I. Sotiropoulou, Anastasia Structural studies of β-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus Journal Article In: Structural Biology, 2024. @article{noKey,
title = {Structural studies of β-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus},
author = {I. Sotiropoulou, Anastasia},
url = {https://journals.iucr.org/d/issues/2024/10/00/gm5108/index.html},
doi = {https://doi.org/10.1107/S2059798324009252},
year = {2024},
date = {2024-09-30},
journal = {Structural Biology},
abstract = {β-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus (Bgl1) has been denoted as having an attractive catalytic profile for various industrial applications. Bgl1 catalyses the final step of in the decomposition of cellulose, an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere. With the aim of enhancing the thermostability of Bgl1 for a broad spectrum of biotechnological processes, it has been subjected to structural studies. Crystal structures of Bgl1 and its complex with glucose were determined at 1.47 and 1.95 Å resolution, respectively. Bgl1 is a member of glycosyl hydrolase family 1 (GH1 superfamily, EC 3.2.1.21) and the results showed that the 3D structure of Bgl1 follows the overall architecture of the GH1 family, with a classical (β/α)8 TIM-barrel fold. Comparisons of Bgl1 with sequence or structural homologues of β-glucosidase reveal quite similar structures but also unique structural features in Bgl1 with plausible functional roles.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
}
β-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus (Bgl1) has been denoted as having an attractive catalytic profile for various industrial applications. Bgl1 catalyses the final step of in the decomposition of cellulose, an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere. With the aim of enhancing the thermostability of Bgl1 for a broad spectrum of biotechnological processes, it has been subjected to structural studies. Crystal structures of Bgl1 and its complex with glucose were determined at 1.47 and 1.95 Å resolution, respectively. Bgl1 is a member of glycosyl hydrolase family 1 (GH1 superfamily, EC 3.2.1.21) and the results showed that the 3D structure of Bgl1 follows the overall architecture of the GH1 family, with a classical (β/α)8 TIM-barrel fold. Comparisons of Bgl1 with sequence or structural homologues of β-glucosidase reveal quite similar structures but also unique structural features in Bgl1 with plausible functional roles. |
Ishii, Kenta Crystal structure of Alzheimer's disease phospholipase D3 provides a molecular basis for understanding its normal and pathological functions Journal Article In: The FEBS journal, 2024. @article{noKey,
title = {Crystal structure of Alzheimer's disease phospholipase D3 provides a molecular basis for understanding its normal and pathological functions},
author = {Ishii, Kenta},
url = {https://febs.onlinelibrary.wiley.com/doi/full/10.1111/febs.17277},
doi = {https://doi.org/10.1111/febs.17277},
year = {2024},
date = {2024-09-25},
journal = {The FEBS journal},
abstract = {Human 5′-3′ exonuclease PLD3, a member of the phospholipase D family of enzymes, has been validated as a therapeutic target for treating Alzheimer's disease. Here, we have determined the crystal structure of the luminal domain of the enzyme at 2.3 Å resolution, revealing a bilobal structure with a catalytic site located between the lobes. We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues, previously shown to be key for phospholipase activity, are not conserved or, are absent. This led us to test whether the enzyme is actually a phospholipase. We could not measure any phospholipase activity but the enzyme shows robust nuclease activity. Finally, we have mapped key single nucleotide polymorphisms onto the structure which reveals plausible reasons as to why they have an impact on Alzheimer's disease.},
keywords = {NT8®, Rock Imager®},
pubstate = {published},
tppubtype = {article}
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Human 5′-3′ exonuclease PLD3, a member of the phospholipase D family of enzymes, has been validated as a therapeutic target for treating Alzheimer's disease. Here, we have determined the crystal structure of the luminal domain of the enzyme at 2.3 Å resolution, revealing a bilobal structure with a catalytic site located between the lobes. We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues, previously shown to be key for phospholipase activity, are not conserved or, are absent. This led us to test whether the enzyme is actually a phospholipase. We could not measure any phospholipase activity but the enzyme shows robust nuclease activity. Finally, we have mapped key single nucleotide polymorphisms onto the structure which reveals plausible reasons as to why they have an impact on Alzheimer's disease. |
A. Lee, Alpha Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response Journal Article In: Preprint, 2024. @article{noKey,
title = {Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response},
author = {A. Lee, Alpha},
url = {https://www.biorxiv.org/content/10.1101/2024.08.19.608619v1.abstract},
doi = {https://doi.org/10.1101/2024.08.19.608619},
year = {2024},
date = {2024-08-20},
journal = {Preprint},
abstract = {A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunitydriven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
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A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunitydriven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development. |
Aschenbrenner, Jasmin Crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P1 21 1 Journal Article In: Protocols part of Springer nature, 2024. @article{noKey,
title = {Crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P1 21 1},
author = {Aschenbrenner, Jasmin},
url = {https://www.protocols.io/view/crystallisation-protocol-for-sars-cov-2-nsp3-macro-df6a3rae.html},
doi = {https://dx.doi.org/10.17504/protocols.io.261ge5ej7g47/v1},
year = {2024},
date = {2024-08-18},
journal = {Protocols part of Springer nature},
abstract = {The COVID-19 pandemic has demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This protocol details an optimized crystallization method for the SARS-CoV-2 nsp3 macrodomain, a potential drug target. Using sitting drop vapor diffusion with seeding, we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening. The method yields crystals that diffract to an average resolution of 1.5 Å, enabling high-resolution structural studies and can also be used for compound development through co-crystallization experiments.
All structures solved during the development of tool compounds for the SARS-CoV-2 nsp3 macrodomain are deposited on the PDB (Group deposition: G_1002283).},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
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The COVID-19 pandemic has demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This protocol details an optimized crystallization method for the SARS-CoV-2 nsp3 macrodomain, a potential drug target. Using sitting drop vapor diffusion with seeding, we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening. The method yields crystals that diffract to an average resolution of 1.5 Å, enabling high-resolution structural studies and can also be used for compound development through co-crystallization experiments.
All structures solved during the development of tool compounds for the SARS-CoV-2 nsp3 macrodomain are deposited on the PDB (Group deposition: G_1002283). |
Aschenbrenner, Jasmin Crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P43 Journal Article In: Protocols part of Springer nature, 2024. @article{noKey,
title = {Crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P43},
author = {Aschenbrenner, Jasmin},
url = {https://www.protocols.io/view/crystallisation-protocol-for-sars-cov-2-nsp3-macro-djdn4i5e.html},
doi = {https://dx.doi.org/10.17504/protocols.io.e6nvw1qb2lmk/v1},
year = {2024},
date = {2024-08-18},
journal = {Protocols part of Springer nature},
abstract = {The COVID-19 pandemic has demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This protocol details an optimized crystallization method for the SARS-CoV-2 nsp3 macrodomain, a potential drug target. Using sitting drop vapor diffusion, we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening. The method yields crystals that diffract to an average resolution of 1.2 Å, enabling high-resolution structural studies.
All structures solved during the development of tool compounds for the SARS-CoV-2 nsp3 macrodomain are deposited on the PDB (Group deposition: G_1002283).},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
}
The COVID-19 pandemic has demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This protocol details an optimized crystallization method for the SARS-CoV-2 nsp3 macrodomain, a potential drug target. Using sitting drop vapor diffusion, we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening. The method yields crystals that diffract to an average resolution of 1.2 Å, enabling high-resolution structural studies.
All structures solved during the development of tool compounds for the SARS-CoV-2 nsp3 macrodomain are deposited on the PDB (Group deposition: G_1002283). |
Aschenbrenner, Jasmin READDI protocol: Crystallisation of CHIKV nsP3 macrodomain Journal Article In: Protocols part of Springer nature, 2024. @article{noKey,
title = {READDI protocol: Crystallisation of CHIKV nsP3 macrodomain},
author = {Aschenbrenner, Jasmin},
url = {https://www.protocols.io/view/readdi-protocol-crystallisation-of-chikv-nsp3-macr-dcr62v9e.html},
doi = {https://dx.doi.org/10.17504/protocols.io.x54v92jzzl3e/v1},
year = {2024},
date = {2024-07-01},
journal = {Protocols part of Springer nature},
abstract = {Chikungunya virus (CHIKV) causes severe fever, rash and debilitating joint pain that can last for months or even years. Millions of people have been infected with CHIKV, mostly in low- and middle-income countries, and the virus continues to spread into new areas due to the geographical expansion of its mosquito hosts. The crystallization protocol and buffer conditions used to obtain reproducible Chikungunya Virus nsP3 macrodomain crystals suitable for XChem fragment screening.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
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Chikungunya virus (CHIKV) causes severe fever, rash and debilitating joint pain that can last for months or even years. Millions of people have been infected with CHIKV, mostly in low- and middle-income countries, and the virus continues to spread into new areas due to the geographical expansion of its mosquito hosts. The crystallization protocol and buffer conditions used to obtain reproducible Chikungunya Virus nsP3 macrodomain crystals suitable for XChem fragment screening. |
Lei, Li Structure and Stability of Ago2 MID-Nucleotide Complexes: All-in-One (Drop) His6-SUMO Tag Removal, Nucleotide Binding, and Crystal Growth Journal Article In: Current Protocols, 2024. @article{noKey,
title = {Structure and Stability of Ago2 MID-Nucleotide Complexes: All-in-One (Drop) His6-SUMO Tag Removal, Nucleotide Binding, and Crystal Growth},
author = {Lei, Li},
url = {https://currentprotocols.onlinelibrary.wiley.com/doi/pdf/10.1002/cpz1.1088},
doi = {https://doi.org/10.1002/cpz1.1088},
year = {2024},
date = {2024-06-23},
journal = {Current Protocols},
abstract = {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.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
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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. |
D. Clarke, John The impact of exchanging the light and heavy chains on the structures of bovine ultralong antibodies Journal Article In: Structural Biology Communications, 2024. @article{noKey,
title = {The impact of exchanging the light and heavy chains on the structures of bovine ultralong antibodies},
author = {D. Clarke, John},
url = {chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://journals.iucr.org/f/issues/2024/07/00/rl5199/rl5199.pdf},
doi = {https://doi.org/10.1107/S2053230X2400606X},
year = {2024},
date = {2024-06-20},
journal = {Structural Biology Communications},
abstract = {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.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
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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. |
N. F. King, Oliver CHiMP: Deep Learning Tools Trained on Protein Crystallisation Micrographs to Enable Automation of Experiments Journal Article In: Preprint, 2024. @article{noKey,
title = {CHiMP: Deep Learning Tools Trained on Protein Crystallisation Micrographs to Enable Automation of Experiments},
author = {N. F. King, Oliver},
url = {https://www.biorxiv.org/content/10.1101/2024.05.22.595345v1.abstract},
doi = {https://doi.org/10.1101/2024.05.22.595345},
year = {2024},
date = {2024-05-22},
journal = {Preprint},
abstract = {A group of three deep learning tools, referred to collectively as CHiMP (Crystal Hits in My Plate) were created for analysis of micrographs of protein crystallisation experiments at the Diamond Light Source (DLS) synchrotron, UK. The first tool, a classification network, assigns images into categories relating to experimental outcomes. The other two tools are networks that perform both object detection and instance segmentation, resulting in masks of individual crystals in the first case, and masks of crystallisation droplets in addition to crystals in the second case, allowing positions and sizes of these entities to be recorded. Creation of these tools used transfer learning, where weights from a pre-trained deep learning network were used as a starting point and re-purposed by further training on a relatively small set of data. Two of the tools are now integrated at the VMXi macromolecular crystallography beamline at DLS where they absolve the need for any user input both for monitoring crystallisation experiments and for triggering in situ data collections. The third is being integrated into the XChem fragment-based drug discovery screening platform, also at DLS, to allow automatic targeting of acoustic compound dispensing into crystallisation droplets.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
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A group of three deep learning tools, referred to collectively as CHiMP (Crystal Hits in My Plate) were created for analysis of micrographs of protein crystallisation experiments at the Diamond Light Source (DLS) synchrotron, UK. The first tool, a classification network, assigns images into categories relating to experimental outcomes. The other two tools are networks that perform both object detection and instance segmentation, resulting in masks of individual crystals in the first case, and masks of crystallisation droplets in addition to crystals in the second case, allowing positions and sizes of these entities to be recorded. Creation of these tools used transfer learning, where weights from a pre-trained deep learning network were used as a starting point and re-purposed by further training on a relatively small set of data. Two of the tools are now integrated at the VMXi macromolecular crystallography beamline at DLS where they absolve the need for any user input both for monitoring crystallisation experiments and for triggering in situ data collections. The third is being integrated into the XChem fragment-based drug discovery screening platform, also at DLS, to allow automatic targeting of acoustic compound dispensing into crystallisation droplets. |
Henriette Wegner, Christina Spectroscopic insights into multi-phase protein crystallization in complex lysate using Raman spectroscopy and a particle-free bypass Journal Article In: Frontiers in Bioengineering and Biotechnology, 2024. @article{noKey,
title = {Spectroscopic insights into multi-phase protein crystallization in complex lysate using Raman spectroscopy and a particle-free bypass},
author = {Henriette Wegner, Christina},
url = {https://www.frontiersin.org/articles/10.3389/fbioe.2024.1397465/full#h3},
doi = {https://doi.org/10.3389/fbioe.2024.1397465},
year = {2024},
date = {2024-05-14},
journal = {Frontiers in Bioengineering and Biotechnology},
abstract = {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.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
}
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. |
Xiao, Han High Resolution Crystal Structure of the Pyruvate Kinase Tetramer in Complex with the Allosteric Activator Mitapivat/AG-348 Journal Article In: Crystals, 2024. @article{noKey,
title = {High Resolution Crystal Structure of the Pyruvate Kinase Tetramer in Complex with the Allosteric Activator Mitapivat/AG-348},
author = {Xiao, Han},
url = {https://www.mdpi.com/2073-4352/14/5/441},
doi = {https://doi.org/10.3390/cryst14050441},
year = {2024},
date = {2024-05-04},
journal = {Crystals},
abstract = {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.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
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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. |
M Lithgo, Ryan Crystallographic Fragment Screen of Coxsackievirus A16 2A Protease identifies new opportunities for the development of broad-spectrum anti-enterovirals Journal Article In: Preprint, 2024. @article{noKey,
title = {Crystallographic Fragment Screen of Coxsackievirus A16 2A Protease identifies new opportunities for the development of broad-spectrum anti-enterovirals},
author = {M Lithgo, Ryan},
url = {https://www.biorxiv.org/content/10.1101/2024.04.29.591684v1.full.pdf},
doi = {https://doi.org/10.1101/2024.04.29.591684},
year = {2024},
date = {2024-04-28},
journal = {Preprint},
abstract = {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.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
}
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. |
Schutzer de Godoy, Andre Schutzer de Godoy Crystallization of Zika virus NS3 helicase Journal Article In: Protocols part of Springer nature, 2024. @article{noKey,
title = {Crystallization of Zika virus NS3 helicase},
author = {Schutzer de Godoy, Andre Schutzer de Godoy},
url = {https://www.protocols.io/view/crystallization-of-zika-virus-ns3-helicase-dcrw2v7e.html},
doi = {https://dx.doi.org/10.17504/protocols.io.e6nvw152dlmk/v1},
year = {2024},
date = {2024-04-25},
journal = {Protocols part of Springer nature},
abstract = {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.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
}
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. |
Lithgo, Ryan Crystallization of Enterovirus D68 3C protease Journal Article In: Protocols part of Springer nature, 2024. @article{noKey,
title = {Crystallization of Enterovirus D68 3C protease},
author = {Lithgo, Ryan},
url = {https://www.protocols.io/view/crystallization-of-enterovirus-d68-3c-protease-dcry2v7w.html},
doi = {https://dx.doi.org/10.17504/protocols.io.5qpvoky29l4o/v1},
year = {2024},
date = {2024-04-25},
journal = {Protocols part of Springer nature},
abstract = {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 D68 (EV-D68) which primarily spreads through respiratory routes causing mostly mild to severe respiratory illness but, in severe cases, acute flaccid myelitis. The 3C protease of EV-D68 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 D68 3C ProB crystals that were applied high-throughput crystallographic follow up compound screening on D68 3C.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
}
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 D68 (EV-D68) which primarily spreads through respiratory routes causing mostly mild to severe respiratory illness but, in severe cases, acute flaccid myelitis. The 3C protease of EV-D68 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 D68 3C ProB crystals that were applied high-throughput crystallographic follow up compound screening on D68 3C. |
Marples, Peter Crystallization of SARS-CoV-2 N Protein Journal Article In: Protocols part of Springer nature, 2024. @article{noKey,
title = {Crystallization of SARS-CoV-2 N Protein},
author = {Marples, Peter},
url = {https://www.protocols.io/view/crystallization-of-sars-cov-2-n-protein-4r3l2q4b3l1y/v1},
doi = {https://dx.doi.org/10.17504/protocols.io.4r3l2q4b3l1y/v1},
year = {2024},
date = {2024-04-25},
journal = {Protocols part of Springer nature},
abstract = {The crystallization protocol and buffer conditions used to obtain reproducible SARS C0V-2 Nucelocapsid crystals suitable for XChem fragment screening},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
}
The crystallization protocol and buffer conditions used to obtain reproducible SARS C0V-2 Nucelocapsid crystals suitable for XChem fragment screening |
Moreira Vacilotto, Milena Two-domain GH30 xylanase from human gut microbiota as a tool for enzymatic production of xylooligosaccharides: Crystallographic structure and a synergy with GH11 xylosidase Journal Article In: Carbohydrate Polymers, 2024. @article{noKey,
title = {Two-domain GH30 xylanase from human gut microbiota as a tool for enzymatic production of xylooligosaccharides: Crystallographic structure and a synergy with GH11 xylosidase},
author = {Moreira Vacilotto, Milena},
url = {https://www.sciencedirect.com/science/article/pii/S0144861724003679#s0010},
doi = {https://doi.org/10.1016/j.carbpol.2024.122141},
year = {2024},
date = {2024-04-11},
journal = {Carbohydrate Polymers},
abstract = {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.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
}
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. |
Metz, A. HEIDI: an experiment-management platform enabling high-throughput fragment and compound screening Journal Article In: Structural Biology, 2024. @article{noKey,
title = {HEIDI: an experiment-management platform enabling high-throughput fragment and compound screening},
author = {Metz, A.},
url = {https://journals.iucr.org/d/issues/2024/05/00/nz5016/index.html},
doi = {https://doi.org/10.1107/S2059798324002833},
year = {2024},
date = {2024-04-11},
journal = {Structural Biology},
abstract = {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.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
}
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. |
Namadurai, Sivakumar The crystal structure of human Navβ3-Ig domain and its implications Journal Article In: Thesis/Desertation, 2024. @article{noKey,
title = {The crystal structure of human Navβ3-Ig domain and its implications},
author = {Namadurai, Sivakumar},
url = {https://www.repository.cam.ac.uk/items/350806bd-6397-4ee3-8d68-d42e69ade1db},
doi = {https://doi.org/10.17863/CAM.107561},
year = {2024},
date = {2024-04-11},
journal = {Thesis/Desertation},
abstract = {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 homotrimer
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 fulllength
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.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
}
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 homotrimer
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 fulllength
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. |
Trëndelina, Rrustemi Exploring Pathogenic Mutations at Phosphorylation Sites through a Peptide-Based Proteomics Screen Journal Article In: Thesis/ Dessertation, 2024. @article{noKey,
title = {Exploring Pathogenic Mutations at Phosphorylation Sites through a Peptide-Based Proteomics Screen},
author = {Trëndelina, Rrustemi},
url = {https://edoc.hu-berlin.de/items/c8b80158-15a3-4a61-ad97-e63e1d9c9a1c},
doi = {http://dx.doi.org/10.18452/28699},
year = {2024},
date = {2024-04-10},
journal = {Thesis/ Dessertation},
abstract = {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.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
}
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. |
Rrustemi, Trendelina Pathogenic mutations of human phosphorylation sites affect protein–protein interactions Journal Article In: Nature Communications, 2024. @article{noKey,
title = {Pathogenic mutations of human phosphorylation sites affect protein–protein interactions},
author = {Rrustemi, Trendelina},
url = {https://www.nature.com/articles/s41467-024-46794-8},
doi = {https://doi.org/10.1038/s41467-024-46794-8},
year = {2024},
date = {2024-04-10},
journal = {Nature Communications},
abstract = {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.},
keywords = {Rock Imager®},
pubstate = {published},
tppubtype = {article}
}
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. |
