419 Citations
The middle MID domain of eukaryotic Argonaute Ago proteins and archaeal and bacterial homologues mediates the interaction with the -terminal nucleotide of miRNA and siRNA guide strands The MID domain of human Ago hAgo is comprised of amino acids with a molecular weight of kDa MID adopts a Rossman-like beta -alpha -beta -alpha -beta -alpha -beta -alpha fold with a nucleotide specificity loop between beta and alpha Multiple crystal structures of nucleotides bound to hAgo MID have been reported whereby complexes were obtained by soaking ligands into crystals of MID domain alone This protocol describes a simplified one-step approach to ... More
The middle (MID) domain of eukaryotic Argonaute (Ago) proteins and archaeal and bacterial homologues mediates the interaction with the 5′-terminal nucleotide of miRNA and siRNA guide strands. The MID domain of human Ago2 (hAgo2) is comprised of 139 amino acids with a molecular weight of 15.56 kDa. MID adopts a Rossman-like beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-alpha4 fold with a nucleotide specificity loop between beta3 and alpha3. Multiple crystal structures of nucleotides bound to hAgo2 MID have been reported, whereby complexes were obtained by soaking ligands into crystals of MID domain alone. This protocol describes a simplified one-step approach to grow well-diffracting crystals of hAgo2 MID-nucleotide complexes by mixing purified His6-SUMO-MID fusion protein, Ulp1 protease, and excess nucleotide in the presence of buffer and precipitant. The crystal structures of MID complexes with UMP, UTP and 2′-3′ linked α-L-threofuranosyl thymidine-3′-triphosphate (tTTP) are presented. This article also describes fluorescence-based assays to measure dissociation constants (Kd) of MID-nucleotide interactions for nucleoside 5′-monophosphates and nucleoside 3′,5′-bisphosphates. Less
The impact of exchanging the light and heavy chains on the structures of bovine ultralong antibodies
The third complementary-determining regions of the heavy-chain CDR H variable regions VH of some cattle antibodies are highly extended consisting of or more residues These ultralong CDR Hs form -ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops The structure of the Fab fragment of a naturally paired bovine ultralong antibody D identified by single B-cell sequencing has been determined to A resolution By swapping the D native light chain with that of an unrelated antigen-unknown ultralong antibody it is shown ... More
The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These ‘ultralong’ CDR3Hs form �-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6 A ˚ resolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing. Less
Protein crystallization as opposed to well-established chromatography processes has the benefits to reduce production costs while reaching a comparable high purity However monitoring crystallization processes remains a challenge as the produced crystals may interfere with analytical measurements Especially for capturing proteins from complex feedstock containing various impurities establishing reliable process analytical technology PAT to monitor protein crystallization processes can be complicated In heterogeneous mixtures important product characteristics can be found by multivariate analysis and chemometrics thus contributing to the development of a thorough process understanding In this project an analytical set-up is established combining offline analytics on-line ultraviolet visible light ... More
Protein crystallization as opposed to well-established chromatography processes has the benefits to reduce production costs while reaching a comparable high purity. However, monitoring crystallization processes remains a challenge as the produced crystals may interfere with analytical measurements. Especially for capturing proteins from complex feedstock containing various impurities, establishing reliable process analytical technology (PAT) to monitor protein crystallization processes can be complicated. In heterogeneous mixtures, important product characteristics can be found by multivariate analysis and chemometrics, thus contributing to the development of a thorough process understanding. In this project, an analytical set-up is established combining offline analytics, on-line ultraviolet visible light (UV/Vis) spectroscopy, and in-line Raman spectroscopy to monitor a stirred-batch crystallization process with multiple phases and species being present. As an example process, the enzyme Lactobacillus kefir alcohol dehydrogenase (LkADH) was crystallized from clarified Escherichia coli (E. coli) lysate on a 300 mL scale in five distinct experiments, with the experimental conditions changing in terms of the initial lysate solution preparation method and precipitant concentration. Since UV/Vis spectroscopy is sensitive to particles, a cross-flow filtration (cross-flow filtration)-based bypass enabled the on-line analysis of the liquid phase providing information on the lysate composition regarding the nucleic acid to protein ratio. A principal component analysis (PCA) of in situ Raman spectra supported the identification of spectra and wavenumber ranges associated with productspecific information and revealed that the experiments followed a comparable, spectral trend when crystals were present. Based on preprocessed Raman spectra, a partial least squares (PLS) regression model was optimized to monitor the target molecule concentration in real-time. The off-line sample analysis provided information on the crystal number and crystal geometry by automated image analysis as well as the concentration of LkADH and host cell proteins (HCPs) In spite of a complex lysate suspension containing scattering crystals and various impurities, it was possible to monitor the target molecule concentration in a heterogeneous, multi-phase process using spectroscopic methods. With the presented analytical set-up of off-line, particle-sensitive on-line, and in-line analyzers, a crystallization capture process can be characterized better in terms of the geometry, yield, and purity of the crystals. Less
Pyruvate kinase PK deficiency is a rare genetic disorder that affects this critical enzyme within the glycolysis pathway In recent years Mitapivat MTPV AG- has emerged as a notable allosteric activator for treating PK deficiency However the allosteric regulatory effects exerted on PK by MTPV are yet to be comprehensively elucidated To shed light on the molecular mechanisms of the allosteric effects we employed crystallography and biophysical methods Our efforts yielded a high-resolution crystal structure of the PK tetramer complexed with MTPV at resolution Isothermal titration calorimetry measurements revealed that MTPV binds to human PK with an affinity of M ... More
Pyruvate kinase (PK) deficiency is a rare genetic disorder that affects this critical enzyme within the glycolysis pathway. In recent years, Mitapivat (MTPV, AG-348) has emerged as a notable allosteric activator for treating PK deficiency. However, the allosteric regulatory effects exerted on PK by MTPV are yet to be comprehensively elucidated. To shed light on the molecular mechanisms of the allosteric effects, we employed crystallography and biophysical methods. Our efforts yielded a high-resolution crystal structure of the PK tetramer complexed with MTPV at 2.1 Å resolution. Isothermal titration calorimetry measurements revealed that MTPV binds to human PK with an affinity of 1 μM. The enhanced structural details now allow for unambiguous analysis of the MTPV-filled cavity intricately embedded within the enzyme. Finally, the structure suggests that MTPV binding induces an allosteric effect on the B-domain situated proximal to the active site. In summary, our study provides valuable insights into the allosteric regulation of PK by MTPV and paves the way for further structure-based drug optimization for therapeutic interventions in PK deficiency. Less
Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak The A protease of these viruses is responsible for the self-cleavage of the poly protein allowing for correct folding and assembly of capsid proteins in the final stages of viral replication These A proteases are highly conserved between Enterovirus species such as Enterovirus A and Coxsackievirus A Inhibition of the A protease deranges capsid folding and assembly preventing formation of mature virions in host cells and making the protease a valuable target for ... More
Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of these viruses is responsible for the self-cleavage of the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. These 2A proteases are highly conserved between Enterovirus species, such as Enterovirus A71 and Coxsackievirus A16. Inhibition of the 2A protease deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. Herein, we describe a crystallographic fragment screening campaign that identified 75 fragments which bind to the 2A protease including 38 unique compounds shown to bind within the active site. These fragments reveal a path for the development of non-peptidomimetic inhibitors of the 2A protease with broad-spectrum anti-enteroviral activity. Less
The crystallization protocol and buffer conditions used to obtain reproducible SARS C V- Nucelocapsid crystals suitable for XChem fragment screening
The development of effective broad-spectrum antivirals forms an important part of preparing for future pandemics A cause for concern is the currently emerging pathogen Enterovirus D EV-D which primarily spreads through respiratory routes causing mostly mild to severe respiratory illness but in severe cases acute flaccid myelitis The C protease of EV-D is a potential target for the development of antiviral drugs due to its essential role in the viral life cycle and high sequence conservation This protocol was used to grow D C ProB crystals that were applied high-throughput crystallographic follow up compound screening on D C
The crystallization protocol and buffer conditions used to obtain Zika NS helicase crystals suitable for XChem fragment screening The Zika virus ZIKV discovered in Africa in swiftly spread across continents causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barr syndrome in adults Despite a decrease in prevalence the potential for a resurgence remains necessitating urgent therapeutic interventions Like other flaviviruses ZIKV presents promising drug targets within its replication machinery notably the NS helicase NS Hel protein which plays critical roles in viral replication However a lack of structural information impedes the development of specific inhibitors ... More
The crystallization protocol and buffer conditions used to obtain Zika NS3 helicase crystals suitable for XChem fragment screening. The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. This protocol was used to grow Zika NS3 crystals that were applied high-throughput crystallographic fragment screening on ZIKV NS3 Helicase. Less
The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening FFCS software suite This framework is further enriched by the option to utilize the Smart Digital User SDU software for automated data collection across the PXI PXII and PXIII beamlines In this work the newly developed HEIDI webpage https heidi psi ch is introduced a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state ... More
The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening (FFCS) software suite. This framework is further enriched by the option to utilize the Smart Digital User (SDU) software for automated data collection across the PXI, PXII and PXIII beamlines. In this work, the newly developed HEIDI webpage (https://heidi.psi.ch) is introduced: a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments. The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state transfer application programming interface (REST API). The migration of the local FFCS MongoDB instance to the cloud is highlighted and detailed. This transition ensures secure, encrypted and consistently accessible data through a robust and reliable REST API tailored for the FFCS software suite. Collectively, these advancements not only significantly elevate the user experience, but also pave the way for future expansions and improvements in the capabilities of the system. Less
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy This includes valorization of hemicellulosic fraction of plant biomass the second most abundant biopolymer from plant cell walls aiming to produce prebiotic oligosaccharides widely explored in food and feed industries In this work we conducted biochemical and biophysical characterization of a prokaryotic two-domain R champanellensis xylanase from glycoside hydrolase GH family RcXyn A and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH and GH families and a GH xylobiohydrolase RcXyn A liberates ... More
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R. champanellensis xylanase from glycoside hydrolase (GH) family 30 (RcXyn30A), and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH10 and GH11 families and a GH11 xylobiohydrolase. RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and is inefficient in cleaving unbranched oligosaccharides. Crystallographic structure of RcXyn30A catalytic domain was solved and refined to 1.37 Å resolution. Structural analysis of the catalytic domain releveled that its high affinity for glucuronic acid substituted xylan is due to the coordination of the substrate decoration by several hydrogen bonds and ionic interactions in the subsite −2. Furthermore, the protein has a larger β5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3) of the catalytic site. Finally, RcXyn30A activity is synergic to that of GH11 xylobiohydrolase. Less
With the rapid advancements in sequencing technologies the identification of single nucleotide mutations has surged surpassing our capacity for functional characterization Remarkably approximately of these disease-linked point mutations are situated within protein regions devoid of a well-defined D structure known as intrinsically disordered regions IDRs These IDRs are recognized for their pivotal roles in the regulation signaling and control of biological processes They can harbor short linear motifs SLiMs that act as mediators in protein-protein interactions PPIs often subject to regulation through post-translational modifications such as phosphorylation Investigating the impact of these IDR mutations on protein-protein interactions is essential for ... More
With the rapid advancements in sequencing technologies, the identification of
single nucleotide mutations has surged, surpassing our capacity for functional
characterization. Remarkably, approximately 20% of these disease-linked point
mutations are situated within protein regions devoid of a well-defined 3D structure,
known as intrinsically disordered regions (IDRs). These IDRs are recognized for
their pivotal roles in the regulation, signaling, and control of biological processes.
They can harbor short linear motifs (SLiMs) that act as mediators in protein-protein
interactions (PPIs), often subject to regulation through post-translational
modifications such as phosphorylation. Investigating the impact of these IDR
mutations on protein-protein interactions is essential for comprehending the
molecular mechanisms underlying human diseases.
In this doctoral thesis, I present a comprehensive exploration of a peptide-based
proteomics screen, employed to scrutinize 36 disease-associated mutations that
impair phosphorylation sites within IDRs. This approach entailed the immobilization
of synthetic peptides, corresponding to the mutated regions, onto a cellulose
membrane. These peptides were then utilized to capture interacting proteins from
cellular extracts. This method facilitated the simultaneous comparison of
interaction partners among wild-type, phosphorylated, and mutated peptide forms,
enabling the functional assessment of individual mutations. Our analysis
uncovered significant disparities
between the interactomes of phosphorylated and non-phosphorylated peptides,
changes frequently attributed to the disruption of phosphorylation-dependent
SLiMs.
Building on our findings, we placed particular emphasis on the S102P mutation
within the transcription factor GATAD1, a mutation associated with dilated
cardiomyopathy. Our screening demonstrated that this mutation disrupts a crucial
phosphorylation site responsible for 14-3-3 protein binding. To delve deeper into
this interaction, we conducted a thorough investigation, employing techniques such
as isothermal titration calorimetry, X-ray crystallography, and alanine scanning
coupled with mass spectrometry. Our meticulous analyses hinted at the regulatory
role of 14-3-3 binding in GATAD1's nucleocytoplasmic transport, achieved by
masking its nuclear localization signal.
In conclusion, this doctoral thesis focuses on the profound impact of pathogenic
mutations within human phosphorylation sites on protein-protein interactions. The
insights from our research shed fresh light on potential molecular mechanisms
underpinning the development of various human diseases, offering a promising
avenue for further investigation and therapeutic exploration. Less
single nucleotide mutations has surged, surpassing our capacity for functional
characterization. Remarkably, approximately 20% of these disease-linked point
mutations are situated within protein regions devoid of a well-defined 3D structure,
known as intrinsically disordered regions (IDRs). These IDRs are recognized for
their pivotal roles in the regulation, signaling, and control of biological processes.
They can harbor short linear motifs (SLiMs) that act as mediators in protein-protein
interactions (PPIs), often subject to regulation through post-translational
modifications such as phosphorylation. Investigating the impact of these IDR
mutations on protein-protein interactions is essential for comprehending the
molecular mechanisms underlying human diseases.
In this doctoral thesis, I present a comprehensive exploration of a peptide-based
proteomics screen, employed to scrutinize 36 disease-associated mutations that
impair phosphorylation sites within IDRs. This approach entailed the immobilization
of synthetic peptides, corresponding to the mutated regions, onto a cellulose
membrane. These peptides were then utilized to capture interacting proteins from
cellular extracts. This method facilitated the simultaneous comparison of
interaction partners among wild-type, phosphorylated, and mutated peptide forms,
enabling the functional assessment of individual mutations. Our analysis
uncovered significant disparities
between the interactomes of phosphorylated and non-phosphorylated peptides,
changes frequently attributed to the disruption of phosphorylation-dependent
SLiMs.
Building on our findings, we placed particular emphasis on the S102P mutation
within the transcription factor GATAD1, a mutation associated with dilated
cardiomyopathy. Our screening demonstrated that this mutation disrupts a crucial
phosphorylation site responsible for 14-3-3 protein binding. To delve deeper into
this interaction, we conducted a thorough investigation, employing techniques such
as isothermal titration calorimetry, X-ray crystallography, and alanine scanning
coupled with mass spectrometry. Our meticulous analyses hinted at the regulatory
role of 14-3-3 binding in GATAD1's nucleocytoplasmic transport, achieved by
masking its nuclear localization signal.
In conclusion, this doctoral thesis focuses on the profound impact of pathogenic
mutations within human phosphorylation sites on protein-protein interactions. The
insights from our research shed fresh light on potential molecular mechanisms
underpinning the development of various human diseases, offering a promising
avenue for further investigation and therapeutic exploration. Less
Despite their lack of a defined D structure intrinsically disordered regions IDRs of proteins play important biological roles Many IDRs contain short linear motifs SLiMs that mediate protein-protein interactions PPIs which can be regulated by post-translational modifications like phosphorylation of pathogenic missense mutations are found in IDRs and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms Here we employ peptide-based interaction proteomics to investigate disease-associated mutations affecting phosphorylation sites Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides often due to disrupted phosphorylation-dependent SLiMs We focused on a mutation of a serine phosphorylation ... More
Despite their lack of a defined 3D structure, intrinsically disordered regions (IDRs) of proteins play important biological roles. Many IDRs contain short linear motifs (SLiMs) that mediate protein-protein interactions (PPIs), which can be regulated by post-translational modifications like phosphorylation. 20% of pathogenic missense mutations are found in IDRs, and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms. Here, we employ peptide-based interaction proteomics to investigate 36 disease-associated mutations affecting phosphorylation sites. Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides, often due to disrupted phosphorylation-dependent SLiMs. We focused on a mutation of a serine phosphorylation site in the transcription factor GATAD1, which causes dilated cardiomyopathy. We find that this phosphorylation site mediates interaction with 14-3-3 family proteins. Follow-up experiments reveal the structural basis of this interaction and suggest that 14-3-3 binding affects GATAD1 nucleocytoplasmic transport by masking a nuclear localisation signal. Our results demonstrate that pathogenic mutations of human phosphorylation sites can significantly impact protein-protein interactions, offering insights into potential molecular mechanisms underlying pathogenesis. Less
Carbohydrate-active enzymes from the glycoside hydrolase family GH play a key role in processing lignocellulosic biomass Although the structural features of some GH enzymes are known the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel A enzyme utilizes to depolymerize cellulosic substrates we used a combination of biochemical assays X-ray crystallography small-angle X-ray scattering and molecular dynamics simulations The results reveal that BlCel A breaks down cellulosic substrates releasing cellobiose and glucose as the major products but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose ... More
Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations. The results reveal that BlCel9A breaks down cellulosic substrates, releasing cellobiose and glucose as the major products, but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose. In addition, fungal lytic polysaccharide oxygenase (LPMO) TtLPMO9H enhances depolymerization of crystalline cellulose by BlCel9A, while exhibiting minimal impact on amorphous cellulose. The crystal structures of BlCel9A in both apo form and bound to cellotriose and cellohexaose were elucidated, unveiling the interactions of BlCel9A with the ligands and their contribution to substrate binding and products release. MD simulation analysis reveals that BlCel9A exhibits higher interdomain flexibility under acidic conditions, and SAXS experiments indicate that the enzyme flexibility is induced by pH and/or temperature. Our findings provide new insights into BlCel9A substrate specificity and binding, and synergy with the LPMOs. Less
A novel monoacylglycerol MAG has been produced for use in the in meso lipid cubic phase crystallization of membrane proteins and complexes MAG differs from monoolein the most extensively used lipid for in meso crystallization in that it is shorter in chain length by one methylene and its cis olefinic bond is two carbons closer to the glycerol headgroup These changes in structure alter the phase behavior of the hydrated lipid and the microstructure of the corresponding mesophases formed Temperature composition phase diagrams for MAG have been constructed using small- and wide-angle X-ray scattering over a range of temperatures and ... More
A novel monoacylglycerol, 7.10 MAG, has been produced for use in the in meso (lipid cubic phase) crystallization of membrane proteins and complexes. 7.10 MAG differs from monoolein, the most extensively used lipid for in meso crystallization, in that it is shorter in chain length by one methylene and its cis olefinic bond is two carbons closer to the glycerol headgroup. These changes in structure alter the phase behavior of the hydrated lipid and the microstructure of the corresponding mesophases formed. Temperature–composition phase diagrams for 7.10 MAG have been constructed using small- and wide-angle X-ray scattering over a range of temperatures and hydration levels that span those used for crystallization. The phase diagrams include lamellar crystalline, fluid isotropic, lamellar liquid-crystalline, cubic-Ia3d, and cubic-Pn3m phases, as observed with monoolein. Conspicuous by its absence is the inverted hexagonal phase which is rationalized on the basis of 7.10 MAG’s chemical constitution. The cubic phase prepared with the new lipid facilitates the growth of crystals that were used to generate high-resolution structures of intramembrane β-barrel and α-helical proteins. Compatibility of fully hydrated 7.10 MAG with cholesterol and phosphatidylcholine means that these two lipids can be used as additives to optimize crystallogenesis in screening trials with 7.10 MAG as the host lipid. Less
The mammalian orthoreovirus reovirus NS protein is required for formation of replication compartments that support viral genome replication and capsid assembly Despite its functional importance a mechanistic understanding of NS is lacking We conducted structural and biochemical analyses of a NS mutant that forms dimers instead of the higher-order oligomers formed by wildtype WT NS The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT NS filaments in complex with RNA observed using cryo-EM The interior of the helical assembly is of appropriate diameter to bind RNA The helical assembly ... More
The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication. Less
Pacak-Zhuang syndrome is caused by mutations in the EPAS gene which encodes for one of the three hypoxia-inducible factor alpha HIF paralogs HIF and is associated with defined but varied phenotypic presentations including neuroendocrine tumors and polycythemia However the mechanisms underlying the complex genotype-phenotype correlations remain incompletely understood Here we devised a quantitative method for determining the dissociation constant Kd of the HIF peptides containing disease-associated mutations and the catalytic domain of prolyl-hydroxylase PHD using microscale thermophoresis MST and showed that neuroendocrine-associated Class HIF mutants have distinctly higher Kd than the exclusively polycythemia-associated Class HIF mutants Based on the co-crystal ... More
Pacak-Zhuang syndrome is caused by mutations in the EPAS1 gene, which encodes for one of the three hypoxia-inducible factor alpha (HIFα) paralogs HIF2α and is associated with defined but varied phenotypic presentations including neuroendocrine tumors and polycythemia. However, the mechanisms underlying the complex genotype-phenotype correlations remain incompletely understood. Here, we devised a quantitative method for determining the dissociation constant (Kd) of the HIF2α peptides containing disease-associated mutations and the catalytic domain of prolyl-hydroxylase (PHD2) using microscale thermophoresis (MST) and showed that neuroendocrine-associated Class 1 HIF2α mutants have distinctly higher Kd than the exclusively polycythemia-associated Class 2 HIF2α mutants. Based on the co-crystal structure of PHD2/HIF2α peptide complex at 1.8 Å resolution, we showed that the Class 1 mutated residues are localized to the critical interface between HIF2α and PHD2, adjacent to the PHD2 active catalytic site, while Class 2 mutated residues are localized to the more flexible region of HIF2α that makes less contact with PHD2. Concordantly, Class 1 mutations were found to significantly increase HIF2α-mediated transcriptional activation in cellulo compared to Class 2 counterparts. These results reveal a structural mechanism in which the strength of the interaction between HIF2α and PHD2 is at the root of the general genotype-phenotype correlations observed in Pacak-Zhuang syndrome. Less
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy especially considering the anthropogenic effects in the global warming and natural resources depletion This includes the valorization of the hemicellulosic fraction of plant biomass aiming to produce prebiotic oligosaccharides widely explored in food and feed industries In the present work we conducted biochemical and biophysical characterization of two prokaryotic xylanases of family from Bacillus pumilus and Ruminococcus champanellensis and assessed their applicability for xylooligosaccharides production using alkaline pretreated corn cob and eucalyptus sawdust collected from a local market and ... More
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy, especially considering the anthropogenic effects in the global warming and natural resources depletion. This includes the valorization of the hemicellulosic fraction of plant biomass, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In the present work, we conducted biochemical and biophysical characterization of two prokaryotic xylanases of family 30_8 from Bacillus pumilus and Ruminococcus champanellensis, and assessed their applicability for xylooligosaccharides production using alkaline pretreated corn cob and eucalyptus sawdust collected from a local market and a sawmill shop in Araraquara, respectively. Mass spectrometry and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis revealed that RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and proved to be highly inefficient in the cleavage of X4, X5 and X6, whereas BpXyn30A produces both linear and branched oligosaccharides. Crystallographic structure of BpXyn30A and RcXyn30A catalytic domain were solved and refined to 2.16 Å and 1.37Å resolution, respectively. Structural analysis of the enzymes binding cleft showed a conserved set of amino acids interacting with glucuronic acid substitution in the subsite -2b by several hydrogen bonds and ionic interactions, a characteristic shared between true glucunoxylanases. Furthermore, RcXyn30A has a larger β5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3). Finally, B. pumilus xylanase obtained higher conversion yields from pretreated biomasses than RcXyn30A, although the latter presents a specific activity against glucuronoxylan 9 times higher than the former. Less
To identify starting points for therapeutics targeting SARS-CoV- the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket Of soaked fragments a total of hits bound either in the active site hits a remote binding pocket three hits or at crystal-packing interfaces two hits Notably two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue The structures ... More
To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography. Less
The tripartite ATP-independent periplasmic TRAP transporters use an extra cytoplasmic substrate binding protein SBP to transport a wide variety of substrates in bacteria and archaea The SBP can adopt an open- or closed state depending on the presence of substrate The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient Insights from experimental structures structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein Here we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM ... More
The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, is locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, is locked in either its assumed inward- or outward-facing states. We characterize the disulfide-locked constructs and use single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa. Less
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold They are generally described as electron carriers that rely on a T copper centre coordinated by four ligands provided by the folded polypeptide The discovery of novel cupredoxins demonstrates the high diversity of this family with variations in terms of copper-binding ligands copper centre geometry redox potential as well as biological function AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans AcoP presents original features including high resistance to acidic pH and a constrained green-type copper centre of high ... More
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper centre coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in terms of copper-binding ligands, copper centre geometry, redox potential, as well as biological function. AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features, including high resistance to acidic pH and a constrained green-type copper centre of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structures, including native reduced AcoP at 1.65 Å resolution, unveil a typical cupredoxin fold. The presence of extended loops, never observed in previously characterized cupredoxins, might account for the interaction of AcoP with physiological partners. The Cu-ligand distances, determined by both X-ray diffraction and EXAFS, show that the AcoP metal centre seems to present both T1 and T1.5 features, in turn suggesting that AcoP might not fit well to the coupled distortion model. The crystal structures of two AcoP mutants confirm that the active centre of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active centre rigidity due to the presence of an extensive hydrogen bond network. Finally, we show that other cupredoxins do not perfectly follow the coupled distortion model as well, raising the suspicion that further alternative models to describe copper centre geometries need to be developed, while the importance of rack-induced contributions should not be underestimated. Less
The phospholipase D PLD family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids PLD is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes yielding a soluble active form The deficiency of PLD leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors While the mechanism of PLD phospholipase activity has been extensively characterized not much is known about how PLDs bind and hydrolyze nucleic acids Here we determined the high-resolution crystal structure of the luminal N-glycosylated domain of ... More
The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3. Less
The pathogen Paracoccidioides lutzii Pb is found in South America countries Colombia Ecuador Venezuela and Brazil especially in the central west and north regions of the latter It belongs to the Ajellomycetaceae family Onygenales order and is typically thermodimorphic presenting yeast cells when it grows in animal tissues but mycelia when in the environment where it produces the infectious propagule This fungus is one of the etiologic agents of Paracoccidioidomycosis PCM the most important endemic fungal infection in Latin America Investigations on its genome have contributed to a better understanding about its metabolism and revealed the complexity of several metabolic ... More
The pathogen Paracoccidioides lutzii (Pb01) is found in South America countries Colombia, Ecuador, Venezuela and Brazil, especially in the central, west, and north regions of the latter. It belongs to the Ajellomycetaceae family, Onygenales order, and is typically thermodimorphic, presenting yeast cells when it grows in animal tissues, but mycelia when in the environment, where it produces the infectious propagule. This fungus is one of the etiologic agents of Paracoccidioidomycosis (PCM), the most important endemic fungal infection in Latin America. Investigations on its genome have contributed to a better understanding about its metabolism and revealed the complexity of several metabolic glycolytic pathways. Glyceraldehyde-3-Phosphate Dehydrogenase from Paracoccidioides lutzii (PlGAPDH) is considered a moonlighting protein and participates in several biological processes of this pathogen. The enzyme was expressed and purified, as seen in SDS-PAGE gel, crystallized and had its three dimensional structure (3D) determined in complex with NAD+, a sulphate ion and d-galactonic acid, therefore, a type of ‘GAA site’. It is the first GAPDH structure to show this chemical type in this site and how this protein can bind an acid derived from oxidation of a linear hexose. Less
Gonorrhea a prevalent sexually transmitted infection affecting millions annually worldwide is caused by Neisseria gonorrhoeae Ngo a superbug resistant to all antibiotic classes Compounding the challenge the absence of protective immunity upon infection allows for reinfection and a viable vaccine against gonococcal infection remains elusive In response to infection the human host deploys nutritional immunity sequestering essential metals like iron from invading bacteria using metal binding proteins To overcome this Ngo employs outer-membrane TonB-dependent transporters TdTs like HpuAB to acquire iron from host metal binding proteins such as hemoglobin Hb Part of our study focused on HpuA the lipoprotein component ... More
Gonorrhea, a prevalent sexually transmitted infection affecting millions annually worldwide, is caused by Neisseria gonorrhoeae (Ngo), a "superbug" resistant to all antibiotic classes. Compounding the challenge, the absence of protective immunity upon infection allows for reinfection, and a viable vaccine against gonococcal infection remains elusive.
In response to infection, the human host deploys nutritional immunity, sequestering essential metals like iron from invading bacteria, using metal binding proteins. To overcome this, Ngo employs outer-membrane TonB-dependent transporters (TdTs), like HpuAB, to acquire iron from host metal binding proteins, such as hemoglobin (Hb).
Part of our study focused on HpuA, the lipoprotein component of the HpuAB system. Mutations targeting hydrophobic residues crucial for Hb interaction were studied. Results demonstrated that without HpuB, strains failed to grow, emphasizing HpuB's role in iron internalization. Notably, when HpuB is produced, deletion and insertion mutations in loop 2 of HpuA affected growth and binding to Hb.
Further investigations into HpuB, the transmembrane protein of the HpuAB system, uncovered essential loop regions for binding and growth on Hb as a sole iron source. Deletion mutations in loops 2, 3, and 4 facilitated binding and growth independently of HpuA production. Intriguingly, mutations in loop 7 abrogated binding and impaired growth in the absence of HpuA, but partial growth and full binding recovery occurred when HpuA was present. This highlighted the importance of loop 7 in iron acquisition and suggested a potential role for both HpuA and HpuB in the binding Hb.
As a combination of non-binding TdT mutants is hypothesized to have the potential to improve vaccine efficacy and provide protection, identifying non-binding HpuB mutants could be important. In summary, this research sheds light on the intricacies of the HpuAB system, contributing valuable insights that could inform the development of an effective gonorrhea vaccine. Less
In response to infection, the human host deploys nutritional immunity, sequestering essential metals like iron from invading bacteria, using metal binding proteins. To overcome this, Ngo employs outer-membrane TonB-dependent transporters (TdTs), like HpuAB, to acquire iron from host metal binding proteins, such as hemoglobin (Hb).
Part of our study focused on HpuA, the lipoprotein component of the HpuAB system. Mutations targeting hydrophobic residues crucial for Hb interaction were studied. Results demonstrated that without HpuB, strains failed to grow, emphasizing HpuB's role in iron internalization. Notably, when HpuB is produced, deletion and insertion mutations in loop 2 of HpuA affected growth and binding to Hb.
Further investigations into HpuB, the transmembrane protein of the HpuAB system, uncovered essential loop regions for binding and growth on Hb as a sole iron source. Deletion mutations in loops 2, 3, and 4 facilitated binding and growth independently of HpuA production. Intriguingly, mutations in loop 7 abrogated binding and impaired growth in the absence of HpuA, but partial growth and full binding recovery occurred when HpuA was present. This highlighted the importance of loop 7 in iron acquisition and suggested a potential role for both HpuA and HpuB in the binding Hb.
As a combination of non-binding TdT mutants is hypothesized to have the potential to improve vaccine efficacy and provide protection, identifying non-binding HpuB mutants could be important. In summary, this research sheds light on the intricacies of the HpuAB system, contributing valuable insights that could inform the development of an effective gonorrhea vaccine. Less
MbtI from Mycobacterium tuberculosis Mtb is a Mg -dependent salicylate synthase belonging to the chorismate-utilizing enzyme CUE family As a fundamental player in iron acquisition MbtI promotes the survival and pathogenicity of Mtb in the infected host Hence it has emerged in the last decade as an innovative potential target for the anti-virulence therapy of tuberculosis In this context -phenylfuran- -carboxylic acids have been identified as potent MbtI inhibitors The first co-crystal structure of MbtI in complex with a member of this class was described in showing the enzyme adopting an open configuration Due to the high mobility of the ... More
MbtI from Mycobacterium tuberculosis (Mtb) is a Mg2+-dependent salicylate synthase, belonging to the chorismate-utilizing enzyme (CUE) family. As a fundamental player in iron acquisition, MbtI promotes the survival and pathogenicity of Mtb in the infected host. Hence, it has emerged in the last decade as an innovative, potential target for the anti-virulence therapy of tuberculosis. In this context, 5-phenylfuran-2-carboxylic acids have been identified as potent MbtI inhibitors. The first co-crystal structure of MbtI in complex with a member of this class was described in 2020, showing the enzyme adopting an open configuration. Due to the high mobility of the loop adjacent to the binding pocket, large portions of the amino acid chain were not defined in the electron density map, hindering computational efforts aimed at structure-driven ligand optimization. Herein, we report a new, high-resolution co-crystal structure of MbtI with a furan-based derivative, in which the closed configuration of the enzyme allowed tracing the entirety of the active site pocket in the presence of the bound inhibitor. Moreover, we describe a new crystal structure of MbtI in open conformation and in complex with the known inhibitor methyl-AMT, suggesting that in vitro potency is not related to the observed enzyme conformation. These findings will prove fundamental to enhance the potency of this series via rational structure-based drug-design approaches. Less
The mammalian Voltage-gated sodium Nav channel is composed of a single subunit kDa a multi-pass membrane protein that renders ion selectivity and two or more Nav subunits kDa that are Type I single-pass membrane proteins and regulate Nav subunit function These subunits are assembled on the plasma membrane of electrically-excitable cells as an intrinsic membrane protein complex and help to initiate and propagate the action potential The four major mammalian Nav -subunit isoforms Nav proteins possess an N-terminal extracellular Immunoglobulin Ig domain ECD a single transmembrane -helix and an intracellular C-terminal region ICD This thesis is mainly focused on the ... More
The mammalian Voltage-gated sodium (Nav) channel is composed of a single α subunit (~ 260 kDa), a multi-pass membrane protein that renders ion selectivity and two or more Navβ subunits (25‒40 kDa), that are Type I single-pass membrane proteins and regulate Navα subunit function. These subunits are assembled on the plasma membrane of electrically-excitable cells as an intrinsic membrane protein complex and help to initiate and propagate the action potential. The four major mammalian Navβ-subunit isoforms, Navβ1‒4 proteins possess an N-terminal extracellular Immunoglobulin (Ig) domain (ECD), a single transmembrane α-helix, and an intracellular C-terminal region (ICD).
This thesis is mainly focused on the structural biology aspects of the human Navβ3 subunit. It reports the atomic structure of the Navβ3-Ig domain as determined by X-ray crystallography. Interestingly, the Navβ3-Ig domain is observed as a trimer in the crystal structure. The homo-trimer assembly interface lies at the N-terminus and is constrained by a disulphide bond not normally present in Ig domains. The Navβ3 subunit Ig domain is known to be glycosylated and contains four potential N-linked glycosylation sites. However, the X-ray crystallography was conducted on deglycosylated protein. Using computational modelling, it is shown that glycan addition would not interfere with Navβ3-Ig domain trimerization. Independent evidence gathered using Analytical Ultracentrifugation (crosslinked, glycosylated Navβ3-Ig domain, in vitro), Proximity Ligation Assay (full-length Navβ3, in vivo), Atomic Force Microscopy (isolated full-length Navβ3, in vitro) and Photo-activated Localisation Microscopic experiments (full-length Navβ3, in situ) support the view that the Navβ3 subunit can form trimers when expressed in cells. The biological significance of Navβ3 subunit trimerization is discussed.
Strategies to express and purify the Navβ1/β2/β4-Ig domains were made. Wild type Navβ2- and Navβ4-Ig domains exist as monomers and dimers, simultaneously in solution, although crystals that diffracted to the necessary resolution were not produced. Less
This thesis is mainly focused on the structural biology aspects of the human Navβ3 subunit. It reports the atomic structure of the Navβ3-Ig domain as determined by X-ray crystallography. Interestingly, the Navβ3-Ig domain is observed as a trimer in the crystal structure. The homo-trimer assembly interface lies at the N-terminus and is constrained by a disulphide bond not normally present in Ig domains. The Navβ3 subunit Ig domain is known to be glycosylated and contains four potential N-linked glycosylation sites. However, the X-ray crystallography was conducted on deglycosylated protein. Using computational modelling, it is shown that glycan addition would not interfere with Navβ3-Ig domain trimerization. Independent evidence gathered using Analytical Ultracentrifugation (crosslinked, glycosylated Navβ3-Ig domain, in vitro), Proximity Ligation Assay (full-length Navβ3, in vivo), Atomic Force Microscopy (isolated full-length Navβ3, in vitro) and Photo-activated Localisation Microscopic experiments (full-length Navβ3, in situ) support the view that the Navβ3 subunit can form trimers when expressed in cells. The biological significance of Navβ3 subunit trimerization is discussed.
Strategies to express and purify the Navβ1/β2/β4-Ig domains were made. Wild type Navβ2- and Navβ4-Ig domains exist as monomers and dimers, simultaneously in solution, although crystals that diffracted to the necessary resolution were not produced. Less
Norovirus is the leading cause of viral gastroenteritis worldwide and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI noroviruses despite most global outbreaks being caused by GII noroviruses Here we determined the crystal structures of the GII Sydney ligand-free norovirus protease at and at with a covalently bound high-affinity IC M protease inhibitor NV- We show that the active sites of the ligand-free protease structure are present in both open and closed conformations as determined by their Arg side ... More
Norovirus is the leading cause of viral gastroenteritis worldwide, and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections. The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI.1 noroviruses, despite most global outbreaks being caused by GII.4 noroviruses. Here, we determined the crystal structures of the GII.4 Sydney 2012 ligand-free norovirus protease at 2.79 Å and at 1.83 Å with a covalently bound high-affinity (IC50 = 0.37 µM) protease inhibitor (NV-004). We show that the active sites of the ligand-free protease structure are present in both open and closed conformations, as determined by their Arg112 side chain orientation. A comparative analysis of the ligand-free and ligand-bound protease structures reveals significant structural differences in the active site cleft and substrate-binding pockets when an inhibitor is covalently bound. We also report a second molecule of NV-004 non-covalently bound within the S4 substrate binding pocket via hydrophobic contacts and a water-mediated hydrogen bond. These new insights can guide structure-aided drug design against the GII.4 genogroup of noroviruses. Less
Acute gastroenteritis caused by human noroviruses HuNoVs is a significant global health and economic burden and is without licensed vaccines or antiviral drugs The GII HuNoV causes most epidemics worldwide This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens HBGA the determinants of cell attachment and susceptibility hampering the development of immunotherapeutics Here we show that a llama-derived nanobody M neutralizes multiple GII variants with high potency in human intestinal enteroids The crystal structure of M complexed with the protruding domain of the GII capsid protein VP revealed ... More
Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide. This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens (HBGA), the determinants of cell attachment and susceptibility, hampering the development of immunotherapeutics. Here, we show that a llama-derived nanobody M4 neutralizes multiple GII.4 variants with high potency in human intestinal enteroids. The crystal structure of M4 complexed with the protruding domain of the GII.4 capsid protein VP1 revealed a conserved epitope, away from the HBGA binding site, fully accessible only when VP1 transitions to a “raised” conformation in the capsid. Together with dynamic light scattering and electron microscopy of the GII.4 VLPs, our studies suggest a mechanism in which M4 accesses the epitope by altering the conformational dynamics of the capsid and triggering its disassembly to neutralize GII.4 infection. Less
Septins are membrane-associated GTP-binding proteins that are present in most eukaryotes They polymerize to play important roles as scaffolds and or diffusion barriers as part of the cytoskeleton -Helical coiled-coil domains are believed to contribute to septin assembly and those observed in both human SEPT and SEPT form antiparallel homodimers These are not compatible with their parallel heterodimeric organization expected from the current model for protofilament assembly but they could explain the interfilament cross-bridges observed by microscopy Here the first structure of a heterodimeric septin coiled coil is presented that between SEPT and SEPT the former is a SEPT SEPT ... More
Septins are membrane-associated, GTP-binding proteins that are present in most eukaryotes. They polymerize to play important roles as scaffolds and/or diffusion barriers as part of the cytoskeleton. α-Helical coiled-coil domains are believed to contribute to septin assembly, and those observed in both human SEPT6 and SEPT8 form antiparallel homodimers. These are not compatible with their parallel heterodimeric organization expected from the current model for protofilament assembly, but they could explain the interfilament cross-bridges observed by microscopy. Here, the first structure of a heterodimeric septin coiled coil is presented, that between SEPT14 and SEPT7; the former is a SEPT6/SEPT8 homolog. This new structure is parallel, with two long helices that are axially shifted by a full helical turn with reference to their sequence alignment. The structure also has unusual knobs-into-holes packing of side chains. Both standard seven-residue (heptad) and the less common 11-residue (hendecad) repeats are present, creating two distinct regions with opposite supercoiling, which gives rise to an overall straight coiled coil. Part of the hendecad region is required for heterodimerization and therefore may be crucial for selective septin recognition. These unconventional sequences and structural features produce a metastable heterocomplex that nonetheless has enough specificity to promote correct protofilament assembly. For instance, the lack of supercoiling may facilitate unzipping and transitioning to the antiparallel homodimeric state. Less
Biomolecular condensates are membrane-less cellular foci formed via liquid-liquid-phase separation LLPS of specific biological macromolecules to provide specialized compartments for regulating cellular functions Many viral proteins undergo LLPS to form such condensates to support viral replication and evade host antiviral responses and thus these condensates are potential targets for designing antivirals Human noroviruses HuNoV cause epidemic and sporadic gastroenteritis worldwide and are of significant health and economic burden Here we show that the RNA-dependent-RNA polymerase RdRp of the pandemic GII HuNoV which is essential for viral replication forms distinct condensates capable of recruiting other viral replication components Confocal microscopy and ... More
Biomolecular condensates are membrane-less cellular foci formed via liquid-liquid-phase separation (LLPS) of specific biological macromolecules to provide specialized compartments for regulating cellular functions. Many viral proteins undergo LLPS to form such condensates to support viral replication and evade host antiviral responses, and thus, these condensates are potential targets for designing antivirals. Human noroviruses (HuNoV) cause epidemic and sporadic gastroenteritis worldwide and are of significant health and economic burden. Here, we show that the RNA-dependent-RNA polymerase (RdRp) of the pandemic GII.4 HuNoV, which is essential for viral replication, forms distinct condensates capable of recruiting other viral replication components. Confocal microscopy and light scattering experiments show that RdRp phase separates into dynamic liquid-like condensates at physiological conditions. These condensates exhibit all the signature features of LLPS, including fluorescence recovery after photo-bleaching, droplet-fusion, surface wetting, and dripping in vitro and in live cells. More importantly, within these condensates, the RdRp exhibits a significant time-dependent increase in its enzymatic activity and recruits other components, such as RNA and the viral genome-linked protein (VPg), which are essential for viral replication. Such condensates, recognized by anti-RdRp antibodies, are observed in HuNoV-infected human intestinal enteroid cultures. Together, our studies demonstrate a hitherto unsuspected activity of HuNoV RdRp to form LLPS, which we suggest provides distinct cellular sites for efficient viral replication and its regulation. Less
Schistosomiasis is caused by parasites of the genus Schistosoma which infect more than million people While praziquantel PZQ has been the main drug for controlling schistosomiasis for over four decades PZQ drug resistant strains have already been reported highlighting the need to search for new schistosomicidal drugs S mansoni survival relies on the redox enzyme thioredoxin glutathione reductase Sm TGR a validated target for the development of new anti-schistosomal drugs Here we report a fragment screening campaign of compounds against Sm TGR using X-ray crystallography and our efforts to optimize the hits found into potent inhibitors We observed binding events ... More
Schistosomiasis is caused by parasites of the genus Schistosoma , which infect more than 200 million people. While praziquantel (PZQ) has been the main drug for controlling schistosomiasis for over four decades, PZQ drug resistant strains have already been reported, highlighting the need to search for new schistosomicidal drugs. S. mansoni survival relies on the redox enzyme thioredoxin glutathione reductase ( Sm TGR), a validated target for the development of new anti-schistosomal drugs. Here we report a fragment screening campaign of 768 compounds against Sm TGR using X-ray crystallography and our efforts to optimize the hits found into potent inhibitors. We observed 49 binding events involving 35 distinct molecular fragments which were found to be distributed across 16 binding sites. Most sites are described for the first time within Sm TGR, a noteworthy exception being the “door stop pocket” near the NADPH binding site. Fragments binding to the latter were prioritized to undergo a “SAR by catalog” strategy for optimization into potential inhibitors. A search for compounds containing any of the prioritized fragments as a substructure was made in commercial databases. The ability of these compounds to inhibit Sm TGR was predicted based on a binary ML classification model, followed by an analysis of the putative binding mode by molecular docking. The 38 best ranked compounds were purchased and experimentally evaluated for Sm TGR inhibition. Compound 14 inhibited 63.6% of enzyme activity at 100 µM and presented an estimated IC 50 of 33 µM against Sm TGR. Less
Several hydrolases have been described to degrade polyethylene terephthalate PET at moderate temperatures ranging from C to C These mesophilic PET hydrolases PETases are less efficient in degrading this plastic polymer than their thermophilic homologs and have therefore been the subject of many protein engineering campaigns However enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations Additionally evaluating the improvements in both activity and stability requires screening numerous variants either individually or using high-throughput screening methods Here we utilize instead the design of chimeras ... More
Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25°C to 40°C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic homologs and have, therefore, been the subject of many protein engineering campaigns. However, enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations. Additionally, evaluating the improvements in both activity and stability requires screening numerous variants, either individually or using high-throughput screening methods. Here, we utilize instead the design of chimeras as a protein engineering strategy to increase the activity and stability of Mors1, an Antarctic PETase active at 25°C. First, we obtained the crystal structure of Mors1 at 1.6 Å resolution, which we used as a scaffold for structure- and sequence-based chimeric design. Then, we designed a Mors1 chimera via loop exchange of a highly divergent active site loop from the thermophilic leaf-branch compost cutinase (LCC) into the equivalent region in Mors1. After restitution of an active site disulfide bond into this chimera, the enzyme exhibited a shift in optimal temperature for activity to 45°C and an increase in fivefold in PET hydrolysis when compared with wild-type Mors1 at 25°C. Our results serve as a proof of concept of the utility of chimeric design to further improve the activity and stability of PETases active at moderate temperatures. Less
The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte TIL therapy We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors TCRs following successful TIL therapy for stage IV malignant melanoma Remarkably individual TCRs could target multiple different tumor types via the HLA A -restricted epitopes EAAGIGILTV LLLGIGILVL and NLSALGIFST from Melan A BST and IMP respectively Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A G-I L-G-I-x-x-x recognition motif Multi-epitope targeting allows individual ... More
The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such “multipronged” T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies. Less
Arc is an immediate early gene that regulates synaptic plasticity in glutamatergic neurons The formation of new long-term memories requires functioning Arc protein Arc is both a protein interaction hub at the dendritic spines and is able to encapsulate its own mRNA in virus-like capsids that transfect nearby cells Relatively little is known about the structure of the mammalian Arc protein It consists of mainly -helical structures that make up the N- and Cterminal domain which are connected by a flexible linker and flanked by flexible N- and Cterminal tails Arc is found in many oligomeric states ranging from dimers ... More
Arc is an immediate early gene that regulates synaptic plasticity in glutamatergic neurons. The
formation of new long-term memories requires functioning Arc protein. Arc is both a protein
interaction hub at the dendritic spines and is able to encapsulate its own mRNA in virus-like
capsids that transfect nearby cells. Relatively little is known about the structure of the
mammalian Arc protein. It consists of mainly α-helical structures that make up the N- and Cterminal
domain, which are connected by a flexible linker and flanked by flexible N- and Cterminal
tails. Arc is found in many oligomeric states ranging from dimers to the predicted 140-
subunit capsids. This study aimed to solve the full-length structure of dimeric mammalian Arc
using X-ray crystallography and single-particle cryo-EM. Nanobodies that bind to Arc with
high affinity were used to stabilize and enlarge the dimeric complex. Structural information
about the Arc-nanobody complex was gathered using SAXS and compared with structures
predicted using AlphaFold. The results show that AlphaFold struggles to predict structures that
match the low-resolution structures of Arc in solution obtained from SAXS, likely due to Arc’s
structural flexibility and tendency to oligomerize. The study has also provided insight into the
binding dynamics of these nanobodies to Arc and highlighted their many uses in addition to
structural chaperones. Optimization of the sample preparation and data collection were
performed for the use in single-particle cryo-EM to solve the dimeric structure of full-length
Arc, although the data collection and processing have not been finished as of this moment. This
data could provide valuable new structural information about Arc, which will help better the
understanding of Arc’s functions and roles in disease. Less
formation of new long-term memories requires functioning Arc protein. Arc is both a protein
interaction hub at the dendritic spines and is able to encapsulate its own mRNA in virus-like
capsids that transfect nearby cells. Relatively little is known about the structure of the
mammalian Arc protein. It consists of mainly α-helical structures that make up the N- and Cterminal
domain, which are connected by a flexible linker and flanked by flexible N- and Cterminal
tails. Arc is found in many oligomeric states ranging from dimers to the predicted 140-
subunit capsids. This study aimed to solve the full-length structure of dimeric mammalian Arc
using X-ray crystallography and single-particle cryo-EM. Nanobodies that bind to Arc with
high affinity were used to stabilize and enlarge the dimeric complex. Structural information
about the Arc-nanobody complex was gathered using SAXS and compared with structures
predicted using AlphaFold. The results show that AlphaFold struggles to predict structures that
match the low-resolution structures of Arc in solution obtained from SAXS, likely due to Arc’s
structural flexibility and tendency to oligomerize. The study has also provided insight into the
binding dynamics of these nanobodies to Arc and highlighted their many uses in addition to
structural chaperones. Optimization of the sample preparation and data collection were
performed for the use in single-particle cryo-EM to solve the dimeric structure of full-length
Arc, although the data collection and processing have not been finished as of this moment. This
data could provide valuable new structural information about Arc, which will help better the
understanding of Arc’s functions and roles in disease. Less
Latrophilins ADGRLs are conserved adhesion-type G protein-coupled receptors associated with early embryonic morphogenesis defects lethality and sterility across multiple model organisms However their mechanistic roles in embryogenesis and the identity of their binding ligands remain unknown Here we identified a cell-surface receptor TOL- the sole Toll-like receptor in C elegans as a novel ligand for the C elegans Latrophilin LAT- The extracellular lectin domain of LAT- directly binds to the second leucine-rich repeat domain of TOL- The highresolution crystal structure and the cryo-EM density map of the LAT- TOL- ectodomain complex reveal a previously-unobserved mode of one-to-one interaction enabled by ... More
Latrophilins/ADGRLs are conserved adhesion-type G protein-coupled receptors associated with early embryonic morphogenesis defects, lethality, and sterility across multiple model organisms. However, their mechanistic roles in embryogenesis and the identity of their binding ligands remain unknown. Here, we identified a cell-surface receptor, TOL-1, the sole Toll-like receptor in C. elegans, as a novel ligand for the C. elegans Latrophilin, LAT-1. The extracellular lectin domain of LAT-1 directly binds to the second leucine-rich repeat domain of TOL-1. The highresolution crystal structure and the cryo-EM density map of the LAT-1–TOL-1 ectodomain complex reveal a previously-unobserved mode of one-to-one interaction enabled by a large interface. CRISPR/Cas9-mediated mutation of key interface residues selectively disrupted the endogenous LAT-1–TOL-1 interaction in C. elegans, leading to partial sterility, lethality, and malformed embryos. Thus, TOL-1 binding to LAT-1 represents a receptor-ligand axis essential for animal morphogenesis. Less
Studying mechanisms of bacterial biofilm generation is of vital importance to understanding bacterial cell cell communication multicellular cohabitation principles and the higher resilience of microorganisms in a biofilm against antibiotics Biofilms of the nonpathogenic gram-positive soil bacterium Bacillus subtilis serve as a model system with biotechnological potential toward plant protection Its major extracellular matrix protein components are TasA and TapA The nature of TasA filaments has been of debate and several forms amyloidic and non-Thioflavin T-stainable have been observed Here we present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of nonamyloidic TasA filaments By analytical ... More
Studying mechanisms of bacterial biofilm generation is of vital importance to understanding bacterial cell–cell communication, multicellular cohabitation principles, and the higher resilience of microorganisms in a biofilm against antibiotics. Biofilms of the nonpathogenic, gram-positive soil bacterium Bacillus subtilis serve as a model system with biotechnological potential toward plant protection. Its major extracellular matrix protein components are TasA and TapA. The nature of TasA filaments has been of debate, and several forms, amyloidic and non-Thioflavin T-stainable have been observed. Here, we present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of nonamyloidic TasA filaments. By analytical ultracentrifugation and NMR, we demonstrate TapA-dependent acceleration of filament formation from solutions of folded TasA. Solid-state NMR revealed intercalation of the N-terminal TasA peptide segment into subsequent protomers to form a filament composed of β-sandwich subunits. The secondary structure around the intercalated N-terminal strand β0 is conserved between filamentous TasA and the Fim and Pap proteins, which form bacterial type I pili, demonstrating such construction principles in a gram-positive organism. Analogous to the chaperones of the chaperone-usher pathway, the role of TapA is in donating its N terminus to serve for TasA folding into an Ig domain-similar filament structure by donor-strand complementation. According to NMR and since the V-set Ig fold of TapA is already complete, its participation within a filament beyond initiation is unlikely. Intriguingly, the most conserved residues in TasA-like proteins (camelysines) of Bacillaceae are located within the protomer interface.
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The modified nucleosides -deoxy- -cyano- and -deoxy- -amido- -deazaguanosine dPreQ and dADG respectively recently discovered in DNA are the products of the bacterial queuosine tRNA modification pathway and the dpd gene cluster the latter of which encodes proteins that comprise the elaborate Dpd restriction modification system present in diverse bacteria Recent genetic studies implicated the dpdA dpdB and dpdC genes as encoding proteins necessary for DNA modification with dpdD dpdK contributing to the restriction phenotype Here we report the in vitro reconstitution of the Dpd modification machinery from Salmonella enterica serovar Montevideo the elucidation of the roles of each protein ... More
The modified nucleosides 2′-deoxy-7-cyano- and 2′-deoxy-7-amido-7-deazaguanosine (dPreQ0 and dADG, respectively) recently discovered in DNA are the products of the bacterial queuosine tRNA modification pathway and the dpd gene cluster, the latter of which encodes proteins that comprise the elaborate Dpd restriction–modification system present in diverse bacteria. Recent genetic studies implicated the dpdA, dpdB and dpdC genes as encoding proteins necessary for DNA modification, with dpdD–dpdK contributing to the restriction phenotype. Here we report the in vitro reconstitution of the Dpd modification machinery from Salmonella enterica serovar Montevideo, the elucidation of the roles of each protein and the X-ray crystal structure of DpdA supported by small-angle X-ray scattering analysis of DpdA and DpdB, the former bound to DNA. While the homology of DpdA with the tRNA-dependent tRNA-guanine transglycosylase enzymes (TGT) in the queuosine pathway suggested a similar transglycosylase activity responsible for the exchange of a guanine base in the DNA for 7-cyano-7-deazaguanine (preQ0), we demonstrate an unexpected ATPase activity in DpdB necessary for insertion of preQ0 into DNA, and identify several catalytically essential active site residues in DpdA involved in the transglycosylation reaction. Further, we identify a modification site for DpdA activity and demonstrate that DpdC functions independently of DpdA/B in converting preQ0-modified DNA to ADG-modified DNA. Less
G-protein metallochaperone MeaB in bacteria methylmalonic aciduria type A MMAA in humans is responsible for facilitating the delivery of adenosylcobalamin AdoCbl to methylmalonyl-CoA mutase MCM the only AdoCbl-dependent enzyme in humans Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids Here we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate GTP analog guanosine- - -methyleno triphosphate GMPPCP with the Cbl-binding domain of its target mutase enzyme MeMCMcbl This structure provides an explanation for the stimulation of the ... More
G-protein metallochaperone MeaB in bacteria [methylmalonic aciduria type A (MMAA) in humans] is responsible for facilitating the delivery of adenosylcobalamin (AdoCbl) to methylmalonyl-CoA mutase (MCM), the only AdoCbl-dependent enzyme in humans. Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids. Here, we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate (GTP) analog guanosine-5′-[(β,γ)-methyleno]triphosphate (GMPPCP) with the Cbl-binding domain of its target mutase enzyme (MeMCMcbl). This structure provides an explanation for the stimulation of the GTP hydrolyase activity of MeaB afforded by target protein binding. We find that upon MCMcbl association, one protomer of the MeaB dimer rotates ~180°, such that the inactive state of MeaB is converted to an active state in which the nucleotide substrate is now surrounded by catalytic residues. Importantly, it is the switch III region that undergoes the largest change, rearranging to make direct contacts with the terminal phosphate of GMPPCP. These structural data additionally provide insights into the molecular basis by which this metallochaperone contributes to AdoCbl delivery without directly binding the cofactor. Our data suggest a model in which GTP-bound MeaB stabilizes a conformation of MCM that is open for AdoCbl insertion, and GTP hydrolysis, as signaled by switch III residues, allows MCM to close and trap its cofactor. Substitutions of switch III residues destabilize the active state of MeaB through loss of protein:nucleotide and protein:protein interactions at the dimer interface, thus uncoupling GTP hydrolysis from AdoCbl delivery. Less
X-ray crystallography has long been a key method in solving the three-dimensional structure of proteins Structural information is essential for unraveling the molecular function of proteins and structure-based drug design However there are several obstacles associated with the structural determination of proteins using X-ray crystallography such as the generation of a large amount of protein samples instability of purified proteins and difficulty in obtaining large and well-diffracting crystals all of which can prolong the process of determining the crystal structure from months to years Over the past decade new techniques and strategies have been developed to assist X-ray crystallographers in ... More
X-ray crystallography has long been a key method in solving the three-dimensional structure of proteins. Structural information is essential for unraveling the molecular function of proteins and structure-based drug design. However, there are several obstacles associated with the structural determination of proteins using X-ray crystallography, such as the generation of a large amount of protein samples, instability of purified proteins, and difficulty in obtaining large and well-diffracting crystals, all of which can prolong the process of determining the crystal structure, from months to years. Over the past decade, new techniques and strategies have been developed to assist X-ray crystallographers in overcoming some of these obstacles. In this chapter, we discuss some of these technological advances. Familiarity with these new developments would benefit researchers in both academic and industrial environments who study macromolecular structural dynamics using X-ray crystallography. Less
Voltage-gated sodium ion channels Nav are central to action potential initiation through regulating the entry of sodium ions Na into excitable cells including cardiomyocytes and neurones The -subunit of Nav consists of four homologous domains DI-DIV each consisting of six transmembrane helices S -S Helices S -S of each domain forms the lining of the central pore through which sodium ions Na enters the cell upon channel activation Helices S -S of each domain form the voltage sensor which becomes displaced in response to changes in intracellular potential Additionally Nav channels include an extracellular turret region whose role in channel ... More
Voltage-gated sodium ion channels (Nav) are central to action potential initiation through regulating the entry of sodium ions (Na+) into excitable cells including cardiomyocytes and neurones. The α-subunit of Nav consists of four homologous domains (DI-DIV), each consisting of six transmembrane helices (S1-S6). Helices S5-S6 of each domain forms the lining of the central pore through which sodium ions (Na+) enters the cell upon channel activation. Helices S1-S4 of each domain form the voltage sensor which becomes displaced in response to changes in intracellular potential. Additionally, Nav channels include an extracellular turret region, whose role in channel function is poorly understood. The C-terminal domain (CTD) of Nav, connected to the DIV-S6, interacts with various other proteins including calmodulin (CaM) and fibroblast growth factor (FGF13) and mediates various regulatory roles. The subtype Nav1.5 is primarily expressed in the heart where it initiates the cardiac action potential whereas Nav1.7 is found in the peripheral nervous system where it is associated with nociception.
Various Nav associated pathologies have been associated with mutations in the extracellular turret region; however, their molecular mechanism is not well understood. In the Nav1.5 structure determined by cryogenic electron microscopy (cryoEM), the wild-type residues that correspond to some of these mutants form a complex salt bridge at the interface between the DII and DIII turret loops. Furthermore, adjacent aromatic residues could potentially form cation-π interactions with the complex salt bridge. This region was examined using site-directed mutagenesis, electrophysiology and in silico modelling, confirming functional roles for the inter-domain salt-bridges and the aromatic residues. Evidence that disruption of these contacts perturbs the geometry of the DEKA selectivity ring and both the outer and inner pore vestibules that are crucial for sodium ion permeability were provided. These findings provide insights into a class of pathological mutations occurring not only in Nav1.5 but also in other sodium channel isoforms.
Further experiments performed preliminary studies that focussed on the CTD of Nav1.5 and Nav1.7, seeking to better understand the role of its regulation by Ca2+ and CaM, using various techniques such as ELISA, isothermal titration calorimetry (ITC) and Bio-Layer interferometry (BLI). The CTDs and CaM recombinant proteins were cloned using the Gateway cloning method, expressed in BL21 (DE3) cells using auto-induction, and purified via affinity chromatography and size exclusion chromatography. Also, attempts were made to determine the yet unresolved structure of Nav1.7 using x-ray crystallography. Finally, using an in-house phage display library of single chain fragment variable (scFv) antibodies, specific binders to the CTD of Nav1.5 and Nav1.7, were found and purified. These scFvs could have gating effects on their Nav channel targets, which might prove therapeutically applicable. Less
Various Nav associated pathologies have been associated with mutations in the extracellular turret region; however, their molecular mechanism is not well understood. In the Nav1.5 structure determined by cryogenic electron microscopy (cryoEM), the wild-type residues that correspond to some of these mutants form a complex salt bridge at the interface between the DII and DIII turret loops. Furthermore, adjacent aromatic residues could potentially form cation-π interactions with the complex salt bridge. This region was examined using site-directed mutagenesis, electrophysiology and in silico modelling, confirming functional roles for the inter-domain salt-bridges and the aromatic residues. Evidence that disruption of these contacts perturbs the geometry of the DEKA selectivity ring and both the outer and inner pore vestibules that are crucial for sodium ion permeability were provided. These findings provide insights into a class of pathological mutations occurring not only in Nav1.5 but also in other sodium channel isoforms.
Further experiments performed preliminary studies that focussed on the CTD of Nav1.5 and Nav1.7, seeking to better understand the role of its regulation by Ca2+ and CaM, using various techniques such as ELISA, isothermal titration calorimetry (ITC) and Bio-Layer interferometry (BLI). The CTDs and CaM recombinant proteins were cloned using the Gateway cloning method, expressed in BL21 (DE3) cells using auto-induction, and purified via affinity chromatography and size exclusion chromatography. Also, attempts were made to determine the yet unresolved structure of Nav1.7 using x-ray crystallography. Finally, using an in-house phage display library of single chain fragment variable (scFv) antibodies, specific binders to the CTD of Nav1.5 and Nav1.7, were found and purified. These scFvs could have gating effects on their Nav channel targets, which might prove therapeutically applicable. Less
Autofluorescence-detected photothermal mid-infrared AF-PTIR microscopy was shown to enable parts-per-million detection of -indomethacin impurity in -indomethacin samples Subtle differences in the photothermal response of the UV-autofluorescence of two indomethacin crystal polymorphs were used for sub-micron chemical discrimination based on fingerprint region mid-IR spectroscopy The AF-PTIR assignment was independently confirmed by second harmonic generation SHG microscopy which was shown to reduce the total analysis time by rapidly identifying the suitable fields of view AF-PTIR microscopy has the potential to assist in the early identification of crystal form impurities in the solid dosage forms development pipeline
Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers Here we show the identification and development of an affinity-enhanced T cell receptor TCR that recognizes a peptide derived from the most common KRAS mutant KRASG D presented in the context of HLA-A The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG D over KRASWT While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG D ... More
Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRASG12D, presented in the context of HLA-A*11:01. The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG12D over KRASWT. While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG12D, thermodynamic analysis and molecular dynamics simulations reveal that TCR specificity is driven by differences in indirect electrostatic interactions. The affinity enhanced TCR, fused to a humanized anti-CD3 scFv, enables selective killing of cancer cells expressing KRASG12D. Our work thus reveals a molecular mechanism that drives TCR selectivity and describes a soluble bispecific molecule with therapeutic potential against cancers harboring a common shared neoantigen. Less
Nonribosomal peptide synthetase heterocyclization Cy domains generate biologically important oxazoline thiazoline groups found in natural products including pharmaceuticals and virulence factors such as some siderophores Cy domains catalyze consecutive condensation and cyclodehydration reactions although the mechanism is unknown To better understand Cy domain catalysis here we report the crystal structure of the second Cy domain Cy of yersiniabactin synthetase from the causative agent of the plague Yersinia pestis Our high-resolution structure of Cy adopts a conformation that enables exploration of interactions with the extended thiazoline-containing cyclodehydration intermediate and the acceptor carrier protein CP to which it is tethered We also ... More
Nonribosomal peptide synthetase heterocyclization (Cy) domains generate biologically important oxazoline/thiazoline groups found in natural products, including pharmaceuticals and virulence factors such as some siderophores. Cy domains catalyze consecutive condensation and cyclodehydration reactions, although the mechanism is unknown. To better understand Cy domain catalysis, here we report the crystal structure of the second Cy domain (Cy2) of yersiniabactin synthetase from the causative agent of the plague, Yersinia pestis. Our high-resolution structure of Cy2 adopts a conformation that enables exploration of interactions with the extended thiazoline-containing cyclodehydration intermediate and the acceptor carrier protein (CP) to which it is tethered. We also report complementary electrostatic interfaces between Cy2 and its donor CP that mediate donor binding. Finally, we explored domain flexibility through normal mode analysis and identified small-molecule fragment-binding sites that may inform future antibiotic design targeting Cy function. Our results suggest how CP binding may influence global Cy conformations, with consequences for active-site remodeling to facilitate the separate condensation and cyclodehydration steps as well as potential inhibitor development. Less
Chickpea is a crop that is known as a source of high-quality proteins CL-AI which belongs to the S globulin and cupin superfamily was initially identified in chickpea seeds CL-AI has recently been shown to inhibit various types of -amylases To determine its molecular mechanism the crystal structure of CL-AI was solved at a final resolution of Structural analysis indicated that each asymmetric unit contains three molecules with threefold symmetry and a head-to-tail association and each molecule is divided into an -chain and a -chain CL-AI has high structural similarity to other S globulins and canonical metal-dependent enzyme-related cupin proteins ... More
Chickpea is a crop that is known as a source of high-quality proteins. CL-AI, which belongs to the 11S globulin and cupin superfamily, was initially identified in chickpea seeds. CL-AI has recently been shown to inhibit various types of α-amylases. To determine its molecular mechanism, the crystal structure of CL-AI was solved at a final resolution of 2.2 Å. Structural analysis indicated that each asymmetric unit contains three molecules with threefold symmetry and a head-to-tail association, and each molecule is divided into an α-chain and a β-chain. CL-AI has high structural similarity to other 11S globulins and canonical metal-dependent enzyme-related cupin proteins, whereas its stimilarity to α-amylase inhibitor from Phaseolus vulgaris is quite low. The structure presented here will provide insight into the function of CL-AI. Less
Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of D protein structures which has recently been further augmented by revolutionary advances in cryoEM However a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts limiting the overview of inherent protein physiological dynamics which play a critical role in the biological functions of proteins Here a room-temperature X-ray crystallo graphic method using temperature as a trigger to record movie-like structural snapshots has been developed The method has been used to show how TL a Da fragment undergoes ... More
Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15 Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery. Less
T-cell receptor TCR immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials The safety of patients is paramount so innovative cell engineering methods are being employed to exploit adaptive immunity while controlling the factors governing antigen receptor ie TCR specificity and cross-reactivity We recently reported a TCR engineering campaign and selectivity profiling assay X-scan targeting a melanoma antigen gene MAGE -A peptide This helped to distinguish between two well-performing TCRs based on cross-reactivity potential during preclinical drug evaluation allowing one to be advanced to T-cell immunotherapeutic clinical trials Here we present three-dimensional structural information on ... More
T-cell receptor (TCR) immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials. The safety of patients is paramount, so innovative cell engineering methods are being employed to exploit adaptive immunity while controlling the factors governing antigen receptor (ie, TCR) specificity and cross-reactivity. We recently reported a TCR engineering campaign and selectivity profiling assay (X-scan) targeting a melanoma antigen gene (MAGE)-A10 peptide. This helped to distinguish between two well-performing TCRs based on cross-reactivity potential during preclinical drug evaluation, allowing one to be advanced to T-cell immunotherapeutic clinical trials. Here, we present three-dimensional structural information on those TCRs, highlighting engineering improvements and molecular mechanisms likely underpinning differential selectivity. Less
Heavy-atom soaking has been a major method for experimental phasing but it has been difficult for membrane proteins partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding The lipid cubic phase LCP has proven to be a successful method for membrane protein crystallization but experimental phasing with LCP-grown crystals remains difficult and so far only such structures were phased experimentally Here the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion SAD phasing of crystals grown in LCP Using a single crystal the structure of the glycerol -phosphate acyltransferase ... More
Heavy-atom soaking has been a major method for experimental phasing, but it has been difficult for membrane proteins, partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding. The lipid cubic phase (LCP) has proven to be a successful method for membrane protein crystallization, but experimental phasing with LCP-grown crystals remains difficult, and so far, only 68 such structures were phased experimentally. Here, the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion (SAD) phasing of crystals grown in LCP. Using a single crystal, the structure of the glycerol 3-phosphate acyltransferase (PlsY, ~21 kDa), a very hydrophobic enzyme with 80% membrane-embedded residues, was solved. Remarkably, a total of 15 Se sites were found in the two monomers of PlsY, translating to one selenourea-binding site per every six residues in the accessible extramembrane protein. Structure analysis reveals that surface-exposed selenourea sites are mostly contributed by mainchain amides and carbonyls. This low-specificity binding pattern may explain its high loading ratio. Importantly, both the crystal diffraction quality and the LCP integrity were unaffected by selenourea soaking. Taken together, selenourea presents a promising and generally useful reagent for heavy-atom soaking of membrane protein crystals grown in LCP. Less
Candida Als family adhesins mediate adhesion to biological and abiotic substrates as well as fungal cell aggregation fungal-bacterial co-aggregation and biofilm formation The activity of at least two family members Als and Als is dependent on amyloid-like protein aggregation that is initiated by shear force Each Als adhesin has a -residue N-terminal Ig-like invasin region The following -residue low complexity threonine-rich T domain unfolds under shear force to expose a critical amyloid-forming segment SNGIVIVATTRTV at the interface between the Ig-like invasin domain and the T domain of Candida albicans Als Amyloid prediction programs identified six potential amyloidogenic sequences in the ... More
Candida Als family adhesins mediate adhesion to biological and abiotic substrates, as well as fungal cell aggregation, fungal-bacterial co-aggregation and biofilm formation. The activity of at least two family members, Als5 and Als1, is dependent on amyloid-like protein aggregation that is initiated by shear force. Each Als adhesin has a ∼300-residue N-terminal Ig-like/invasin region. The following 108-residue, low complexity, threonine-rich (T) domain unfolds under shear force to expose a critical amyloid-forming segment 322SNGIVIVATTRTV334 at the interface between the Ig-like/invasin domain 2 and the T domain of Candida albicans Als5. Amyloid prediction programs identified six potential amyloidogenic sequences in the Ig-like/invasin region and three others in the T domain of C. albicans Als5. Peptides derived from four of these sequences formed fibrils that bound thioflavin T, the amyloid indicator dye, and three of these revealed atomic-resolution structures of cross-β spines. These are the first atomic-level structures for fungal adhesins. One of these segments, from the T domain, revealed kinked β-sheets, similarly to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. Based on the cross-β structures in Als proteins, we use evolutionary arguments to identify functional amyloidogenic sequences in other fungal adhesins, including adhesins from Candida auris. Thus, cross-β structures are often involved in fungal pathogenesis and potentially in antifungal therapy. Less
G protein-coupled receptors GPCRs play vital roles in human physiology and pathophysiology This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics However the production and crystallization of GPCRs for structure determination comes with many challenges In this chapter we provide a comprehensive protocol for expressing and purifying the thromboxane A receptor TPR an attractive therapeutic target for use in structure studies Guidelines for crystallizing the TPR are also included Together these procedures provide a template for generating crystal structures of the TPR and indeed other ... More
G protein-coupled receptors (GPCRs) play vital roles in human physiology and pathophysiology. This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics. However, the production and crystallization of GPCRs for structure determination comes with many challenges.
In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands. Less
In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands. Less
In situ diffraction data collection using crystallization plates has been utilized for macromolecules to evaluate crystal quality without requiring additional sample treatment such as cryocooling Although it is difficult to collect complete data sets using this technique due to the mechanical limitation of crystal rotation recent advances in methods for data collection from multiple crystals have overcome this issue At SPring- an in situ diffraction measurement system was constructed consisting of a goniometer for a plate an articulated robot and plate storage Using this system complete data sets were obtained utilizing the small-wedge measurement method Combining this system with an ... More
In situ diffraction data collection using crystallization plates has been utilized for macromolecules to evaluate crystal quality without requiring additional sample treatment such as cryocooling. Although it is difficult to collect complete data sets using this technique due to the mechanical limitation of crystal rotation, recent advances in methods for data collection from multiple crystals have overcome this issue. At SPring-8, an in situ diffraction measurement system was constructed consisting of a goniometer for a plate, an articulated robot and plate storage. Using this system, complete data sets were obtained utilizing the small-wedge measurement method. Combining this system with an acoustic liquid handler to prepare protein-ligand complex crystals by applying fragment compounds to trypsin crystals for in situ soaking, binding was confirmed for seven out of eight compounds. These results show that the system functioned properly to collect complete data for structural analysis and to expand the capability for ligand screening in combination with a liquid dispenser. Less
Viruses are responsible for some of the most deadly human diseases yet available vaccines and antivirals address only a fraction of the potential viral human pathogens Here we provide a methodology for managing human herpesvirus HHV infection by covalently inactivating the HHV maturational protease via a conserved non-catalytic cysteine C Using human cytomegalovirus protease HCMV Pr as a model we screened a library of disulfides to identify molecules that tether to C and inhibit proteolysis then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs We further developed an optimized tool compound targeted ... More
Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection. Less