901 Citations
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
Interactions between plants and pathogens drive long-term co-evolution through cycles of effector diversification and immune recognition Effectors play central roles in this molecular interplay and zinc-binding folds have been identified in a subset of pathogen effectors yet the contribution of zinc coordination to effector stability and immune recognition remains unclear Here we investigated the structure and recognition of the AvrSr effector from the wheat stem rust fungus Puccinia graminis f sp tritici Pgt Structural and biochemical analyses show that AvrSr adopts a fold containing two zinc-binding sites Transient expression assays in Nicotiana benthamiana show that AvrSr directly interacts with Sr ... More
Interactions between plants and pathogens drive long-term co-evolution through cycles of effector diversification and immune recognition. Effectors play central roles in this molecular interplay, and zinc-binding folds have been identified in a subset of pathogen effectors, yet the contribution of zinc coordination to effector stability and immune recognition remains unclear.
Here, we investigated the structure and recognition of the AvrSr33 effector from the wheat stem rust fungus (Puccinia graminis f. sp. tritici, Pgt). Structural and biochemical analyses show that AvrSr33 adopts a fold containing two zinc-binding sites.
Transient expression assays in Nicotiana benthamiana show that AvrSr33 directly interacts with Sr33, and comparison of recognised and non-recognised AvrSr33 variants identifies a polymorphic loop associated with recognition. This loop is positioned adjacent to one of the zinc-binding sites with its orientation constrained by zinc coordination. Reciprocal mutations of key surface residues within this region alter recognition, whereas mutation of the zinc-binding site abolishes recognition.
Our data suggest that zinc coordination in AvrSr33 provides a structurally constrained scaffold that supports the surface features associated with Sr33 recognition. These findings provide a mechanistic framework for understanding how zinc coordination contributes to effector recognition and may influence the evolutionary trajectories of pathogen effectors. Less
Here, we investigated the structure and recognition of the AvrSr33 effector from the wheat stem rust fungus (Puccinia graminis f. sp. tritici, Pgt). Structural and biochemical analyses show that AvrSr33 adopts a fold containing two zinc-binding sites.
Transient expression assays in Nicotiana benthamiana show that AvrSr33 directly interacts with Sr33, and comparison of recognised and non-recognised AvrSr33 variants identifies a polymorphic loop associated with recognition. This loop is positioned adjacent to one of the zinc-binding sites with its orientation constrained by zinc coordination. Reciprocal mutations of key surface residues within this region alter recognition, whereas mutation of the zinc-binding site abolishes recognition.
Our data suggest that zinc coordination in AvrSr33 provides a structurally constrained scaffold that supports the surface features associated with Sr33 recognition. These findings provide a mechanistic framework for understanding how zinc coordination contributes to effector recognition and may influence the evolutionary trajectories of pathogen effectors. Less
Parasite-derived homologs of the cytokine macrophage migration inhibitory factor MIF function as virulent factors during parasitic infection Recent evidence suggests MIF-like products from multicellular helminthic species can be targeted to ameliorate parasite burden Here we identify a broad contingent of hypothetical MIF-like proteins from genomic helminth repositories and perform structure analysis to validate conserved homology Further we employ a diverse subset of MIF-specific assays to establish cross-species functionality of helminth MIF proteins including native enzymatic activity binding to cognate receptor CD direct interactions with human MIF and signaling through chemokine receptors CXCR and CXCR We demonstrate that MIF-like proteins retain ... More
Parasite-derived homologs of the cytokine macrophage migration inhibitory factor (MIF) function as virulent factors during parasitic infection. Recent evidence suggests MIF-like products from multicellular helminthic species can be targeted to ameliorate parasite burden. Here, we identify a broad contingent of hypothetical MIF-like proteins from genomic helminth repositories and perform structure analysis to validate conserved homology. Further, we employ a diverse subset of MIF-specific assays to establish cross-species functionality of helminth MIF proteins, including native enzymatic activity, binding to cognate receptor CD74, direct interactions with human MIF, and signaling through chemokine receptors CXCR2 and CXCR4. We demonstrate that MIF-like proteins retain a preserved architecture but are capable of diverse physiological outcomes due to small changes in key components of the conserved MIF structure. This work simultaneously provides a mechanistic understanding of helminth virulence upon infection, while broadly examining the potential to neutralize MIF-like proteins for protection against various pathological species. 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
Pyrophosphate-dependent phosphofructokinases provide an alternative route through central carbon metabolism by using inorganic pyrophosphate rather than ATP to phosphorylate fructose- -phosphate These enzymes are absent from mammals but occur in several anaerobic and parasitic organisms where they may support metabolic flexibility and represent potential therapeutic vulnerabilities Here we report three crystal structures of Trichomonas vaginalis PPi-dependent phosphofructokinase TvPPi-PFK a cytosolic enzyme that contributes substantially to parasite glycolytic flux The structures show that TvPPi-PFK adopts the conserved two-domain PFK fold assembles as a tetramer and preserves the canonical PPi MgPPi -binding motifs required for pyrophosphate-dependent catalysis Mass photometry confirms that TvPPi-PFK ... More
Pyrophosphate-dependent phosphofructokinases provide an alternative route through central carbon metabolism by using inorganic pyrophosphate rather than ATP to phosphorylate fructose-6-phosphate. These enzymes are absent from mammals but occur in several anaerobic and parasitic organisms, where they may support metabolic flexibility and represent potential therapeutic vulnerabilities. Here, we report three crystal structures of Trichomonas vaginalis PPi-dependent phosphofructokinase 1 (TvPPi-PFK), a cytosolic enzyme that contributes substantially to parasite glycolytic flux. The structures show that TvPPi-PFK adopts the conserved two-domain PFK fold, assembles as a tetramer, and preserves the canonical PPi/MgPPi²⁻-binding motifs required for pyrophosphate-dependent catalysis. Mass photometry confirms that TvPPi-PFK is predominantly tetrameric in solution and shows that nucleotide- and Mg²⁺/PPi-containing conditions further stabilize the tetrameric assembly. Unexpectedly, the ligand-bound structures reveal ATP, AMP, and sugar-phosphate ligands at a recurring dimer-interface site that is spatially distinct from the canonical PPi/MgPPi²⁻ catalytic donor-binding pocket. This non-canonical interface is more accessible in TvPPi-PFK than in available PPi-PFK structures, suggesting a previously unrecognized ligand-recognition feature. These findings expand the structural landscape of the PFK superfamily and provide a framework for testing how nucleotide and sugar-phosphate binding may influence TvPPi-PFK function, regulation, and future antitrichomonal drug-discovery efforts. Less
Chemical topology has emerged as a unique dimension in protein engineering motivating the pursuit of topologically nontrivial protein architectures for functional advantages such as enhanced stability and rich dynamics However the structural diversity of artificial mechanically interlocked proteins remains limited Here we report the computational design and cellular synthesis of a pair of topological isomers via symmetric assembly of orthogonal entangling motifs By fusing two C symmetric entangling motifs i e p dim and HP in specific arrangements we programmed the formation of multiple crossings which upon cyclization yielded a protein Solomon link and a protein three-twist knot The fusion ... More
Chemical topology has emerged as a unique dimension in protein engineering, motivating the pursuit of topologically nontrivial protein architectures for functional advantages, such as enhanced stability and rich dynamics. However, the structural diversity of artificial mechanically interlocked proteins remains limited. Here, we report the computational design and cellular synthesis of a pair of topological isomers via symmetric assembly of orthogonal entangling motifs. By fusing two C2 symmetric entangling motifs, i.e., p53dim and HP0242, in specific arrangements, we programmed the formation of multiple crossings, which upon cyclization yielded a protein Solomon link and a protein three-twist knot. The fusion patterns and linker lengths were systematically optimized to direct the formation of the intended topologies. Their successful cellular synthesis was validated through biophysical and structural analyses, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, size exclusion chromatography, and liquid chromatography-mass spectrometry. Notably, we report the crystal structure of an artificial protein three-twist knot. Both the Solomon link and the three-twist knot displayed increased structural compactness and stability relative to their controls with lower topological complexity (e.g., Hopf link, trefoil knot, and linear forms), as evidenced by their superior thermal stability and resistance to chemical denaturation. This modular design strategy provides a rational and extensible route to diverse mechanically interlocked proteins and could be generalized to access even more complex architectures, such as protein chainmail-like nanocages and woven protein frameworks. 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
Structural biology has fundamentally influenced pharmaceutical research and development at Merck Sharp Dohme LLC Rahway New Jersey USA progressing from pioneering macromolecular crystallography in the s to a fully integrated platform that today encompasses X-ray crystallography cryo-electron microscopy micro-electron diffraction and cryo-electron tomography In this review we present a comprehensive overview of how these complementary methods have advanced drug discovery across diverse therapeutic areas We illustrate how atomic to cellular structural insights inform drug discovery and development from target identification and validation hit finding lead identification through lead optimization and clinical progression Furthermore we describe how structural biology techniques aid ... More
Structural biology has fundamentally influenced pharmaceutical research and development at Merck Sharp & Dohme LLC, Rahway, New Jersey, USA, progressing from pioneering macromolecular crystallography in the 1980s to a fully integrated platform that today encompasses X-ray crystallography, cryo-electron microscopy, micro-electron diffraction and cryo-electron tomography. In this review, we present a comprehensive overview of how these complementary methods have advanced drug discovery across diverse therapeutic areas. We illustrate how atomic to cellular structural insights inform drug discovery and development, from target identification and validation, hit finding, lead identification through lead optimization and clinical progression. Furthermore, we describe how structural biology techniques aid in formulation strategies of antibodies and vaccines. Finally, we highlight the growing integration of ex situ and in situ approaches as a paradigm shift towards elucidating drug mechanisms in native cellular contexts, a transition poised to accelerate the discovery and development of next-generation therapeutics. 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
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
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
TL A is a proinflammatory cytokine in the tumor necrosis factor TNF superfamily that signals via DR on T helper cells innate lymphoid cells and fibroblasts Dysregulated TL A signaling has been hypothesized to affect multiple immune-mediated diseases with clinical proof of concept demonstrated in ulcerative colitis and Crohn s disease We characterized the binding affinity specificity structure pharmacodynamics pharmacokinetics and toxicity profiles of SPY and SPY two novel extended half-life monoclonal antibodies that inhibit TL A SPY and SPY demonstrated selective high-affinity binding to human TL A KD pM and potent functional inhibition of DR signaling Based on Fc ... More
TL1A is a proinflammatory cytokine in the tumor necrosis factor (TNF) superfamily that signals via DR3 on T helper cells, innate lymphoid cells, and fibroblasts. Dysregulated TL1A signaling has been hypothesized to affect multiple immune-mediated diseases, with clinical proof of concept demonstrated in ulcerative colitis and Crohn’s disease. We characterized the binding affinity, specificity, structure, pharmacodynamics, pharmacokinetics, and toxicity profiles of SPY002 and SPY072, two novel extended half-life monoclonal antibodies that inhibit TL1A. SPY002 and SPY072 demonstrated selective, high-affinity binding to human TL1A (KD ≈ 31–35 pM) and potent functional inhibition of DR3 signaling. Based on Fc modifications, SPY002 and SPY072 showed attenuated Fc effector function and increased FcRn binding at acidic pH (5.8). Both antibodies exhibited enhanced PK profiles in nonhuman primates, resulting in predicted human half-lives that support quarterly or biannual dosing. In toxicity studies, no drug-related adverse effects were observed with either antibody at exposures >10 times those anticipated in clinical trials. In a rat collagen-induced arthritis model, anti-TL1A antibody treatment effectively reduced arthritis severity, with similar efficacy to the TNF antagonist etanercept. In humanized mouse Imiquimod-induced psoriasis and 2,4,6‑trinitrobenzene sulfonic acid colitis models, anti-TL1A demonstrated similar efficacy to anti–IL-23 and anti-TNF antibodies. These findings characterize two novel extended half-life TL1A antibodies and support the ongoing Phase 2 clinical development of SPY002 and SPY072 for immune-mediated diseases such as inflammatory bowel disease and rheumatic diseases. Less
Background Calcium-dependent protein kinase CDPK has emerged as a protozoan specific target for the treatment of cryptosporidiosis A previous study identified pyridopyrimidinones as new Cryptosporidium parvum Cp CDPK inhibitors with potent growth inhibition against C parvum and C hominis Docking analyses suggested the unique positioning of the kinase s Chelix could present refinement opportunities Methods Compounds designed to optimize the pyridopyrimidinones focused on the back-pocket region predicted to be proximal to the C-helix the solvent exposed region and the ATP ribose-binding site Designed derivatives were synthesized and assessed for CpCDPK and Src kinase inhibition and for Cryptosporidium spp growth inhibition ... More
Background: Calcium-dependent protein kinase 1 (CDPK1) has emerged as a protozoan specific
target for the treatment of cryptosporidiosis. A previous study identified pyridopyrimidinones as
new Cryptosporidium parvum (Cp) CDPK1 inhibitors with potent growth inhibition against C.
parvum and C. hominis. Docking analyses suggested the unique positioning of the kinase’s αChelix could present refinement opportunities.
Methods: Compounds designed to optimize the pyridopyrimidinones focused on the back-pocket region predicted to be proximal to the αC-helix, the solvent exposed region and the ATP ribose-binding site. Designed derivatives were synthesized and assessed for CpCDPK1 and Src kinase inhibition and for Cryptosporidium spp., growth inhibition in mammalian cells. AMP/Mg+2 and three inhibitors were co-crystalized with CpCDPK1, and two inhibitors were profiled for kinase selectivity.
Results: WIN 4-88 was identified with CpCDPK1 (IC50 = 0.056 μM), and growth inhibition of zoonotic C. parvum (NLuc EC50 = 0.042 μM), anthroponotic C. parvum (Tu114 EC50 = 0.030 μM), and C. hominis Tu502 (EC50 = 0.062 μM), as well as enhanced kinome selectivity. The crystal structures confirmed the predicted binding mode, indicating key interactions with hinge residue Y155, similar orientations of the solvent expose moieties, occupancy of the back-pocket near the αC-helix and for one inhibitor containing a solubilizing hydroxyethyl attached to the central heterocycle extension into the ATP ribose-binding site.
Conclusions: The expanded structure-activity relationship and structural insights will potentially be applicable to other chemotypes with similar binding modes and will enhance development of CpCDPK1 inhibitors for the treatment of cryptosporidiosis. Less
target for the treatment of cryptosporidiosis. A previous study identified pyridopyrimidinones as
new Cryptosporidium parvum (Cp) CDPK1 inhibitors with potent growth inhibition against C.
parvum and C. hominis. Docking analyses suggested the unique positioning of the kinase’s αChelix could present refinement opportunities.
Methods: Compounds designed to optimize the pyridopyrimidinones focused on the back-pocket region predicted to be proximal to the αC-helix, the solvent exposed region and the ATP ribose-binding site. Designed derivatives were synthesized and assessed for CpCDPK1 and Src kinase inhibition and for Cryptosporidium spp., growth inhibition in mammalian cells. AMP/Mg+2 and three inhibitors were co-crystalized with CpCDPK1, and two inhibitors were profiled for kinase selectivity.
Results: WIN 4-88 was identified with CpCDPK1 (IC50 = 0.056 μM), and growth inhibition of zoonotic C. parvum (NLuc EC50 = 0.042 μM), anthroponotic C. parvum (Tu114 EC50 = 0.030 μM), and C. hominis Tu502 (EC50 = 0.062 μM), as well as enhanced kinome selectivity. The crystal structures confirmed the predicted binding mode, indicating key interactions with hinge residue Y155, similar orientations of the solvent expose moieties, occupancy of the back-pocket near the αC-helix and for one inhibitor containing a solubilizing hydroxyethyl attached to the central heterocycle extension into the ATP ribose-binding site.
Conclusions: The expanded structure-activity relationship and structural insights will potentially be applicable to other chemotypes with similar binding modes and will enhance development of CpCDPK1 inhibitors for the treatment of cryptosporidiosis. Less
Insomnia is a widespread sleep disorder that significantly impairs quality of life and imposes a societal burden Although benzodiazepines and Z-drugs are commonly used for treating insomnia these drugs often have side effects such as excessive muscle relaxation and dependency The orexin signaling pathway has emerged as a promising therapeutic target for insomnia with dual orexin receptor antagonists DORAs offering an alternative approach to treatment To reduce the potential of these drugs for next-day residual effects we developed vornorexant a novel DORA with a high receptor affinity and a short elimination half-life In this study we investigated the molecular interactions ... More
Insomnia is a widespread sleep disorder that significantly impairs quality of life and imposes a societal burden. Although benzodiazepines and Z-drugs are commonly used for treating insomnia, these drugs often have side effects, such as excessive muscle relaxation and dependency. The orexin signaling pathway has emerged as a promising therapeutic target for insomnia, with dual orexin receptor antagonists (DORAs) offering an alternative approach to treatment. To reduce the potential of these drugs for next-day residual effects, we developed vornorexant, a novel DORA with a high receptor affinity and a short elimination half-life. In this study, we investigated the molecular interactions of this drug with human orexin receptors through crystal structure analysis of its binding to orexin type 2 receptor (OX2R) and a docking simulation of the drug with orexin type 1 receptor (OX1R). The crystal structure of the OX2R–vornorexant complex revealed a conserved U-shaped conformation stabilized by hydrophobic and hydrogen-bonding interactions, including key contacts with Asn324, His350 and Pro131. OX1R–vornorexant docking simulations indicated a similar binding mode to OX1R, with no steric hindrance observed, supporting a balanced dual antagonism. These results provide a structural basis for the high-affinity dual antagonism of vornorexant and offer insights for the design of orexin receptor antagonists. 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 precise and selective transport of protons across cellular membranes relies on the dynamic formation and dissipation of hydrogen-bonding networks involving water molecules protein sidechains and backbone carbonyls As in aqueous solution protons are conducted over long distances along chains of hydrogen-bonded water molecules within narrow protein pores To engineer proton-conductive pathways therefore we must explicitly account for the dynamic behavior of these networks In previous work we showed that incorporation of polar Gln residues into hydrophobic pores drives formation of transient single-file water wires that enable proton-selective transport Here we sought to enhance conduction by introducing targeted Ile-to-Ser substitutions ... More
The precise and selective transport of protons across cellular membranes relies on the dynamic formation and dissipation of hydrogen-bonding networks involving water molecules, protein sidechains, and backbone carbonyls. As in aqueous solution, protons are conducted over long distances along chains of hydrogen-bonded water molecules within narrow protein pores. To engineer proton-conductive pathways, therefore, we must explicitly account for the dynamic behavior of these networks. In previous work, we showed that incorporation of polar Gln residues into hydrophobic pores drives formation of transient, single-file water wires that enable proton-selective transport. Here, we sought to enhance conduction by introducing targeted Ile-to-Ser substitutions to extend connectivity across the pore. We find that the position of Ser relative to Gln modulates sidechain dynamics and, in turn, channel hydration. Although increased polarity reduces hydrophobic length and enhances hydration, these effects alone do not explain the observed conduction rates. Instead, asymmetry in the arrangement and dynamics of polar sidechains emerges as a key determinant of proton conductivity. Together, these results demonstrate that proton conduction is governed not only by pore polarity and hydration, but also by the dynamic and asymmetric organization of hydrogen-bonding networks. This work establishes design principles for engineering proton-selective channels and reveals how asymmetry enables efficient proton transport across biological membranes. 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
Constant changes in SARS-CoV- in human populations as well as potential future spillovers from animal coronaviruses have provided the impetus for the development of additional direct-acting antivirals We describe herein the discovery of a new class of broad-spectrum inhibitors of coronavirus C-like protease CLpro a cysteine protease essential for viral replication and a validated drug target that incorporate in their structure a -oxazaphospholidin- -one scaffold Inhibitors and were found to have EC values of and nM against SARS-CoV- CLpro respectively and CC values M These compounds also potently inhibited MERS-CoV CLpro IC nM and nM respectively Importantly several of the ... More
Constant changes in SARS-CoV-2 in human populations as well as potential future spillovers from animal coronaviruses have provided the impetus for the development of additional direct-acting antivirals. We describe herein the discovery of a new class of broad-spectrum inhibitors of coronavirus 3C-like protease (3CLpro), a cysteine protease essential for viral replication and a validated drug target, that incorporate in their structure a 1,3,2-oxazaphospholidin-3-one scaffold. Inhibitors 1 and 2 were found to have EC50 values of 60 and 50 nM against SARS-CoV-2 3CLpro, respectively, and CC50 values >100 µM. These compounds also potently inhibited MERS-CoV 3CLpro (IC50 120 nM and 90 nM, respectively). Importantly, several of the synthesized compounds inhibited recombinant human cathepsin L with IC50 values in the low nM to sub-nM range. Thus, the compounds can potentially exhibit high antiviral potency by abrogating viral entry via the inhibition of cathepsin L and viral replication by inhibition of 3CLpro. High resolution cocrystal structures were determined to elucidate the mechanism of action, identify the molecular determinants associated with binding, and to inform the optimization process. Less
Structural analyses of Trichomonas vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK)
Trichomonas vaginalis causes trichomoniasis the most common non-viral sexually transmitted disease in humans T vaginalis pyrophosphate-dependent phosphofructokinase TvPPi-PFK is a putative target for rational structure-based drug discovery given its absence in mammals and its importance for parasite survival TvPPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose- -phosphate using pyrophosphate PPi as the phosphoryl donor This reversible reaction catalyzed by TvPPi-PFK is the first committed step in glycolysis Its reverse reaction is vital for gluconeogenesis in T vaginalis The purification crystallization structure determination and preliminary structure-functional analyses of three crystal structures of TvPPi-PFK are presented All three structures ... More
Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease in humans. T. vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK) is a putative target for rational, structure-based drug discovery, given its absence in mammals and its importance for parasite survival. TvPPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose-6-phosphate using pyrophosphate (PPi) as the phosphoryl donor. This reversible reaction, catalyzed by TvPPi-PFK, is the first committed step in glycolysis. Its reverse reaction is vital for gluconeogenesis in T. vaginalis. The purification, crystallization, structure determination, and preliminary structure-functional analyses of three crystal structures of TvPPi-PFK are presented. All three structures organize as tetramers with the conserved motifs essential for pyrophosphate binding and PPi-PFK catalytic activity. Comparative analysis with structural neighbors from other organisms demonstrated that despite sharing <29% sequence identity, TvPPi-PFK’s protomer shares overall topology with both PPi- and ATP-dependent PFKs. Mass photometry confirmed that TvPPi-PFK formed tetramers under near-physiological conditions. Unexpectedly, TvPPi-PFK crystals dephosphorylate ATP to AMP during soaking. In all three structures, either ATP or AMP is bound at the enzyme’s dimer interface, typical of ATP-PFKs, but a novel finding for PPi-PFKs. Furthermore, a sugar phosphate binding site was observed in proximity to the ATP-binding site. Thus, the three reported TvPPi-PFK structures validate its established PPi-dependent activity while revealing previously unreported ATP and sugar phosphate binding. This study also lays a foundation for future research into putative ATP-dependent activity of TvPPi-PFK and for evaluating known phosphofructokinase inhibitors as potential therapeutics for trichomoniasis. These findings expand our understanding of PFK superfamily diversity and support the continued exploration of TvPPi-PFK as a drug target for trichomoniasis Less
The ToxRS system belongs to a family of co-component transmembrane transcription regulators that act as sensors of environmental cues and regulate virulence gene expression in several bacterial pathogens These systems are thought to operate by sensing environmental stimuli and transmitting signals through periplasmic domains to activate DNA-binding transcription factors In the enteric pathogens Vibrio parahaemolyticus and Vibrio cholerae the ToxRS system regulates virulence factors responsible for severe gastrointestinal symptoms in humans ToxR is a DNA-binding regulator associated in the periplasm with ToxS a protein of poorly understood function ToxS modulates the activity of its binding partner ToxR in the presence ... More
The ToxRS system belongs to a family of co-component transmembrane transcription regulators that act as sensors of environmental cues and regulate virulence gene expression in several bacterial pathogens. These systems are thought to operate by sensing environmental stimuli and transmitting signals through periplasmic domains to activate DNA-binding transcription factors. In the enteric pathogens Vibrio parahaemolyticus and Vibrio cholerae, the ToxRS system regulates virulence factors responsible for severe gastrointestinal symptoms in humans. ToxR is a DNA-binding regulator associated in the periplasm with ToxS, a protein of poorly understood function. ToxS modulates the activity of its binding partner ToxR in the presence of bile salts, antimicrobial cholesterol metabolites secreted into the gut. To date, the molecular mechanism underlying this regulation remains unclear. We present crystal structures of the V. parahaemolyticus ToxS periplasmic domain (ToxSp) with and without the bile salt glycocholate. ToxSp forms an 8-stranded broken β-barrel with a central α-helix and is structurally homologous to a group of chaperone proteins. ToxSp has a highly conserved hydrophobic core that stabilizes the β-barrel fold, while the binding pocket tolerates substantial variation, consistent with binding hydrophobic ligands. Strikingly, we discovered that Vp-ToxSp binds three molecules of glycocholate and the presence of this bile salt leads to the formation a strand-swapped ToxS homodimer. Finally, modeling two ToxR periplasmic domains in complex with the glycocholate-bound ToxSp homodimer provides a structure-based model for bile salt-mediated heterotetramerization of the ToxRS system. Overall, our study addresses a major longstanding question in the field of Vibrio virulence regulation providing a scenario that could apply to other pathogens that utilize these membrane-bound family transcriptional regulators. Less
Recoverin is a key calcium sensor that controls the desensitization of the visual rhodopsin by GRK Previous studies have traditionally been conducted on bovine protein bRec while data on human ortholog hRec remain scarce Here we combine X-ray crystallography Xray absorption spectroscopy XANES quantum mechanical calculations molecular dynamics and functional assays to provide an integrated characterization of hRec The Ca -bound hRec structure was solved at showing that unlike bRec hRec interacts with ROS membranes at physiologically relevant submicromolar Ca levels due to a speciesspecific charge distribution that might influence membrane interactions Both recoverins form a set of Ca Zn ... More
Recoverin is a key calcium sensor that controls the desensitization of the visual rhodopsin
by GRK1. Previous studies have traditionally been conducted on bovine protein (bRec), while
data on human ortholog (hRec) remain scarce. Here, we combine X-ray crystallography, Xray absorption spectroscopy (XANES), quantum mechanical calculations, molecular
dynamics, and functional assays to provide an integrated characterization of hRec. The
2Ca2+-bound hRec structure was solved at 1.60 Å, showing that, unlike bRec, hRec interacts
with ROS membranes at physiologically relevant submicromolar Ca2+ levels, due to a speciesspecific charge distribution that might influence membrane interactions. Both recoverins
form a set of Ca2+/Zn2+-bound conformers with improved functional performance. X-ray
crystallography (1.85 Å) and XANES revealed a specific tetrahedral Zn2+ site in 1Ca2+-bound
hRec, the first such site reported in the NCS family. In 1Ca2+-bound hRec, zinc promotes the
formation of active state, whereas in 2Ca2+-state of bRec, it significantly enhances GRK1
binding, as the latter can complement the Zn2+ coordination. These data refine our
understanding of recoverin function in humans and highlight its role as a key link between
calcium and zinc signaling in mammalian photoreceptors under normal and pathological
conditions. Less
by GRK1. Previous studies have traditionally been conducted on bovine protein (bRec), while
data on human ortholog (hRec) remain scarce. Here, we combine X-ray crystallography, Xray absorption spectroscopy (XANES), quantum mechanical calculations, molecular
dynamics, and functional assays to provide an integrated characterization of hRec. The
2Ca2+-bound hRec structure was solved at 1.60 Å, showing that, unlike bRec, hRec interacts
with ROS membranes at physiologically relevant submicromolar Ca2+ levels, due to a speciesspecific charge distribution that might influence membrane interactions. Both recoverins
form a set of Ca2+/Zn2+-bound conformers with improved functional performance. X-ray
crystallography (1.85 Å) and XANES revealed a specific tetrahedral Zn2+ site in 1Ca2+-bound
hRec, the first such site reported in the NCS family. In 1Ca2+-bound hRec, zinc promotes the
formation of active state, whereas in 2Ca2+-state of bRec, it significantly enhances GRK1
binding, as the latter can complement the Zn2+ coordination. These data refine our
understanding of recoverin function in humans and highlight its role as a key link between
calcium and zinc signaling in mammalian photoreceptors under normal and pathological
conditions. 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
UreE is a nickel chaperone that is required for the safe and efficient delivery of nickel to the active site of the metalloenzyme urease which is a key virulence factor of the urinary-tract pathogen Proteus mirabilis We investigated the structural features of P mirabilis UreE PmUreE using protein X-ray crystallography and its nickel-binding capacity by inductively coupled plasma mass spectrometry Here we report a resolution crystal structure of homodimeric PmUreE and show that it has the capacity to bind five Ni II ions per dimer Truncation of the histidine-rich C-terminus reduced the nickel-binding capacity by two Ni II ions per ... More
UreE is a nickel chaperone that is required for the safe and efficient delivery of nickel to the active site of the metalloenzyme urease, which is a key virulence factor of the urinary-tract pathogen Proteus mirabilis. We investigated the structural features of P. mirabilis UreE (PmUreE) using protein X-ray crystallography and its nickel-binding capacity by inductively coupled plasma mass spectrometry. Here, we report a 2.0 Å resolution crystal structure of homodimeric PmUreE and show that it has the capacity to bind five Ni(II) ions per dimer. Truncation of the histidine-rich C-terminus reduced the nickel-binding capacity by two Ni(II) ions per dimer, and comparison with homologous UreE structures allowed the assignment of putative nickel-binding sites within the PmUreE structure. These findings increase our understanding of how PmUreE binds nickel and ultimately prevents this toxic metal from causing significant cellular damage in P. mirabilis. 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
-Hemolysin Ahly is a major Staphylococcus aureus virulence determinant implicated in tissue injury and immune dysregulation antibody inhibitors have reached clinical trials but alternatives with improved ease of manufacture and tissue penetration are desirable Here we demonstrate that phage-derived bicyclic peptides can serve as compact chemically tractable Ahly neutralisers Using TATB-scaffolded M phage libraries we identified WNP-motif containing bicyclic binders with a lead hit of Peptide KD nM and progressed the lead by iterative affinity maturation to Peptide KD nM and by incorporation of strategically chosen non-canonical amino acids to yield Peptide KD nM A co-crystal structure with AhlyH A ... More
α-Hemolysin (Ahly) is a major Staphylococcus aureus virulence determinant implicated in tissue injury and immune dysregulation; antibody inhibitors have reached clinical trials but alternatives with improved ease of manufacture and tissue penetration are desirable. Here we demonstrate that phage-derived bicyclic peptides can serve as compact, chemically tractable Ahly neutralisers. Using TATB-scaffolded M13 phage libraries we identified WNP-motif containing bicyclic binders, with a lead hit of Peptide 14 (KD = 1792nM) and progressed the lead by iterative affinity maturation to Peptide 20 (KD = 609 nM) and by incorporation of strategically chosen non-canonical amino acids to yield Peptide 88 (KD = 96 nM). A 2.2 Å co-crystal structure with AhlyH35A locates the binding footprint on the rim domain and explains the critical role of the WNP motif in target engagement. Functional assays show that the Peptide 88 blocks Ahly mediated hemolysis, inhibits Ahly driven ADAM10 activation, and elucidate its inhibitory mechanism of preventing Ahly binding to human A549 epithelial cells. Peptide 88 protects A549 cells from recombinant toxin and attenuates cytotoxicity in S. aureus co-culture experiments, whilst showing no toxicity to A549 cells. Bicyclic peptides thus represent a new and promising anti-virulence modality: small, synthetically accessible molecules that mimic antibody recognition, with therapeutic potential against S. aureus infections. Less
Safe and effective vaccines against co-circulating mosquito-borne orthoflaviviruses such as Zika virus ZikV and the four serotypes of Dengue virus DenV - must elicit broadly neutralizing antibodies bnAbs to prevent the risk of enhancement of infection by non-neutralizing antibodies We recently discovered new orthoflavivirus-directed bnAbs including F S which neutralizes DenV - and ZikV with comparable or superior potency to the previously characterized E dimer epitope EDE bnAbs Here we used cryoEM and X-ray crystallography to understand the basis of cross-neutralization of F S at the molecular level We obtained a cryoEM structure of F S Fab bound to a ... More
Safe and effective vaccines against co-circulating mosquito-borne orthoflaviviruses such as Zika virus (ZikV) and the four serotypes of Dengue virus (DenV1-4) must elicit broadly neutralizing antibodies (bnAbs) to prevent the risk of enhancement of infection by non-neutralizing antibodies. We recently discovered new orthoflavivirus-directed bnAbs, including F25.S02, which neutralizes DenV1-4 and ZikV with comparable or superior potency to the previously characterized E dimer epitope (EDE) bnAbs. Here, we used cryoEM and X-ray crystallography to understand the basis of cross-neutralization of F25.S02 at the molecular level. We obtained a ~ 4.2 Å cryoEM structure of F25.S02 Fab bound to a stabilized DenV3 soluble E protein dimer and a 2.3 Å crystal structure of F25.S02 Fab bound to ZikV soluble E protein dimer. Like previously described EDE1 bnAbs, the structural epitope of F25.S02 is at the E dimer interface, encompassing predominantly conserved regions in domain II, including the fusion loop. However, unlike EDE1 bnAbs, F25.S02 binding is almost entirely dependent on the heavy chain and is shifted slightly away from the dimer symmetry axis. Our findings emphasize the importance of this cross-neutralizing site of vulnerability for DenV and ZikV that can facilitate rational design of vaccines and therapeutics. Less
Improving health and quality of life in our society is a key focus of drug development Methods for drug discovery are being optimized in multiple ways to reduce costs and timelines Crystallographic fragment screening CFS is increasingly being employed as an early screening method in drug discovery projects Here we demonstrate that selecting the optimal protein crystal form can significantly impact hit rates Two CFS campaigns are carried out against the two crystal forms of the SARS-CoV- main protease using the same fragment library and an almost identical experimental setup Although both crystal forms exhibit similar diffraction properties the observed ... More
Improving health and quality of life in our society is a key focus of drug development. Methods for drug discovery are being optimized in multiple ways to reduce costs and timelines. Crystallographic fragment screening (CFS) is increasingly being employed as an early screening method in drug discovery projects. Here, we demonstrate that selecting the optimal protein crystal form can significantly impact hit rates. Two CFS campaigns are carried out against the two crystal forms of the SARS-CoV-2 main protease, using the same fragment library and an almost identical experimental setup. Although both crystal forms exhibit similar diffraction properties, the observed hit rates in the two campaigns differ significantly. A hit rate of 3% is determined for the monoclinic crystals, while a hit rate of 16% is observed for the orthorhombic crystals. These findings are consistent with the more open molecular packing in the orthorhombic crystals, where the solvent channels leading to the active sites are approximately twice the size of those in the monoclinic crystal form. Our results highlight the critical importance of the crystal form in a crystallographic screening, identifying it as one of the most important parameters to optimize when preparing a CFS campaign. Less
Complement and pathogenic antibodies act independently and together to mediate the pathology of many autoimmune diseases To address these drivers of disease we generated a monoclonal antibody mAb CSL that binds and inhibits both complement and the neonatal Fc fragment crystallizable receptor FcRn The fragment antigen binding Fab portion of CSL was engineered to bind both human C huC zymogen and the active fragment huC b to inhibit the classical and lectin complement pathways in vitro and C b deposition on primary lung endothelial cells using a -dimensional microvascular model system Engineering of a triple amino acid mutation YPY motif ... More
Complement and pathogenic antibodies act independently and together to mediate the pathology of many autoimmune diseases. To address these drivers of disease, we generated a monoclonal antibody (mAb), CSL305, that binds and inhibits both complement and the neonatal Fc (fragment crystallizable) receptor FcRn. The fragment antigen binding (Fab) portion of CSL305 was engineered to bind both human C2 (huC2) zymogen and the active fragment huC2b to inhibit the classical and lectin complement pathways in vitro, and C3b deposition on primary lung endothelial cells using a 3-dimensional microvascular model system. Engineering of a triple amino acid mutation (“YPY” motif) into the Fc region of CSL305 increased its affinity to FcRn at both acidic and neutral pH, allowing it to also act as a potent FcRn antagonist. Intracellular trafficking experiments demonstrated that CSL305, but not the wild-type (WT) mAb lacking the YPY motif, was able to block immunoglobulin G (IgG) recycling in vitro. The generation of a high resolution 2.6Å crystal structure of CSL305 Fab region bound to huC2b showed that the epitope lies directly over the huC2b catalytic triad, providing evidence of its complement mechanism of action as a neutralising mAb. Early pharmacokinetic (PK)/pharmacodynamic (PD) studies using CSL305 in cynomolgus monkeys demonstrated both complement inhibition and FcRn antagonism in vivo, with reductions in complement classical pathway activity and endogenous IgG observed following single intravenous (IV) administration. CSL305 thus represents a dual-functional mAb as a potential therapeutic candidate. Less
Light Oxygen Voltage LOV domains are important widespread receptors of blue light that also found applications in optogenetics and imaging While LOV domains from mesophiles are relatively well characterized their counterparts from thermophilic microorganisms remain understudied Here we express two constructs of a LOV domain belonging to a histidine kinase from Meiothermus ruber MrLOV and MrLOVe and show that they are photoactive with recovery time values of and min respectively and thermostable Crystal structures reveal that MrLOV which lacks helices A and J forms a parallel dimer whereas MrLOVe is a tetramer organized as an antiparallel dimer of two parallel ... More
Light Oxygen Voltage (LOV) domains are important widespread receptors of blue light that also found applications in optogenetics and imaging. While LOV domains from mesophiles are relatively well characterized, their counterparts from thermophilic microorganisms remain understudied. Here, we express two constructs of a LOV domain belonging to a histidine kinase from Meiothermus ruber, MrLOV and MrLOVe, and show that they are photoactive, with recovery time values of 21 and 27 min, respectively, and thermostable. Crystal structures reveal that MrLOV, which lacks helices A’α and Jα, forms a parallel dimer, whereas MrLOVe is a tetramer organized as an antiparallel dimer of two parallel dimers interacting via helices Jα. One MrLOVe dimer is symmetric, and the other is asymmetric, with conformational differences mirroring activation-related changes in other LOV domains. Our data provide the structural basis for understanding and engineering of thermophilic LOVs and pave the way for development of thermostable and photostable LOV-derived optogenetic tools and flavin-based fluorescent proteins. Less
Gene expression is governed by dynamic switches between repressive and activating transcriptional states Among the molecules mediating these transitions chromatin readers and transcription factors play pivotal roles However how they assemble with regulatory machineries to enable crosstalk between gene repression and activation remains unknown Here we use an integrative structural dynamics approach combining cryo-EM crosslinking mass spectrometry fragment-resolved protein interactome mapping and crystallography to show how the dual-role chromatin reader Cti and transcription factors Ash and Ume engage the Sin deacetylase complex a major regulatory hub in eukaryotes We find that Cti competes with Ash to drive its dynamic recruitment ... More
Gene expression is governed by dynamic switches between repressive and activating transcriptional states1,2. Among the molecules mediating these transitions, chromatin readers and transcription factors play pivotal roles3,4. However, how they assemble with regulatory machineries to enable crosstalk between gene repression and activation remains unknown. Here, we use an integrative structural dynamics approach – combining cryo-EM, crosslinking mass spectrometry, fragment-resolved protein interactome mapping and crystallography – to show how the dual-role chromatin reader Cti6 and transcription factors Ash1 and Ume6 engage the Sin3 deacetylase complex, a major regulatory hub in eukaryotes5. We find that Cti6 competes with Ash1 to drive its dynamic recruitment to a shared peripheral module, while Ume6 engages the Sin3 scaffold through a defined, minimal interface. Using high-throughput mutational scanning, we reveal deleterious and gain-of-function mutations in Sin3, identifying evolutionarily conserved residues essential for anchoring transcription factors. Together, these results provide structural and functional insights into how dual-role regulators engage the central Sin3 complex, revealing subtle assembly principles that may facilitate crosstalk between gene repression and activation. They also establish an integrative multidisciplinary framework to dissect the dynamics of macromolecular assemblies across biological systems. 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
Epstein-Barr virus EBV causes infectious mononucleosis and contributes to neurodegenerative disorders and malignancies particularly in immune-compromised hosts Transplant patients face high risk of post-transplant lymphoproliferative disease a life-threatening EBV-driven lymphoma There are no EBV-specific vaccines or treatments however neutralizing antibodies against EBV glycoproteins may offer utility as therapeutic agents EBV entry into B cells involves gp which binds complement receptors and gp which engages HLA class II to trigger fusion Most existing monoclonal antibodies mAbs against these antigens are non-human limiting clinical use Using a transgenic mouse model we generate two gp and eight gp genetically human neutralizing mAbs that ... More
Epstein-Barr virus (EBV) causes infectious mononucleosis and contributes to neurodegenerative disorders and malignancies, particularly in immune-compromised hosts. Transplant patients face high risk of post-transplant lymphoproliferative disease, a life-threatening EBV-driven lymphoma. There are no EBV-specific vaccines or treatments; however, neutralizing antibodies against EBV glycoproteins may offer utility as therapeutic agents. EBV entry into B cells involves gp350, which binds complement receptors, and gp42, which engages HLA class II to trigger fusion. Most existing monoclonal antibodies (mAbs) against these antigens are non-human, limiting clinical use. Using a transgenic mouse model, we generate two gp350 and eight gp42 genetically human neutralizing mAbs that block receptor binding. Structural analyses reveal extended sites of vulnerability relevant to vaccine development. Delivery of a gp42 mAb protects humanized mice from EBV challenge, while a gp350 mAb provides partial protection. These mAbs highlight the utility of transgenic mice to produce therapeutic mAbs for preventing EBV-driven disease. Less
HECT E ligases regulate many cellular processes yet how they recognise their substrates and synthesise specific types of poly-ubiquitin chains is still incompletely understood HECTD a member of the other HECT family is implicated in the regulation of inflammation apoptosis and infection and highly expressed in several cancers These functions are largely attributed to its ligase activity and modification of diverse substrates with different types of ubiquitin chains We present a detailed analysis of the ligase activity of HECTD including its ubiquitin linkage preferences oligomeric state and substrate ubiquitination Using cryo-EM we provide the full-length structures of HECTD in both ... More
HECT E3 ligases regulate many cellular processes, yet how they recognise their substrates and synthesise specific types of poly-ubiquitin chains is still incompletely understood. HECTD3, a member of the “other HECT” family, is implicated in the regulation of inflammation, apoptosis, and infection and highly expressed in several cancers. These functions are largely attributed to its ligase activity and modification of diverse substrates with different types of ubiquitin chains. We present a detailed analysis of the ligase activity of HECTD3, including its ubiquitin linkage preferences, oligomeric state and substrate ubiquitination. Using cryo-EM, we provide the full-length structures of HECTD3 in both apo and ubiquitin-loaded forms, revealing key insights into its domain organisation, including discovery of a distinct fold of the N-terminal region, and mechanistic features. Some of these are shared with other HECT ligases, while others are unique to HECTD3 and contribute to differences in its catalytic mechanisms and functional diversity. Less
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
Natural resistance-associated macrophage proteins Nramps are divalent transition metal transporters found in most organisms typically coupling metal uptake to proton co-transport How this coupling evolved however remains unclear We present structural functional and evolutionary analyses of a clade B Nramp from the gut bacterium Bacteroides fragilis BfraNramp Phylogenetic reconstruction positions clade B as the most basal group of canonical Nramps retaining conserved metal-binding motifs while lacking most residues that form the canonical proton pathway We show that BfraNramp efficiently transports Mn and Cd with high apparent affinity but without proton co-transport or dependence on membrane potential or pH Structures of ... More
Natural resistance-associated macrophage proteins (Nramps) are divalent transition metal transporters found in most organisms, typically coupling metal uptake to proton co-transport. How this coupling evolved, however, remains unclear. We present structural, functional, and evolutionary analyses of a clade B Nramp from the gut bacterium Bacteroides fragilis (BfraNramp). Phylogenetic reconstruction positions clade B as the most basal group of canonical Nramps, retaining conserved metal-binding motifs while lacking most residues that form the canonical proton pathway. We show that BfraNramp efficiently transports Mn²⁺ and Cd²⁺ with high apparent affinity but without proton co-transport or dependence on membrane potential or pH. Structures of metal-free and Mn²⁺-bound BfraNramp reveal an inward-open conformation and a distinct metal coordination geometry involving a conserved glutamate on transmembrane helix 3. Together, these results identify clade B Nramps as proton-independent transition metal uniporters and suggest that proton coupling emerged later in Nramp evolution, following establishment of the metal-binding site. Less
The recent discovery of the isonitrile biosynthetic enzyme ScoE expanded the catalytic repertoire of the Fe II KG-dependent dioxygenase enzyme family ScoE synthesizes an isonitrile functional group from a glycyl-fatty acid adduct with both the isonitrile nitrogen and carbon atoms coming from the glycyl moiety This challenging chemistry cannot be performed in a single step Instead the mechanism appears to require two half reactions each involving KG cleavage to generate a highly reactive iron-oxygen species Here we report sixteen crystal structures that provide snapshots along the reaction trajectory of Rv a ScoE homolog from Mycobacterium tuberculosis These structures which are ... More
The recent discovery of the isonitrile biosynthetic enzyme ScoE expanded the catalytic repertoire of the Fe(II)/αKG-dependent dioxygenase enzyme family. ScoE synthesizes an isonitrile functional group from a glycyl-fatty acid adduct, with both the isonitrile nitrogen and carbon atoms coming from the glycyl moiety. This challenging chemistry cannot be performed in a single step. Instead, the mechanism appears to require two half reactions, each involving αKG cleavage to generate a highly reactive iron-oxygen species. Here, we report sixteen crystal structures that provide snapshots along the reaction trajectory of Rv0097, a ScoE homolog from Mycobacterium tuberculosis. These structures, which are both of wild-type and Rv0097 variants, include a substrate 3-((carboxymethyl)amino)decanoic acid (CADA)-bound structure, an αKG-bound structure, and a structure with both CADA and αKG bound. These structural data reveal how Rv0097 employs conformational rearrangements to protect the unstable CADA-reaction intermediate that is formed in the first half reaction while swapping out αKG cleavage products for a second molecule of αKG. Additionally, these structures, together with data from site-directed mutagenesis, provide insight into Rv0097’s preference for substrates with long alkyl chains, potentially facilitating efforts to re-engineer ScoE/Rv0097 to synthesize isonitrile functional groups on a wider range of small molecules. 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
The ToxRS system is a member of a two-protein transmembrane transcriptional regulator family of proteins that act as critical environmental stress sensors and regulate virulence gene expression in some bacterial pathogens These systems are thought to operate by sensing environmental stimuli and transmitting signals through periplasmic domains to activate DNA-binding transcription factors In Vibrio parahaemolyticus and Vibrio cholerae the ToxRS system regulates virulence factors responsible for severe gastrointestinal symptoms in humans ToxS has been shown to modulate the activity of its binding partner ToxR by binding bile salts antimicrobial cholesterol metabolites secreted into the human gut However the molecular mechanism ... More
The ToxRS system is a member of a two-protein transmembrane transcriptional regulator family of proteins that act as critical environmental stress sensors and regulate virulence gene expression in some bacterial pathogens. These systems are thought to operate by sensing environmental stimuli and transmitting signals through periplasmic domains to activate DNA-binding transcription factors. In Vibrio parahaemolyticus and Vibrio cholerae, the ToxRS system regulates virulence factors responsible for severe gastrointestinal symptoms in humans. ToxS has been shown to modulate the activity of its binding partner ToxR by binding bile salts, antimicrobial cholesterol metabolites secreted into the human gut. However, the molecular mechanism underlying this regulation is unclear. Here, we present the crystal structures of the V. parahaemolyticus ToxS periplasmic domain (ToxSp) with and without the bile salt glycocholate. ToxSp forms an 8-stranded broken β-barrel with a central α-helix and is structurally homologous to a group of chaperone proteins. Notably, the glycocholate-bound ToxSp structure forms a strand-swapped homodimer containing three bound glycocholate molecules. Modeling two ToxR periplasmic domains in complex with the glycocholate-bound ToxSp dimer provides a structure-based model for bile salt activation of the ToxRS system and suggests that ToxRS homologs may be regulated in a similar manner across diverse bacterial species. Less
Iron homeostasis in various pathogenic bacteria is regulated by bacterioferritins Bfr which function to store Fe and release Fe as needed for metabolic processes The Bfr structure consists of kDa subunits in which dimer pairs bind a heme molecule and are assembled into a highly symmetrical -meric spherical structure with an internal core diameter of approximately Release of iron is facilitated by the binding of a kDa Fe- S ferredoxin Bfd to specific sites on the surface of Bfr which transfers electrons to the core thereby reducing the stored Fe to Fe for mobilization The crystal structures of Bfr from ... More
Iron homeostasis in various pathogenic bacteria is regulated by bacterioferritins (Bfr) which function to store Fe3+ and release Fe2+ as needed for metabolic processes. The Bfr structure consists of 18 kDa subunits in which dimer pairs bind a heme molecule and are assembled into a highly symmetrical 24-meric spherical structure with an internal core diameter of approximately 80 Å. Release of iron is facilitated by the binding of a 7 kDa [2Fe-2S] ferredoxin (Bfd) to specific sites on the surface of Bfr which transfers electrons to the core thereby reducing the stored Fe3+ to Fe2+ for mobilization. The crystal structures of Bfr from Brucella abortus (Ba) in the apo and iron bound forms are presented and compared with those from Acinetobacter baumannii (Ab) and Pseudomonas aeruginosa (Pa). Additionally, models of the Bfr:Bfd complexes for Ba and Ab are provided and compared with the Pa complex. Finally, compounds known to target the Bfr:Bfd interaction in Pa were docked to the Ba and Ab structures which provided insight regarding the potential binding mode and inhibitory mechanism. Less
UreE is a nickel chaperone required for the safe and efficient delivery of nickel to the active site of the metalloenzyme urease a key virulence factor of the urinary tract pathogen Proteus mirabilis We investigated the structural features of P mirabilis UreE using protein X-ray crystallography and its nickel-binding capacity by inductively coupled plasma-mass spectrometry Here we report a crystal structure of homodimeric PmUreE and show it has capacity to bind five nickel ions per dimer Truncation of the histidine-rich C-terminus reduced nickel binding capacity by two nickel ions per dimer and comparison with homologous UreE structures allowed the assignment ... More
UreE is a nickel chaperone required for the safe and efficient delivery of nickel to the active site of the metalloenzyme, urease; a key virulence factor of the urinary tract pathogen, Proteus mirabilis. We investigated the structural features of P. mirabilis UreE using protein X-ray crystallography and its nickel-binding capacity by inductively coupled plasma-mass spectrometry. Here, we report a 2.0 Å crystal structure of homodimeric PmUreE and show it has capacity to bind five nickel ions per dimer. Truncation of the histidine-rich C-terminus reduced nickel binding capacity by two nickel ions per dimer and comparison with homologous UreE structures allowed the assignment of putative nickel binding sites within the PmUreE structure. These findings increase our understanding of how PmUreE binds nickel and ultimately prevents this toxic metal from causing significant cellular damage in P. mirabilis. Less
Human T-cell Leukemia Virus type HTLV- is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology especially when compared to the closely related virus HIV A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host Importantly the first in class anti-capsid drug approved lenacapavir can provide long-acting pre-exposure prophylaxis Such a property would provide a means to prevent the transmission of HTLV- but its capsid has not previously ... More
Human T-cell Leukemia Virus type 1 (HTLV-1) is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions. Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology, especially when compared to the closely related virus, HIV. A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host. Importantly, the first in class anti-capsid drug approved, lenacapavir, can provide long-acting pre-exposure prophylaxis. Such a property would provide a means to prevent the transmission of HTLV-1, but its capsid has not previously been considered as a drug target. Here we describe the first high-resolution crystal structures of the HTLV-1 capsid protein, define essential lattice interfaces, and identify a previously unknown ligand-binding pocket. We show that this pocket is essential for virus infectivity, providing a potential target for future anti-capsid drug development. Less
Photobleaching of fluorescent proteins often limits the acquisition of high-quality images in microscopy StayGold a novel dimeric GFP recently monomerized through sequence engineering addresses this challenge with its high photostability There is now a focus on producing different colored StayGold derivatives to facilitate concurrent tagging of multiple targets The unnatural amino acid -aminotyrosine has previously been shown to redshift superfolder GFP upon incorporation into its chromophore via genetic code expansion Here we apply the same strategy to redshift StayGold through substitution of tyrosine- with -aminotyrosine The resultant red fluorescent protein StayRose shows an excitation wavelength maximum of nm and an ... More
Photobleaching of fluorescent proteins often limits the acquisition of high-quality images in microscopy. StayGold, a novel dimeric GFP recently monomerized through sequence engineering, addresses this challenge with its high photostability. There is now a focus on producing different colored StayGold derivatives to facilitate concurrent tagging of multiple targets. The unnatural amino acid 3-aminotyrosine has previously been shown to redshift superfolder GFP upon incorporation into its chromophore via genetic code expansion. Here, we apply the same strategy to redshift StayGold through substitution of tyrosine-58 with 3-aminotyrosine. The resultant red fluorescent protein, StayRose, shows an excitation wavelength maximum of 530 nm and an emission wavelength maximum of 588 nm. Importantly, the monomeric mStayRose retains the favorable photostability in vivo in Escherichia coli and zebrafish embryos. A high-resolution crystal structure of StayRose confirms the modified structure of the amino chromophore within an unperturbed 3D fold. Although reliant on genetic code expansion, StayRose provides an important step toward developing redshifted StayGold derivatives. Less
Heliorhodopsins HeRs the third rhodopsin family are characterized by inverted membrane topology and confinement to monoderm organisms yet their biological meaning has so far remained a mystery We report the first crystal structure of a eukaryotic HeR supported by structural modeling and comparative analyses across all domains of life A conserved carotenoid-binding site reminiscent of secondary antennae in some microbial rhodopsins is identified and found to be common among HeRs We show that inverted topology allows recruitment of exogenous xanthophylls inaccessible in diderm cells explaining HeRs distinctive orientation and distribution These findings reveal a previously unrecognized light-harvesting mechanism of HeRs ... More
Heliorhodopsins (HeRs), the third rhodopsin family, are characterized by inverted membrane topology and confinement to monoderm organisms, yet their biological meaning has so far remained a mystery. We report the first crystal structure of a eukaryotic HeR, supported by structural modeling and comparative analyses across all domains of life. A conserved carotenoid-binding site, reminiscent of secondary antennae in some microbial rhodopsins, is identified and found to be common among HeRs. We show that inverted topology allows recruitment of exogenous xanthophylls, inaccessible in diderm cells, explaining HeRs’ distinctive orientation and distribution. These findings reveal a previously unrecognized light-harvesting mechanism of HeRs, expand the known repertoire of microbial phototrophy, and suggest evolutionary constraints linking membrane topology to environmental metabolite accessibility. Less
The composition of the primordial genetic material remains uncertain Studies of duplex structure and stability and of nonenzymatic template copying chemistry provide insight into the viability of potentially primordial genetic polymers Recent work suggests that - deoxyribo-purine nucleotides may have been generated together with ribonucleotides on the early Earth Since DNA RNA duplexes are known to be less stable than RNA RNA duplexes we have examined the impact of dA dI and dG substitutions on RNA structure and nonenzymatic template copying We find that single -deoxyribo-purine substitutions reduce RNA duplex stability as expected Crystallographic studies show that such substitutions lead ... More
The composition of the primordial genetic material remains uncertain. Studies of duplex structure and stability, and of nonenzymatic template copying chemistry, provide insight into the viability of potentially primordial genetic polymers. Recent work suggests that 2′- deoxyribo-purine nucleotides may have been generated together with ribonucleotides on the early Earth. Since DNA/RNA duplexes are known to be less stable than RNA/RNA duplexes, we have examined the impact of dA, dI, and dG substitutions on RNA structure and nonenzymatic template copying. We find that single 2′-deoxyribo-purine substitutions reduce RNA duplex stability, as expected. Crystallographic studies show that such substitutions lead to minimal structural changes but point to diminished solvation as a likely reason for duplex destabilization. Kinetic studies show that dI and dG substrates exhibit slightly weaker template binding and slower rates of template-directed primer extension than the corresponding ribo-purine substrates. In contrast, dA substrates exhibit much slower reaction kinetics but higher template affinity than rA substrates. Our results suggest that a mixed RNA/DNA primordial genetic polymer would have suffered from moderately slower rates of template copying, but that this could have been offset by an advantage due to more facile strand separation or exchange. Less
C domains are ubiquitous membrane-binding modules of residues in eukaryotes that are often associated with proteins involved in membrane trafficking and lipid modification The genome of Trichomonas vaginalis the most common non-viral sexually transmitted human pathogen encodes eight genes that contain a N-terminal C module linked to a XYPPX-repeat domain of more than four XYPPX repeats C -XYPPX While the function of the XYPPX-repeat domain remains unknown its multiple association with C domains in T vaginalis suggests it is important The C domain from one of these C -XYPPX-repeat proteins Tv-C - was structurally and physically characterized using X-ray crystallography ... More
C2 domains are ubiquitous membrane-binding modules of ∼130 residues in eukaryotes that are often associated with proteins involved in membrane trafficking and lipid modification. The genome of Trichomonas vaginalis, the most common, non-viral, sexually transmitted human pathogen, encodes eight genes that contain a N-terminal C2 module linked to a XYPPX-repeat domain of more than four XYPPX repeats (C2-XYPPX). While the function of the XYPPX-repeat domain remains unknown, its multiple association with C2 domains in T. vaginalis suggests it is important. The C2 domain from one of these C2-XYPPX-repeat proteins, Tv-C2-1, was structurally and physically characterized using X-ray crystallography and NMR spectroscopy. The crystal structure for Tv-C2-1 shows that this domain shares a fold common to all C2 domains, a compact Greek-key motif composed of eight anti-parallel β-strands in the type-2 topology. An NMR chemical shift perturbation study with Ca2+ showed that Tv-C2-1 bound two Ca2+ atoms primarily via two loops (loop-1 and loop-3) on the predicted calcium binding face of the protein with Kds of 58.0 ± 0.1 μM and 232 ± 6 μM. Estimations of the overall rotational correlation time, τc, in the apo (11.1 ns) and Ca2+-bound (9.2 ns) state suggests the protein becomes more compact upon Ca2+ binding, consistent with a decrease in dynamics in loop-3 and marginally in loop-1 suggested by amide 15N heteronuclear steady-state {1H}-15N NOEs. Showing Tv-C2-1 binds calcium and adopts a compact Greek-key motif structure, two primary features of C2 domains, suggests understanding the function of the XYPPX-repeat domain may be warranted. Less
Spermine a pivotal player in biomolecular condensation and diverse cellular processes has emerged as a focus of investigation in aging neurodegeneration and other diseases Despite its significance the mechanistic details of spermine remain incompletely understood Here we describe the distinct modulation by spermine on Alzheimer s Tau and Parkinson s -synuclein elucidating their condensation behaviors in vitro and in vivo Using biophysical techniques including time-resolved SAXS and NMR we trace electrostatically driven transitions from atomic-scale conformational changes to mesoscopic structures Notably spermine extends lifespan ameliorates movement deficits and restores mitochondrial function in C elegans models expressing Tau and -synuclein Acting ... More
Spermine, a pivotal player in biomolecular condensation and diverse cellular processes, has emerged as a focus of investigation in aging, neurodegeneration, and other diseases. Despite its significance, the mechanistic details of spermine remain incompletely understood. Here, we describe the distinct modulation by spermine on Alzheimer’s Tau and Parkinson’s α-synuclein, elucidating their condensation behaviors in vitro and in vivo. Using biophysical techniques including time-resolved SAXS and NMR, we trace electrostatically driven transitions from atomic-scale conformational changes to mesoscopic structures. Notably, spermine extends lifespan, ameliorates movement deficits, and restores mitochondrial function in C. elegans models expressing Tau and α-synuclein. Acting as a molecular glue, spermine orchestrates in vivo condensation of α-synuclein, influences condensate mobility, and promotes degradation via autophagy, specifically through autophagosome expansion. This study unveils the interplay between spermine, protein condensation, and functional outcomes, advancing our understanding of neurodegenerative diseases and paving the way for therapeutic development. Less
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