888 Citations
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
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
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
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
-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
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
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
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
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
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
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 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
Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design Nowadays the use of robotic systems for crystal growth and diffraction analysis is widespread and high throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field However the identification of crystals is still largely carried out through manual inspection sometimes involving tens of thousands of images which represents a bottleneck in an otherwise highly automated process Here we describe AXIS an AI-based Crystal Identification System combining the DINOv computer vision model state-of-the-art transfer learning and MARCO the largest crystallization dataset available to date ... More
Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design. Nowadays, the use of robotic systems for crystal growth and diffraction analysis is widespread and high throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field. However, the identification of crystals is still largely carried out through manual inspection, sometimes involving tens of thousands of images, which represents a bottleneck in an otherwise highly automated process. Here we describe AXIS, an AI-based Crystal Identification System combining the DINOv2 computer vision model, state-of-the-art transfer learning and MARCO, the largest crystallization dataset available to date, for automated crystal detection. AXIS can operate both with visible and UV light images and integrates a Lab-In-The-Loop approach combining ML and expert inputs for continuous learning and specialization. AXIS enables automated annotation of large crystallization image datasets with performance and accuracy comparable to that of human experts and the Lab-In-The-Loop approach introduced here enables efficient adaptation to local conditions facilitating widespread application, which has been a major limitation to date. AXIS can help correct human errors in image annotation and removes critical bottlenecks, particularly in the context of extensive crystallization screens or high throughput applications like fragment and ligand screening unlocking the potential for higher levels of automation that are key both in fundamental and translational research. Less
Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase composed of factor Xa fXa and fVa The affinity of fXa for fVa is low with assembly and function dependent on phospholipid PL membranes Some snakes have evolved venom versions of fXa that bind to fVa with high affinity and efficiently activate prothrombin in the absence of PL We created a similar high-affinity PL-independent human prothrombinase with mutations to human fXa M The increase in affinity enabled cryogenic electron microscopy cryo-EM structure determination of M -prothrombinase to a resolution of All protein domains were well resolved ... More
Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase, composed of factor Xa (fXa) and fVa. The affinity of fXa for fVa is low, with assembly and function dependent on phospholipid (PL) membranes. Some snakes have evolved venom versions of fXa that bind to fVa with high affinity and efficiently activate prothrombin in the absence of PL. We created a similar high-affinity, PL-independent human prothrombinase with 17 mutations to human fXa (M17). The increase in affinity enabled cryogenic electron microscopy (cryo-EM) structure determination of M17-prothrombinase to a resolution of 3.3 Å. All protein domains were well resolved in the map, except for the Gla domain of fXa. The main contacts involve the serine protease and EGF2 domains of fXa and the A2 and A3 domains of fVa, resulting in the burying of a total surface area of 4,900 Å2. The map is of sufficient quality to resolve side chain interactions, including several key M17 mutations. To aid in the placement of the loop Cterminal to the A2 domain (a2-loop), we solved a high-resolution crystal structure of fXa in complex with a synthetic a2 peptide. The acidic a2-loop interacts with the basic heparin binding site of fXa, involving a conserved antiparallel -strand interaction. The M17-prothrombinase structure is compatible with data from biochemical and mutagenesis research and provides important new insights into the assembly and function of the prothrombinase complex. Less
Severe acute respiratory syndrome coronavirus SARS-CoV- continues to threaten global health This underpins the need for novel therapeutics against this virus Nonstructural protein Nsp of SARS-CoV- is a multifunctional protein with an essential role in viral replication As such it presents itself as an attractive target for drug discovery Here we describe two crystallographic fragment-screening campaigns against Nsp one using the established F X-Entry Screen and one using a new chemically and structurally diverse fragment library which we call the KIT library Together hits could be identified from screened fragments which constitutes the highest hit rate reported for Nsp to ... More
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten global health. This underpins the need for novel therapeutics against this virus. Nonstructural protein 1 (Nsp1) of SARS-CoV-2 is a multifunctional protein with an essential role in viral replication. As such, it presents itself as an attractive target for drug discovery. Here, we describe two crystallographic fragment-screening campaigns against Nsp1, one using the established F2X-Entry Screen and one using a new, chemically and structurally diverse fragment library, which we call the KIT library. Together, 21 hits could be identified from 192 screened fragments, which constitutes the highest hit rate reported for Nsp1 to date. Many hits bind to a key functional region and interact with residues involved in cellular mRNA cleavage, ribosome binding and viral RNA recognition. Furthermore, most of the identified fragments share a common binding mode, providing promising starting points for further optimization into drug-like compounds that can disrupt the role of Nsp1 in viral replication. Less
Ferritins are a widespread family of proteins involved in iron homeostasis While classic ferritins consist of four -helices and form -meric nanocages related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity Here by analyzing the available genomic data we identify a family of double ferritin-like proteins DFLPs composed of two four-helical domains which arose by duplication of a ferritin fold protein We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens TmDFLP and CaDFLP and show that they form homodimers and bind heme We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase ... More
Ferritins are a widespread family of proteins involved in iron homeostasis. While classic ferritins consist of four α-helices and form 24-meric nanocages, related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity. Here, by analyzing the available genomic data, we identify a family of double ferritin-like proteins (DFLPs) composed of two four-helical domains, which arose by duplication of a ferritin fold protein. We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens, TmDFLP and CaDFLP, and show that they form homodimers and bind heme. We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase activity. Furthermore, we show that some DFLPs, including TmDFLP and CaDFLP, are highly likely to be targeted into encapsulin shells. Our work expands the range of known iron metabolism systems and highlights the power of genome mining for discovery of new proteins. Less
Efficient drug discovery relies on workflows that integrate structural insights with rapid and cost-effective exploration of chemical space Here we present a data-driven fragment-based lead discovery approach to target Neuronal Calcium Sensor NCS- protein-protein interactions PPIs This study represents a complete implementation of a single high-value design-make-test-analyze cycle that directly yields compounds with micromolar affinity with the potential to modulate NCS- interactions with key targets including the G-protein chaperone Ric- A and the dopamine D and cannabinoid CB receptors X-ray crystallographic fragment screening CFS revealed diverse interaction patterns within the NCS- hydrophobic crevice Algorithmically guided fragment evolution and automated synthesis ... More
Efficient drug discovery relies on workflows that integrate structural insights with rapid and cost-effective exploration of chemical space. Here, we present a data-driven fragment-based lead discovery approach to target Neuronal Calcium Sensor 1 (NCS-1) protein-protein interactions (PPIs). This study represents a complete implementation of a single high-value design-make-test-analyze cycle that directly yields compounds with micromolar affinity with the potential to modulate NCS-1 interactions with key targets, including the G-protein chaperone Ric-8A and the dopamine D2 and cannabinoid CB1 receptors. X-ray crystallographic fragment screening (CFS) revealed diverse interaction patterns within the NCS-1 hydrophobic crevice. Algorithmically guided fragment evolution and automated synthesis enabled the rapid generation of over 250 derivatives, with biophysical validation using LC-MS and Grating-coupled interferometry. Structural analyses highlighted key pharmacophores, with selected compounds exhibiting favorable drug-like properties and potential blood-brain barrier penetration, making them promising candidates for neurodegenerative and neurodevelopmental disorders. Our results demonstrate the feasibility of accelerated hit-to-lead development at synchrotrons, demonstrating a robust, scalable platform for PPI-targeting drug discovery. The generated chemically diverse scaffolds provide a strong foundation for future therapeutic optimization. Less
The enzymatic degradation of polyethylene terephthalate PET offers a sustainable solution for PET recycling Over the past two decades more than PETases have been characterized primarily exhibiting similar sequences and structures Here we report new PET-degrading hydrolases including HaloPETase from the marine Halopseudomonas lineage thereby extending the narrow sequence space by novel features at the active site The crystal structure of HaloPETase was determined to a resolution of revealing a unique active site architecture and a lack of the canonical -stacking clamp found in PETases so far Further variations in active site composition and loop structures were observed Additionally we ... More
The enzymatic degradation of polyethylene terephthalate (PET) offers a sustainable solution for PET recycling. Over the past two decades, more than 100 PETases have been characterized, primarily exhibiting similar sequences and structures. Here, we report new PET-degrading α/β hydrolases, including HaloPETase1 from the marine Halopseudomonas lineage, thereby extending the narrow sequence space by novel features at the active site. The crystal structure of HaloPETase1 was determined to a resolution of 1.16 Å, revealing a unique active site architecture and a lack of the canonical π-stacking clamp found in PETases so far. Further, variations in active site composition and loop structures were observed. Additionally, we found five more enzymes from the same lineage, two of which have a high similarity to type IIa bacterial PETases, while the other three resemble HaloPETase1. All these enzymes exhibited high salt tolerance ranging from 2.5 to 5 M NaCl leading to higher total product releases upon PET degradation at 40 or 50 °C. Based on these findings, we propose an extension of the existing PETase classification system to include type III PETases. Less
In more and more drug discovery projects crystallographic fragment screening CFS is employed as an early screening method Here we demonstrate that choosing the right crystal form has a profound influence on the hit rates and hence success and speed of downstream lead generation Two CFS campaigns with the same fragment library and an almost identical experimental setup were carried out against the two crystal forms of the SARS-CoV- main protease While both crystal forms exhibit similar diffraction properties the observed hit rates in the two campaigns were vastly different For the monoclinic crystals a hit rate of was determined ... More
In more and more drug discovery projects, crystallographic fragment screening (CFS) is employed as an early screening method. Here, we demonstrate that choosing the right crystal form has a profound influence on the hit rates and hence success and speed of downstream lead generation. Two CFS campaigns with the same fragment library and an almost identical experimental setup were carried out against the two crystal forms of the SARS-CoV-2 main protease.While both crystal forms exhibit similar diffraction properties, the observed hit rates in the two campaigns were vastly different. For the monoclinic crystals a hit rate of 3% was determined, while a hit rate of 16% was observed for the orthorhombic crystals. These findings align with the more open molecular packing in the orthorhombic crystals where the solvent channels leading to the active sites are about twice larger than in the monoclinic crystal form. Our results highlight the critical importance of the crystal system in a crystallographic fragment-screening campaign and identify this parameter as one of the most important ones to be optimized during preparation of a campaign. Less
Glutarimide analogs such as thalidomide redirect the E ubiquitin ligase CRL CRBN to induce degradation of certain zinc finger ZF proteins Although the core structural motif recognized by CRBN has been characterized it does not fully explain substrate specificity To explore the role of residues adjacent to this core motif we constructed a comprehensive ZF reporter library of reporters derived from human ZF proteins and conducted a library-on-library screen with glutarimide analogs to identify compounds that collectively degrade ZF reporters Cryo-electron microscopy and crystal structures of ZFs in complex with CRBN revealed the importance of interactions beyond the core ZF ... More
Glutarimide analogs, such as thalidomide, redirect the E3 ubiquitin ligase CRL4CRBN to induce degradation of certain zinc finger (ZF) proteins. Although the core structural motif recognized by CRBN has been characterized, it does not fully explain substrate specificity. To explore the role of residues adjacent to this core motif, we constructed a comprehensive ZF reporter library of 9,097 reporters derived from 1,655 human ZF proteins and conducted a library-on-library screen with 29 glutarimide analogs to identify compounds that collectively degrade 38 ZF reporters. Cryo-electron microscopy and crystal structures of ZFs in complex with CRBN revealed the importance of interactions beyond the core ZF degron. We used systematic mutagenesis of ZFs and CRBN to identify modes of neosubstrate recruitment requiring distinct amino acids. Finally, we found subtle chemical variations in glutarimide analogs that alter target scope and selectivity, thus providing a roadmap for their rational design. Less
Deep learning has revolutionized soluble protein design yet de novo transmembrane TM protein engineering remains hindered by scarce structural data complex membrane-specific interactions and conformational dynamics We developed TMDiffusion TMDF a joint all-heavy-atom sequence structure diffusion model trained to capture the full interaction diversity of natural TM proteins including weak and polar contact networks TMDF designs diverse TM architectures associating domains inhibitors and conformational switches in a single step achieving experimental success A crystal structure of designed proteins matches predictions with atomic accuracy Leveraging TMDF we built synthetic single-pass receptors whose de novo TM domains toggle between conformations enabling precise ... More
Deep learning has revolutionized soluble protein design, yet de novo transmembrane (TM) protein engineering remains hindered by scarce structural data, complex membrane-specific interactions and conformational dynamics. We developed TMDiffusion (TMDF), a joint all-heavy-atom sequence–structure diffusion model trained to capture the full interaction diversity of natural TM proteins, including weak and polar contact networks. TMDF designs diverse TM architectures—associating domains, inhibitors, and conformational switches—in a single step, achieving >70% experimental success. A crystal structure of designed proteins matches predictions with atomic accuracy. Leveraging TMDF, we built synthetic single-pass receptors whose de novo TM domains toggle between conformations, enabling precise control of signalling outputs consistent with predicted equilibria. These results show that membrane-adapted DL models can accurately encode and program TM association energetics and conformations. TMDF establishes a general framework for bottom-up design of TM proteins with programmable functions, advancing both mechanistic studies of membrane proteins and development of next-generation therapeutics. Less
Transcription factors TFs regulate gene expression by engaging chromatin remodeling complexes yet the structural principles governing these critical interactions remain poorly defined Here we uncover the molecular mechanism by which lineage-specific pioneer transcription factor PU encoded by SPI directly engages the BAF mSWI SNF chromatin remodeling complex First using a variety of genomic approaches we establish that BAF collaborates with PU to regulate transcription in AML cells Then using a combination of biochemistry and biophysics mass spectrometry-based protein footprinting and crystallography we map the PU -BAF A interface to a disordered region of PU that adopts a helical conformation upon ... More
Transcription factors (TFs) regulate gene expression by engaging chromatin remodeling complexes, yet the structural principles governing these critical interactions remain poorly defined. Here, we uncover the molecular mechanism by which lineage-specific pioneer transcription factor PU.1 (encoded by SPI1) directly engages the BAF (mSWI/SNF) chromatin remodeling complex. First, using a variety of genomic approaches, we establish that BAF collaborates with PU.1 to regulate transcription in AML cells. Then, using a combination of biochemistry and biophysics, mass spectrometry-based protein footprinting, and crystallography, we map the PU.1-BAF60A interface to a disordered region of PU.1 that adopts a helical conformation upon binding to the YEATS-like domain of BAF60A. Disruption of this functionally critical interface via knockdown abrogates the ability of PU.1 to rescue cell viability. Finally, we conducted a high-throughput screen that yielded small molecules which selectively bind BAF60A and disrupt PU.1 binding. Co-crystal structures reveal distinct compound binding modes that converge on a critical PU.1-BAF60A interaction hotspot. These findings define, for the first time, the structural interface between a pioneer transcription factor and the BAF complex and establish a platform that enables targeting transcription factor-chromatin remodeling complex interactions in cancer. Less
Dual-specificity mitogen-activated protein kinase MAPK phosphatases MKPs directly dephosphorylate and inactivate the MAPKs Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored Here we sought to define the molecular mechanism of MKP regulation through an allosteric site within its catalytic domain We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y is required to maintain the structural integrity of the allosteric pocket Along with molecular dynamics simulations these data provide insight into how changes in ... More
Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (MKPs) directly dephosphorylate and inactivate the MAPKs. Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established, a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored. Here, we sought to define the molecular mechanism of MKP5 regulation through an allosteric site within its catalytic domain. We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y435 is required to maintain the structural integrity of the allosteric pocket. Along with molecular dynamics simulations, these data provide insight into how changes in the allosteric pocket propagate conformational flexibility in the surrounding loops to reorganize catalytically crucial residues in the active site. Furthermore, Y435 contributes to the interaction with p38 MAPK and JNK, thereby promoting dephosphorylation. Collectively, these results highlight the role of Y435 in the allosteric site as a novel mode of MKP5 regulation by p38 MAPK and JNK Less
Transport and Golgi Organization Homolog TANGO protein deficiency disorder TDD is a rare autosomal recessive disorder characterized by multi-systemic abnormalities and significant phenotypic variability including neurodevelopmental delay seizures intermittent ataxia hypothyroidism rhabdomyolysis life-threatening metabolic derangements and cardiac arrhythmias Mutations in TANGO result in mitochondrial dysfunction abnormal lipid homeostasis with cardiolipin deficiency and impaired Golgi-ER trafficking in TANGO patient-derived cells Despite the wide recognition of the clinical manifestations of TDD and numerous molecular studies the precise function of TANGO and the pathophysiology of TDD remain poorly understood A computationally derived three-dimensional structure model suggested that TANGO adopts an -fold similar to ... More
Transport and Golgi Organization 2 Homolog (TANGO2) protein deficiency disorder (TDD) is a rare autosomal recessive disorder characterized by multi-systemic abnormalities and significant phenotypic variability including neurodevelopmental delay, seizures, intermittent ataxia, hypothyroidism, rhabdomyolysis, life-threatening metabolic derangements, and cardiac arrhythmias. Mutations in TANGO2 result in mitochondrial dysfunction, abnormal lipid homeostasis with cardiolipin deficiency, and impaired Golgi-ER trafficking in TANGO2 patient-derived cells. Despite the wide recognition of the clinical manifestations of TDD and numerous molecular studies, the precise function of TANGO2 and the pathophysiology of TDD remain poorly understood. A computationally derived three-dimensional structure model suggested that TANGO2 adopts an αββα-fold, similar to the N-terminal nucleophile aminohydrolase (Ntn) superfamily of proteins, but the experimentally verified structure has not been available thus far. Here, we present the first crystal structure of the recombinant human TANGO2, determined at 1.70 Å resolution. The X-ray structure data confirmed its predicted tertiary fold with similarity to the Ntn-hydrolase family of proteins, and the comparative analysis of the active site architecture, including residues involved in catalysis and putative ligand binding site, suggests a potential hydrolase function. Additional examination of the common mutation sites found in TDD patients provides insight regarding their potential effect on protein structure integrity. Less
This protocol describes the crystallization of Enterovirus EV- A protease mutant C A containing the VP - A junction in the active site The crystals form within - hours using a crystallization screen composed of M NaCl and ethanol The crystal structure was determined using X-ray diffraction resulting in hexagonal prism crystals in space group P with unit cell dimensions of and an average resolution of The protein was expressed using the plasmid Enterovirus Coxsackievirus A A protease
This protocol describes the crystallization of Enterovirus EV- A protease mutant C A containing the VP - A junction in the active site The crystals form within - hours using a crystallization screen composed of M NaCl and ethanol The crystal structure was determined using X-ray diffraction resulting in hexagonal prism crystals in space group P with unit cell dimensions of and an average resolution of The protein was expressed using the plasmid Enterovirus Coxsackievirus A A protease