Beyond the coupled distortion model: structural analysis of the single domain cupredoxin AcoP, a green mononuclear copper centre with original features Roger, Magali In: 2024. @article{noKey,
title = {Beyond the coupled distortion model: structural analysis of the single domain cupredoxin AcoP, a green mononuclear copper centre with original features},
author = {Roger, Magali},
url = {https://pubs.rsc.org/en/content/articlehtml/2024/dt/d3dt03372d},
doi = {10.1039/D3DT03372D},
year = {2024},
date = {2024-01-01},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Targeting transcription factors through an IMiD independent zinc finger domain Liu, Bee Hui, Liu, Miao In: 2024. @article{noKey,
title = {Targeting transcription factors through an IMiD independent zinc finger domain},
author = {Liu, Bee Hui, Liu, Miao},
url = {https://www.biorxiv.org/content/10.1101/2024.01.03.574032v1.abstract},
doi = {https://doi.org/10.1101/2024.01.03.574032},
year = {2024},
date = {2024-01-01},
abstract = {Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in four clusters, including an IMiD degron in zinc finger cluster two (ZFC2). Surprisingly, IMiDs do not inhibit growth of SALL4 expressing cancer cells. To overcome this limit, we focused on a non-IMiD degron, SALL4 zinc finger cluster four (ZFC4). By combining AlphaFold and the ZFC4-DNA crystal structure, we identified a potential ZFC4 drug pocket. Utilizing an in silico docking algorithm and cell viability assays, we screened chemical libraries and discovered SH6, which selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 led to significant 62% tumor growth inhibition of SALL4+ xenografts in vivo and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors in cancer.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in four clusters, including an IMiD degron in zinc finger cluster two (ZFC2). Surprisingly, IMiDs do not inhibit growth of SALL4 expressing cancer cells. To overcome this limit, we focused on a non-IMiD degron, SALL4 zinc finger cluster four (ZFC4). By combining AlphaFold and the ZFC4-DNA crystal structure, we identified a potential ZFC4 drug pocket. Utilizing an in silico docking algorithm and cell viability assays, we screened chemical libraries and discovered SH6, which selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 led to significant 62% tumor growth inhibition of SALL4+ xenografts in vivo and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors in cancer. |
Deficiency in PHD2-mediated hydroxylation of HIF2α underlies Pacak-Zhuang syndrome G. Ferens, Fraser, C. Taber, Cassandra In: 2024. @article{noKey,
title = {Deficiency in PHD2-mediated hydroxylation of HIF2α underlies Pacak-Zhuang syndrome},
author = {G. Ferens, Fraser, C. Taber, Cassandra},
url = {https://www.nature.com/articles/s42003-024-05904-4},
doi = {https://doi.org/10.1038/s42003-024-05904-4},
year = {2024},
date = {2024-01-01},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories Zhao, Boyang In: 2024. @article{noKey,
title = {Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories},
author = {Zhao, Boyang},
url = {https://www.nature.com/articles/s41467-024-46627-8#Sec15},
doi = {https://doi.org/10.1038/s41467-024-46627-8},
year = {2024},
date = {2024-01-01},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Molecular mechanism of cellulose depolymerization by the two-domain BlCel9A enzyme from the glycoside hydrolase family 9 Ares de Araújo, Evandro In: 2024. @article{noKey,
title = {Molecular mechanism of cellulose depolymerization by the two-domain BlCel9A enzyme from the glycoside hydrolase family 9},
author = {Ares de Araújo, Evandro},
url = {https://www.sciencedirect.com/science/article/pii/S0144861723012043},
doi = {https://doi.org/10.1016/j.carbpol.2023.121739},
year = {2024},
date = {2024-01-01},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP Peter, Martin F., Ruland, Jan A. In: 2023. @article{noKey,
title = {Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP},
author = {Peter, Martin F., Ruland, Jan A.},
url = {https://www.biorxiv.org/content/10.1101/2023.03.04.531103v1.abstract},
doi = {https://doi.org/10.1101/2023.03.04.531103},
year = {2023},
date = {2023-01-01},
abstract = {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, was locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, was locked in either its predicted inward- or outward-facing states. We characterized the disulfide-locked variants and used 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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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, was locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, was locked in either its predicted inward- or outward-facing states. We characterized the disulfide-locked variants and used 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. |
7-Deazaguanines in DNA: functional and structural elucidation of a DNA modification system Gedara, Samanthi Herath, Wood, Evan In: 2023. @article{noKey,
title = {7-Deazaguanines in DNA: functional and structural elucidation of a DNA modification system},
author = {Gedara, Samanthi Herath, Wood, Evan},
url = {https://academic.oup.com/nar/article/51/8/3836/7079639},
doi = {https://doi.org/10.1093/nar/gkad141},
year = {2023},
date = {2023-01-01},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Structural basis and functional roles for Toll-like receptor binding to Latrophilin adhesion-GPCR in embryo development Rosas, Gabriel Carmona, Li, Jingxian In: 2023. @article{noKey,
title = {Structural basis and functional roles for Toll-like receptor binding to Latrophilin adhesion-GPCR in embryo development},
author = {Rosas, Gabriel Carmona, Li, Jingxian},
url = {https://www.biorxiv.org/content/10.1101/2023.05.04.539414v2.abstract},
doi = {https://doi.org/10.1101/2023.05.04.539414},
year = {2023},
date = {2023-01-01},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Nanobody-aided structural study of the activityregulated cytoskeleton-associated protein (Arc) using synchrotron radiation and cryo-EM Neset, Lasse In: 2023. @article{noKey,
title = {Nanobody-aided structural study of the activityregulated cytoskeleton-associated protein (Arc) using synchrotron radiation and cryo-EM},
author = {Neset, Lasse},
url = {https://bora.uib.no/bora-xmlui/handle/11250/3071867},
doi = {Thesis},
year = {2023},
date = {2023-01-01},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Fragment library screening by X-ray crystallography and hit optimization against thioredoxin glutathione reductase of Schistosoma mansoni Neto, Lauro Ribeiro Souza, Montoya, Bogar Omar In: 2023. @article{noKey,
title = {Fragment library screening by X-ray crystallography and hit optimization against thioredoxin glutathione reductase of Schistosoma mansoni},
author = {Neto, Lauro Ribeiro Souza, Montoya, Bogar Omar},
url = {https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&cad=rja&uact=8&ved=2ahUKEwisrtTU95GBAxVeFVkFHUFFBroQFnoECA0QAQ&url=https%3A%2F%2Fwww.researchsquare.com%2Farticle%2Frs-3253599%2Flatest.pdf&usg=AOvVaw0xuXYut-6ryhQKf6g2o_3v&opi=89978449},
doi = {https://doi.org/10.21203/rs.3.rs-3253599/v1},
year = {2023},
date = {2023-01-01},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as platforms for viral replication Kaundal, Soni, Anish, Ramakrishnan In: 2023. @article{noKey,
title = {RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as platforms for viral replication},
author = {Kaundal, Soni, Anish, Ramakrishnan},
url = {https://www.biorxiv.org/content/10.1101/2023.08.24.554692v2.abstract},
doi = {https://doi.org/10.1101/2023.08.24.554692},
year = {2023},
date = {2023-01-01},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Structure of glyceraldehyde-3-phosphate dehydrogenase from Paracoccidioides lutzii in complex with an aldonic sugar acid Hernández-Prieto, Jonathan Heiler, Martini, Viviane Paula In: 2023. @article{noKey,
title = {Structure of glyceraldehyde-3-phosphate dehydrogenase from Paracoccidioides lutzii in complex with an aldonic sugar acid},
author = {Hernández-Prieto, Jonathan Heiler, Martini, Viviane Paula},
url = {https://www.sciencedirect.com/science/article/pii/S0300908423002407?casa_token=R0uGAzoFfpcAAAAA:y-qY3rzdBagk0iPh26-8fzePogOsR2z2UIga_akXrIhyIxS7YiGc0nSFIEMUVvgobC5ELa7l},
doi = {https://doi.org/10.1016/j.biochi.2023.09.013},
year = {2023},
date = {2023-01-01},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
X-ray structure of the metastable SEPT14–SEPT7 coiled coil reveals a hendecad region crucial for heterodimerization Cavini, Italo A, Winter, Ashley J In: 2023. @article{noKey,
title = {X-ray structure of the metastable SEPT14–SEPT7 coiled coil reveals a hendecad region crucial for heterodimerization},
author = {Cavini, Italo A, Winter, Ashley J},
url = {https://pubmed.ncbi.nlm.nih.gov/37712436/},
doi = {10.1107/S2059798323006514},
year = {2023},
date = {2023-01-01},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Structural Study of a New MbtI-Inhibitor Complex: Towards an Optimized Model for Structure-Based Drug Discovery Mori, Matteo, Villa, Stefania In: 2023. @article{noKey,
title = {Structural Study of a New MbtI-Inhibitor Complex: Towards an Optimized Model for Structure-Based Drug Discovery},
author = {Mori, Matteo, Villa, Stefania},
url = {https://www.mdpi.com/1424-8247/16/11/1559},
doi = {https://doi.org/10.3390/ph16111559},
year = {2023},
date = {2023-01-01},
abstract = {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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Fragment-based screening targeting an open form of the SARS-CoV-2 main protease binding pocket Huang, Chia-Ying, Metz, Alexander In: 2023. @article{noKey,
title = {Fragment-based screening targeting an open form of the SARS-CoV-2 main protease binding pocket},
author = {Huang, Chia-Ying, Metz, Alexander},
url = {https://www.biorxiv.org/content/10.1101/2023.11.15.567102v2.abstract},
doi = {https://doi.org/10.1101/2023.11.15.567102},
year = {2023},
date = {2023-01-01},
abstract = {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 fragments soaked, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (2 hits) or at crystal packing interfaces (3 hits). Notably, two fragments with a pose sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. Our 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.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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 fragments soaked, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (2 hits) or at crystal packing interfaces (3 hits). Notably, two fragments with a pose sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. Our 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. |
Structural Insights into Molecular Recognition by Human Chemokine CCL19 Lewandowski, Eric M., Kroeck, Kyle G. In: 2022. @article{noKey,
title = {Structural Insights into Molecular Recognition by Human Chemokine CCL19},
author = {Lewandowski, Eric M., Kroeck, Kyle G.},
url = {https://pubs.acs.org/doi/full/10.1021/acs.biochem.1c00759},
doi = {https://doi.org/10.1021/acs.biochem.1c00759},
year = {2022},
date = {2022-01-01},
abstract = {The human chemokines CCL19 and CCL21 bind to the G protein-coupled receptor (GPCR) CCR7 and play an important role in the trafficking of immune cells as well as cancer metastasis. Conserved binding sites for sulfotyrosine residues on the receptor contribute significantly to the chemokine/GPCR interaction and have been shown to provide promising targets for new drug-discovery efforts to disrupt the chemokine/GPCR interaction and, consequently, tumor metastasis. Here, we report the first X-ray crystal structure of a truncated CCL19 (residues 7–70) at 2.50 Å resolution, revealing molecular details crucial for protein–protein interactions. Although the overall structure is similar to the previously determined NMR model, there are important variations, particularly near the N terminus and the so-called 30’s and 40’s loops. Computational analysis using the FTMap server indicates the potential importance of these areas in ligand binding and the differences in binding hotspots compared to CCL21. NMR titration experiments using a CCR7-derived peptide (residues 5–11, TDDYIGD) further demonstrate potential receptor recognition sites, such as those near the C terminus and 40’s loop, which consist of both positively charged and hydrophobic residues that may be important for receptor binding. Taken together, the X-ray, NMR, and computational analysis herein provide insights into the overall structure and molecular features of CCL19 and enables investigation into this chemokine’s function and inhibitor development.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
The human chemokines CCL19 and CCL21 bind to the G protein-coupled receptor (GPCR) CCR7 and play an important role in the trafficking of immune cells as well as cancer metastasis. Conserved binding sites for sulfotyrosine residues on the receptor contribute significantly to the chemokine/GPCR interaction and have been shown to provide promising targets for new drug-discovery efforts to disrupt the chemokine/GPCR interaction and, consequently, tumor metastasis. Here, we report the first X-ray crystal structure of a truncated CCL19 (residues 7–70) at 2.50 Å resolution, revealing molecular details crucial for protein–protein interactions. Although the overall structure is similar to the previously determined NMR model, there are important variations, particularly near the N terminus and the so-called 30’s and 40’s loops. Computational analysis using the FTMap server indicates the potential importance of these areas in ligand binding and the differences in binding hotspots compared to CCL21. NMR titration experiments using a CCR7-derived peptide (residues 5–11, TDDYIGD) further demonstrate potential receptor recognition sites, such as those near the C terminus and 40’s loop, which consist of both positively charged and hydrophobic residues that may be important for receptor binding. Taken together, the X-ray, NMR, and computational analysis herein provide insights into the overall structure and molecular features of CCL19 and enables investigation into this chemokine’s function and inhibitor development. |
Fast fragment- and compound-screening pipeline at the Swiss Light Source Kaminski, Jakub W, Vera, Laura In: 2022. @article{noKey,
title = {Fast fragment- and compound-screening pipeline at the Swiss Light Source},
author = {Kaminski, Jakub W, Vera, Laura},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8900825/},
doi = {doi: 10.1107/S2059798322000705.},
year = {2022},
date = {2022-01-01},
abstract = {Over the last two decades, fragment-based drug discovery (FBDD) has emerged as an effective and efficient method to identify new chemical scaffolds for the development of lead compounds. X-ray crystallography can be used in FBDD as a tool to validate and develop fragments identified as binders by other methods. However, it is also often used with great success as a primary screening technique. In recent years, technological advances at macromolecular crystallography beamlines in terms of instrumentation, beam intensity and robotics have enabled the development of dedicated platforms at synchrotron sources for FBDD using X-ray crystallography. Here, the development of the Fast Fragment and Compound Screening (FFCS) platform, an integrated next-generation pipeline for crystal soaking, handling and data collection which allows crystallography-based screening of protein crystals against hundreds of fragments and compounds, at the Swiss Light Source is reported.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Over the last two decades, fragment-based drug discovery (FBDD) has emerged as an effective and efficient method to identify new chemical scaffolds for the development of lead compounds. X-ray crystallography can be used in FBDD as a tool to validate and develop fragments identified as binders by other methods. However, it is also often used with great success as a primary screening technique. In recent years, technological advances at macromolecular crystallography beamlines in terms of instrumentation, beam intensity and robotics have enabled the development of dedicated platforms at synchrotron sources for FBDD using X-ray crystallography. Here, the development of the Fast Fragment and Compound Screening (FFCS) platform, an integrated next-generation pipeline for crystal soaking, handling and data collection which allows crystallography-based screening of protein crystals against hundreds of fragments and compounds, at the Swiss Light Source is reported. |
Three-dimensional structure of the single domain cupredoxin AcoP Roger, Magali, Leone, Philippe In: 2022. @article{noKey,
title = {Three-dimensional structure of the single domain cupredoxin AcoP},
author = {Roger, Magali, Leone, Philippe},
url = {https://europepmc.org/article/ppr/ppr471119},
doi = {https://doi.org/10.1101/2022.03.21.484586},
year = {2022},
date = {2022-01-01},
abstract = {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 center coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in term of copper-binding ligands, copper center geometry, redox potential, as well as biological function. AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans . AcoP presents original features: highly resistant to acidic pH, it possesses a constrained green-type copper center 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 structure of AcoP at 1.65 Å resolution unveils a typical cupredoxin fold with extended loops, never observed in previously characterized cupredoxins, that might be involved in the interaction of AcoP with its physiological partners. Moreover, the structure shows that the green color of AcoP cannot be attributed to nonclassical copper ligands, its green-colored copper center raising from a long Cu-S (Cys) bond, determined by both X-ray diffraction and EXAFS. The crystal structures of two AcoP mutants confirm that the active center 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 center rigidity due to the presence of an extensive hydrogen bond network.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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 center coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in term of copper-binding ligands, copper center geometry, redox potential, as well as biological function. AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans . AcoP presents original features: highly resistant to acidic pH, it possesses a constrained green-type copper center 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 structure of AcoP at 1.65 Å resolution unveils a typical cupredoxin fold with extended loops, never observed in previously characterized cupredoxins, that might be involved in the interaction of AcoP with its physiological partners. Moreover, the structure shows that the green color of AcoP cannot be attributed to nonclassical copper ligands, its green-colored copper center raising from a long Cu-S (Cys) bond, determined by both X-ray diffraction and EXAFS. The crystal structures of two AcoP mutants confirm that the active center 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 center rigidity due to the presence of an extensive hydrogen bond network. |
The Structure of Bilirubin Oxidase from Bacillus pumilus Reveals a Unique Disulfide Bond for Site-Specific Direct Electron Transfer Gihaz, Shalev, Herzallh, Nidaa Shrara In: 2022. @article{noKey,
title = {The Structure of Bilirubin Oxidase from Bacillus pumilus Reveals a Unique Disulfide Bond for Site-Specific Direct Electron Transfer},
author = {Gihaz, Shalev, Herzallh, Nidaa Shrara},
url = {https://www.mdpi.com/2079-6374/12/5/258},
doi = {https://doi.org/10.3390/bios12050258},
year = {2022},
date = {2022-01-01},
abstract = {Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications. Bilirubin oxidase (BOD) is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials. BOD has been extensively investigated over the last few decades. While the enzyme’s internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated, a crystal structure of BOD from bacterial origin has never been determined. Here we present the first crystal structure of BOD from Bacillus pumilus (BpBOD) at 3.5 Å resolution. Overall, BpBOD shows high homology with the fungal enzymes; however, it holds a unique surface-exposed disulfide bond between Cys229 and Cys322 residues. We present methodologies to orient the T1 site towards the electrode by coupling the reduced disulfide bond with maleimide moiety on the electrodes. The developed configurations were further investigated and revealed improved direct electron transfer rates with the electrodes. The work presented here may contribute to the construction of rationally designed bioanodes or biocathode configurations that are based on redox-active enzymes.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications. Bilirubin oxidase (BOD) is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials. BOD has been extensively investigated over the last few decades. While the enzyme’s internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated, a crystal structure of BOD from bacterial origin has never been determined. Here we present the first crystal structure of BOD from Bacillus pumilus (BpBOD) at 3.5 Å resolution. Overall, BpBOD shows high homology with the fungal enzymes; however, it holds a unique surface-exposed disulfide bond between Cys229 and Cys322 residues. We present methodologies to orient the T1 site towards the electrode by coupling the reduced disulfide bond with maleimide moiety on the electrodes. The developed configurations were further investigated and revealed improved direct electron transfer rates with the electrodes. The work presented here may contribute to the construction of rationally designed bioanodes or biocathode configurations that are based on redox-active enzymes. |
Production and Crystallization of Nanobodies in Complex with the Receptor Binding Domain of the SARS-CoV-2 Spike Protein Bas, Audrey Le, Mikolajek, Halina In: 2022. @article{noKey,
title = {Production and Crystallization of Nanobodies in Complex with the Receptor Binding Domain of the SARS-CoV-2 Spike Protein},
author = {Bas, Audrey Le, Mikolajek, Halina},
url = {https://bio-protocol.org/e4406?p=51&way=207},
doi = {https://doi.org/10.21769/BioProtoc.4406},
year = {2022},
date = {2022-01-01},
abstract = {The receptor binding domain of spike protein of SARS-CoV-2 binds angitensin coverting enzyme 2, on the surface of epithelial cells, leading to fusion and entry of virus into the cell. Based on our experience, we described a work flow, for expressing and purifying proteins, and screening conditions for generating diffraction quality crystals, of the complex. Production and crystallization of protein complex take abut twelve days., from construction of vectors to harvesting and freezing crystals for data collection.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
The receptor binding domain of spike protein of SARS-CoV-2 binds angitensin coverting enzyme 2, on the surface of epithelial cells, leading to fusion and entry of virus into the cell. Based on our experience, we described a work flow, for expressing and purifying proteins, and screening conditions for generating diffraction quality crystals, of the complex. Production and crystallization of protein complex take abut twelve days., from construction of vectors to harvesting and freezing crystals for data collection. |
