medicine. Traditional single-crystal X-ray diffraction (SCXRD) has always been restricted by its stringent requirements on the physical state, size, and quality of crystals. This review discusses the arsenal of equipment and theoretical techniques for obtaining single-crystal structures, including SCXRD, PXRD & CSP, and more recently, Micro-ED. It further explores the significant crystal growth techniques based on three foundational methods: solution-based crystallization, melt crystallization, and sublimation crystallization. Detailed discussion is provided on the crystallizability of molecules and the refinement of crystal growth methods. Specifically, for crystallizable analytes, a combination of crystal growth enhancement techniques and high-throughput technologies (under-oil) can compensate for poor crystallinity, small size, and defects under normal conditions. For molecules inherently resistant to crystallization, a “crystallization chaperone”, such as a MOFs as a crystalline sponge or tetraaryladamantane as a cocrystallization chaperone, can determine absolute configurations. Looking ahead, this review emphasizes the potential of artificial intelligence and machine learning approaches for crystal growth and structural prediction. The development of integrated analysis strategies combining SCXRD, PXRD, and Micro-ED is identified as a future trend for providing comprehensive structural insights. This review highlights the significance of advancements in single-crystal structural analysis techniques, paving the way for groundbreaking innovations in molecular design and materials science, and predicts a bright future for the field with new technologies. Less
Filament-forming proteins such as TasA Bacillus subtilis and camelysins CalY CalY Bacillus cereus pose a particular challenge for structural analysis due to their strong tendency to self-association and their polydispersity which severely limits their ability to crystallize or to be a target for NMR-spectroscopy To address this it is necessary to modify the amino acid sequence to prevent filamentation Engineering a series of N- and C-terminal truncated variants by removing flexible parts is often key to success N-terminal extensions are also a powerful tool for obtaining crystals of fiber-forming proteins
Transformation of agro-industrial products into value-added products such as prebiotic oligosaccharides is a key element of the emerging bioeconomy Here we characterized a new GH glucuronoxylanase from Bacillus pumilus BpXyn A for its potential in producing xylooligosaccharides XOS BpXyn A showed tolerance to ethanol and NaCl and released both linear and branched XOS containing MeGlcA at the penultimate nonreducing end residue Its X-ray structure determined at resolution revealed high similarity to other glucuronoxylanases Furthermore BpXyn A achieved higher xylan conversion yields from corn cob and Eucalyptus sawdust than Ruminococcus champanellensisRcXyn A Finally fermentation assays showed that Bifidobacterium adolescentis metabolized neutral ... More
Transformation of agro-industrial products into value-added products, such as prebiotic oligosaccharides, is a key element of the emerging bioeconomy. Here, we characterized a new GH30_8 glucuronoxylanase from Bacillus pumilus (BpXyn30_8A) for its potential in producing xylooligosaccharides (XOS). BpXyn30_8A showed tolerance to ethanol and NaCl and released both linear and branched XOS containing MeGlcA at the penultimate nonreducing end residue. Its X-ray structure, determined at 2.16 Å resolution, revealed high similarity to other glucuronoxylanases. Furthermore, BpXyn30_8A achieved higher xylan conversion yields from corn cob and Eucalyptus sawdust than Ruminococcus champanellensisRcXyn30A. Finally, fermentation assays showed that Bifidobacterium adolescentis metabolized neutral XOS to acetate and lactate, whereas acidic XOS were poorly utilized. These results highlight the potential of BpXyn30_8A as a valuable enzyme for the green transformation of plant biomass into prebiotic oligosaccharides with promising applications in human and animal nutrition, health, and biotechnology. Less
Mitochondrial crista junctions CJs operate as regulated gateways into the cristae microenvironment whose protein metabolite and ion compositions are finely tuned for mitochondrial function The Mic -Mic complex of the mitochondrial contact site and cristae organizing system MICOS complex was suggested to span across CJs and act as a diffusion barrier but little is known of how its dynamic architecture facilitates this task To address this open question we determined the crystal structure of an amino-terminal dimeric helical bundle of human Mic These and previous structural and biochemical data were harnessed in molecular dynamic MD simulations to develop a dynamic ... More
Mitochondrial crista junctions (CJs) operate as regulated gateways into the cristae microenvironment, whose protein, metabolite, and ion compositions are finely tuned for mitochondrial function. The Mic60-Mic19 complex of the mitochondrial contact site and cristae organizing system (MICOS) complex was suggested to span across CJs and act as a diffusion barrier, but little is known of how its dynamic architecture facilitates this task. To address this open question, we determined the crystal structure of an amino-terminal dimeric helical bundle of human Mic60. These and previous structural and biochemical data were harnessed in molecular dynamic (MD) simulations to develop a dynamic model of the human tetrameric Mic60-Mic19 subcomplex in the CJ environment, to validate its architecture using in organello cross-linking data and to computationally characterize its function as a diffusion barrier. Our integrative structural biology approach enables the functional investigation of flexible, multidomain protein complexes which escape conventional structural biology methods. Less
TIR Toll interleukin- receptor domains are found in proteins involved in immunity pathways in organisms ranging from humans and plant to bacteria Bacterial TIR domain-containing proteins have been shown to contribute to pathogenicity and anti-viral activity of bacteria During bacterial infection some TIR domain-containing proteins act as virulence factors to inhibit immune responses by interfering with Toll-like receptor signalling Other bacterial TIR domain-containing proteins are involved in bacterial anti-viral defence Many of TIR domain-containing proteins have been shown to have NAD cleavage activity and relevant to host cell death and bacterial anti-phage defence system In addition some TIR domain-containing proteins ... More
TIR (Toll/interleukin-1 receptor) domains are found in proteins involved in immunity pathways in organisms ranging from humans and plant to bacteria. Bacterial TIR domain-containing proteins have been shown to contribute to pathogenicity and anti-viral activity of bacteria. During bacterial infection, some TIR domain-containing proteins act as virulence factors to inhibit immune responses by interfering with Toll-like receptor signalling. Other bacterial TIR domain-containing proteins are involved in bacterial anti-viral defence. Many of TIR domain-containing proteins have been shown to have NAD+ cleavage activity and relevant to host cell death and bacterial anti-phage defence system. In addition, some TIR domain-containing proteins have been reported that has DNA binding activity. Here, we report our studies on two bacterial TIR domain-containing proteins: AbTir and PumA. AbTir (Acinetobacter baumannii TIR domain-containing) is one of the few bacterial proteins that has been reported to produce a variant of cyclic ADPR (ADP ribose) after NAD+ cleavage. Previous study in our lab determined the crystal structure of AbTir TIR domain in its monomeric form and the chemical structure of the cyclic ADPR it produces (termed 2’cADPR). However, we could not find the significant NAD+ binding pocked in the AbTirTIR crystal structure. PumA is a TIR domain-containing protein from the multi-drug resistant pathogen Pseudomonas aeruginosa PA7 is essential for its virulence. Study shows that PumA can block host’s immune signalling pathway
In Chapter 2, we demonstrated that PumA, like AbTir, has NAD⁺ cleavage activity and forms filaments upon 3AD binding. We used nanobodies to stabilize PumA and successfully obtained several PumA:nanobody complex crystals. NADase assays showed that some nanobodies significantly inhibit its enzymatic activity, offering potential inhibitory tools.
In Chapter 3, we extended the structural analysis of PumA. We solved the crystal structure of a PumA mutant and characterized several nanobody-bound complexes. Cryo-EM and MicroED efforts revealed filament formation in the presence of 3AD, but bundling limited resolution. Nanobodies again showed inhibitory effects on NADase activity.
In Chapter 4, we investigated DNA binding by bacterial TIR proteins. We found that AbTir lacks DNA-binding activity, while PumA and its homologs from other pathogens bind DNA and form filaments upon DNA interaction. Cryo-EM confirmed DNA-induced filament formation by PumA, suggesting a role in nucleic acid sensing.
Overall, my project shows bacterial TIR domain-containing proteins assemblies in bacterial virulence and antiviral defense, and to identify potential small-molecule inhibitors targeting these mechanisms. Less
In Chapter 2, we demonstrated that PumA, like AbTir, has NAD⁺ cleavage activity and forms filaments upon 3AD binding. We used nanobodies to stabilize PumA and successfully obtained several PumA:nanobody complex crystals. NADase assays showed that some nanobodies significantly inhibit its enzymatic activity, offering potential inhibitory tools.
In Chapter 3, we extended the structural analysis of PumA. We solved the crystal structure of a PumA mutant and characterized several nanobody-bound complexes. Cryo-EM and MicroED efforts revealed filament formation in the presence of 3AD, but bundling limited resolution. Nanobodies again showed inhibitory effects on NADase activity.
In Chapter 4, we investigated DNA binding by bacterial TIR proteins. We found that AbTir lacks DNA-binding activity, while PumA and its homologs from other pathogens bind DNA and form filaments upon DNA interaction. Cryo-EM confirmed DNA-induced filament formation by PumA, suggesting a role in nucleic acid sensing.
Overall, my project shows bacterial TIR domain-containing proteins assemblies in bacterial virulence and antiviral defense, and to identify potential small-molecule inhibitors targeting these mechanisms. Less
Spermine a pivotal player in biomolecular condensation and diverse cellular processes has emerged as a focus of investigation in aging neurodegeneration and other diseases Despite its significance the mechanistic details of spermine remain incompletely understood Here we describe the distinct modulation by spermine on Alzheimer s Tau and Parkinson s -synuclein elucidating their condensation behaviors in vitro and in vivo Using biophysical techniques including time-resolved SAXS and NMR we trace electrostatically driven transitions from atomic-scale conformational changes to mesoscopic structures Notably spermine extends lifespan ameliorates movement deficits and restores mitochondrial function in C elegans models expressing Tau and -synuclein Acting ... More
Spermine, a pivotal player in biomolecular condensation and diverse cellular processes, has emerged as a focus of investigation in aging, neurodegeneration, and other diseases. Despite its significance, the mechanistic details of spermine remain incompletely understood. Here, we describe the distinct modulation by spermine on Alzheimer’s Tau and Parkinson’s α-synuclein, elucidating their condensation behaviors in vitro and in vivo. Using biophysical techniques including time-resolved SAXS and NMR, we trace electrostatically driven transitions from atomic-scale conformational changes to mesoscopic structures. Notably, spermine extends lifespan, ameliorates movement deficits, and restores mitochondrial function in C. elegans models expressing Tau and α-synuclein. Acting as a molecular glue, spermine orchestrates in vivo condensation of α-synuclein, influences condensate mobility, and promotes degradation via autophagy, specifically through autophagosome expansion. This study unveils the interplay between spermine, protein condensation, and functional outcomes, advancing our understanding of neurodegenerative diseases and paving the way for therapeutic development. Less
Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design Nowadays the use of robotic systems for crystal growth and diffraction analysis is widespread and high throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field However the identification of crystals is still largely carried out through manual inspection sometimes involving tens of thousands of images which represents a bottleneck in an otherwise highly automated process Here we describe AXIS an AI-based Crystal Identification System combining the DINOv computer vision model state-of-the-art transfer learning and MARCO the largest crystallization dataset available to date ... More
Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design. Nowadays, the use of robotic systems for crystal growth and diffraction analysis is widespread and high throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field. However, the identification of crystals is still largely carried out through manual inspection, sometimes involving tens of thousands of images, which represents a bottleneck in an otherwise highly automated process. Here we describe AXIS, an AI-based Crystal Identification System combining the DINOv2 computer vision model, state-of-the-art transfer learning and MARCO, the largest crystallization dataset available to date, for automated crystal detection. AXIS can operate both with visible and UV light images and integrates a Lab-In-The-Loop approach combining ML and expert inputs for continuous learning and specialization. AXIS enables automated annotation of large crystallization image datasets with performance and accuracy comparable to that of human experts and the Lab-In-The-Loop approach introduced here enables efficient adaptation to local conditions facilitating widespread application, which has been a major limitation to date. AXIS can help correct human errors in image annotation and removes critical bottlenecks, particularly in the context of extensive crystallization screens or high throughput applications like fragment and ligand screening unlocking the potential for higher levels of automation that are key both in fundamental and translational research. Less
Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase composed of factor Xa fXa and fVa The affinity of fXa for fVa is low with assembly and function dependent on phospholipid PL membranes Some snakes have evolved venom versions of fXa that bind to fVa with high affinity and efficiently activate prothrombin in the absence of PL We created a similar high-affinity PL-independent human prothrombinase with mutations to human fXa M The increase in affinity enabled cryogenic electron microscopy cryo-EM structure determination of M -prothrombinase to a resolution of All protein domains were well resolved ... More
Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase, composed of factor Xa (fXa) and fVa. The affinity of fXa for fVa is low, with assembly and function dependent on phospholipid (PL) membranes. Some snakes have evolved venom versions of fXa that bind to fVa with high affinity and efficiently activate prothrombin in the absence of PL. We created a similar high-affinity, PL-independent human prothrombinase with 17 mutations to human fXa (M17). The increase in affinity enabled cryogenic electron microscopy (cryo-EM) structure determination of M17-prothrombinase to a resolution of 3.3 Å. All protein domains were well resolved in the map, except for the Gla domain of fXa. The main contacts involve the serine protease and EGF2 domains of fXa and the A2 and A3 domains of fVa, resulting in the burying of a total surface area of 4,900 Å2. The map is of sufficient quality to resolve side chain interactions, including several key M17 mutations. To aid in the placement of the loop Cterminal to the A2 domain (a2-loop), we solved a high-resolution crystal structure of fXa in complex with a synthetic a2 peptide. The acidic a2-loop interacts with the basic heparin binding site of fXa, involving a conserved antiparallel -strand interaction. The M17-prothrombinase structure is compatible with data from biochemical and mutagenesis research and provides important new insights into the assembly and function of the prothrombinase complex. Less
The enzymatic degradation of polyethylene terephthalate PET offers a sustainable solution for PET recycling Over the past two decades more than PETases have been characterized primarily exhibiting similar sequences and structures Here we report new PET-degrading hydrolases including HaloPETase from the marine Halopseudomonas lineage thereby extending the narrow sequence space by novel features at the active site The crystal structure of HaloPETase was determined to a resolution of revealing a unique active site architecture and a lack of the canonical -stacking clamp found in PETases so far Further variations in active site composition and loop structures were observed Additionally we ... More
The enzymatic degradation of polyethylene terephthalate (PET) offers a sustainable solution for PET recycling. Over the past two decades, more than 100 PETases have been characterized, primarily exhibiting similar sequences and structures. Here, we report new PET-degrading α/β hydrolases, including HaloPETase1 from the marine Halopseudomonas lineage, thereby extending the narrow sequence space by novel features at the active site. The crystal structure of HaloPETase1 was determined to a resolution of 1.16 Å, revealing a unique active site architecture and a lack of the canonical π-stacking clamp found in PETases so far. Further, variations in active site composition and loop structures were observed. Additionally, we found five more enzymes from the same lineage, two of which have a high similarity to type IIa bacterial PETases, while the other three resemble HaloPETase1. All these enzymes exhibited high salt tolerance ranging from 2.5 to 5 M NaCl leading to higher total product releases upon PET degradation at 40 or 50 °C. Based on these findings, we propose an extension of the existing PETase classification system to include type III PETases. Less
Efficient drug discovery relies on workflows that integrate structural insights with rapid and cost-effective exploration of chemical space Here we present a data-driven fragment-based lead discovery approach to target Neuronal Calcium Sensor NCS- protein-protein interactions PPIs This study represents a complete implementation of a single high-value design-make-test-analyze cycle that directly yields compounds with micromolar affinity with the potential to modulate NCS- interactions with key targets including the G-protein chaperone Ric- A and the dopamine D and cannabinoid CB receptors X-ray crystallographic fragment screening CFS revealed diverse interaction patterns within the NCS- hydrophobic crevice Algorithmically guided fragment evolution and automated synthesis ... More
Efficient drug discovery relies on workflows that integrate structural insights with rapid and cost-effective exploration of chemical space. Here, we present a data-driven fragment-based lead discovery approach to target Neuronal Calcium Sensor 1 (NCS-1) protein-protein interactions (PPIs). This study represents a complete implementation of a single high-value design-make-test-analyze cycle that directly yields compounds with micromolar affinity with the potential to modulate NCS-1 interactions with key targets, including the G-protein chaperone Ric-8A and the dopamine D2 and cannabinoid CB1 receptors. X-ray crystallographic fragment screening (CFS) revealed diverse interaction patterns within the NCS-1 hydrophobic crevice. Algorithmically guided fragment evolution and automated synthesis enabled the rapid generation of over 250 derivatives, with biophysical validation using LC-MS and Grating-coupled interferometry. Structural analyses highlighted key pharmacophores, with selected compounds exhibiting favorable drug-like properties and potential blood-brain barrier penetration, making them promising candidates for neurodegenerative and neurodevelopmental disorders. Our results demonstrate the feasibility of accelerated hit-to-lead development at synchrotrons, demonstrating a robust, scalable platform for PPI-targeting drug discovery. The generated chemically diverse scaffolds provide a strong foundation for future therapeutic optimization. Less
Deep learning has revolutionized soluble protein design yet de novo transmembrane TM protein engineering remains hindered by scarce structural data complex membrane-specific interactions and conformational dynamics We developed TMDiffusion TMDF a joint all-heavy-atom sequence structure diffusion model trained to capture the full interaction diversity of natural TM proteins including weak and polar contact networks TMDF designs diverse TM architectures associating domains inhibitors and conformational switches in a single step achieving experimental success A crystal structure of designed proteins matches predictions with atomic accuracy Leveraging TMDF we built synthetic single-pass receptors whose de novo TM domains toggle between conformations enabling precise ... More
Deep learning has revolutionized soluble protein design, yet de novo transmembrane (TM) protein engineering remains hindered by scarce structural data, complex membrane-specific interactions and conformational dynamics. We developed TMDiffusion (TMDF), a joint all-heavy-atom sequence–structure diffusion model trained to capture the full interaction diversity of natural TM proteins, including weak and polar contact networks. TMDF designs diverse TM architectures—associating domains, inhibitors, and conformational switches—in a single step, achieving >70% experimental success. A crystal structure of designed proteins matches predictions with atomic accuracy. Leveraging TMDF, we built synthetic single-pass receptors whose de novo TM domains toggle between conformations, enabling precise control of signalling outputs consistent with predicted equilibria. These results show that membrane-adapted DL models can accurately encode and program TM association energetics and conformations. TMDF establishes a general framework for bottom-up design of TM proteins with programmable functions, advancing both mechanistic studies of membrane proteins and development of next-generation therapeutics. Less
This protocol describes the crystallization of Enterovirus EV- A protease mutant C A containing the VP - A junction in the active site The crystals form within - hours using a crystallization screen composed of M NaCl and ethanol The crystal structure was determined using X-ray diffraction resulting in hexagonal prism crystals in space group P with unit cell dimensions of and an average resolution of The protein was expressed using the plasmid Enterovirus Coxsackievirus A A protease
This protocol describes the crystallization of Enterovirus EV- A protease mutant C A containing the VP - A junction in the active site The crystals form within - hours using a crystallization screen composed of M NaCl and ethanol The crystal structure was determined using X-ray diffraction resulting in hexagonal prism crystals in space group P with unit cell dimensions of and an average resolution of The protein was expressed using the plasmid Enterovirus Coxsackievirus A A protease
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
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
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
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
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
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
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
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
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
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
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
A group of three deep learning tools referred to collectively as CHiMP Crystal Hits in My Plate were created for analysis of micrographs of protein crystallisation experiments at the Diamond Light Source DLS synchrotron UK The first tool a classification network assigns images into categories relating to experimental outcomes The other two tools are networks that perform both object detection and instance segmentation resulting in masks of individual crystals in the first case and masks of crystallisation droplets in addition to crystals in the second case allowing positions and sizes of these entities to be recorded Creation of these tools ... More
A group of three deep learning tools, referred to collectively as CHiMP (Crystal Hits in My Plate) were created for analysis of micrographs of protein crystallisation experiments at the Diamond Light Source (DLS) synchrotron, UK. The first tool, a classification network, assigns images into categories relating to experimental outcomes. The other two tools are networks that perform both object detection and instance segmentation, resulting in masks of individual crystals in the first case, and masks of crystallisation droplets in addition to crystals in the second case, allowing positions and sizes of these entities to be recorded. Creation of these tools used transfer learning, where weights from a pre-trained deep learning network were used as a starting point and re-purposed by further training on a relatively small set of data. Two of the tools are now integrated at the VMXi macromolecular crystallography beamline at DLS where they absolve the need for any user input both for monitoring crystallisation experiments and for triggering in situ data collections. The third is being integrated into the XChem fragment-based drug discovery screening platform, also at DLS, to allow automatic targeting of acoustic compound dispensing into crystallisation droplets. Less
-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharo lyticus Bgl has been denoted as having an attractive catalytic profile for various industrial applications Bgl catalyses the final step of in the decomposition of cellulose an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere With the aim of enhancing the thermostability of Bgl for a broad spectrum of biotechnological processes it has been subjected to structural studies Crystal structures of Bgl and its complex with glucose were determined at and resolution respectively Bgl ... More
β-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus (Bgl1) has been denoted as having an attractive catalytic profile for various industrial applications. Bgl1 catalyses the final step of in the decomposition of cellulose, an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere. With the aim of enhancing the thermostability of Bgl1 for a broad spectrum of biotechnological processes, it has been subjected to structural studies. Crystal structures of Bgl1 and its complex with glucose were determined at 1.47 and 1.95 Å resolution, respectively. Bgl1 is a member of glycosyl hydrolase family 1 (GH1 superfamily, EC 3.2.1.21) and the results showed that the 3D structure of Bgl1 follows the overall architecture of the GH1 family, with a classical (β/α)8 TIM-barrel fold. Comparisons of Bgl1 with sequence or structural homologues of β-glucosidase reveal quite similar structures but also unique structural features in Bgl1 with plausible functional roles. Less