Angelos Stamos, Nikolaos Beyond Barriers, Big Crystallisation Hurdles: Atropisomerism in bRo5 Compounds Explored by Computational and NMR Studies Journal Article In: 2025. @article{noKey,
title = {Beyond Barriers, Big Crystallisation Hurdles: Atropisomerism in bRo5 Compounds Explored by Computational and NMR Studies},
author = {Angelos Stamos, Nikolaos},
url = {https://chemrxiv.org/engage/chemrxiv/article-details/679a3f956dde43c908d979c7},
doi = {https://doi.org/10.26434/chemrxiv-2025-rz4q6},
year = {2025},
date = {2025-02-03},
abstract = {Crystallisation and stereochemical stability are pivotal factors in pharmaceutical development, particularly for complex beyond Rule of 5 (bRo5) compounds. In this study, we explore the intricate interplay between atropisomerism and crystallisation using two model bRo5 compounds, namely ACBI1 and BI201335, both violating three of four Lipinski’s rules. One of the tool compounds exhibits Class 2 atropisomeric behaviour and the other devoid of it. A diverse array of crystallisation methods—including solution-phase crystallisation, cocrystallisation, and salt formation—was applied, revealing the critical role of atropisomerism induced stereochemistry in polymorphism and nucleation outcomes. In-silico torsion profile calculations and NMR studies were employed to elucidate the rotational energy barriers and confirm the presence or absence of atropisomerism. This comprehensive analysis highlights the significance of understanding stereochemical phenomena like atropisomerism in designing and developing bRo5 compounds. By integrating advanced analytical techniques and crystallisation strategies, this work provides novel insights into tailoring pharmaceutical properties for nextgeneration therapeutics.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Crystallisation and stereochemical stability are pivotal factors in pharmaceutical development, particularly for complex beyond Rule of 5 (bRo5) compounds. In this study, we explore the intricate interplay between atropisomerism and crystallisation using two model bRo5 compounds, namely ACBI1 and BI201335, both violating three of four Lipinski’s rules. One of the tool compounds exhibits Class 2 atropisomeric behaviour and the other devoid of it. A diverse array of crystallisation methods—including solution-phase crystallisation, cocrystallisation, and salt formation—was applied, revealing the critical role of atropisomerism induced stereochemistry in polymorphism and nucleation outcomes. In-silico torsion profile calculations and NMR studies were employed to elucidate the rotational energy barriers and confirm the presence or absence of atropisomerism. This comprehensive analysis highlights the significance of understanding stereochemical phenomena like atropisomerism in designing and developing bRo5 compounds. By integrating advanced analytical techniques and crystallisation strategies, this work provides novel insights into tailoring pharmaceutical properties for nextgeneration therapeutics. |
M. Böhm, Jonas From dimer to tetramer: the evolutionary trajectory of C4 photosynthetic-NADP-ME oligomeric state in Poaceae Journal Article In: 2025. @article{noKey,
title = {From dimer to tetramer: the evolutionary trajectory of C4 photosynthetic-NADP-ME oligomeric state in Poaceae},
author = {M. Böhm, Jonas},
url = {https://www.biorxiv.org/content/10.1101/2025.01.05.631420v1.abstract},
doi = {https://doi.org/10.1101/2025.01.05.631420},
year = {2025},
date = {2025-01-06},
abstract = {The C4 carbon concentrating mechanism relies on specialized enzymes that have evolved unique expression patterns and biochemical properties distinct to their ancestral housekeeping forms. In maize and sorghum, the evolution of C4-NADP-malic enzyme (C4-NADP-ME) involved gene duplication and neofunctionalization, leading to the emergence of two plastidic isoforms: C4-NADP-ME and nonC4-NADP-ME, each with distinct kinetic and structural features. While C4-NADP-ME functions primarily as a tetramer, nonC4-NADP-ME exists in an equilibrium between dimeric and tetrameric forms, favoring the dimer in solution. This study shows which evolutionary changes in amino acid sequences influence the structure and function of these isoforms. By integrating X-ray crystallography, cryo-electron microscopy, computational molecular modeling and targeted biochemical analysis of mutant and truncated protein variants, we identify crucial roles for the N- and C-terminal regions and specific amino acid residues in governing isoform oligomerization. Our results reveal that the N-terminal region is essential for stabilizing the dimeric form of nonC4-NADP-ME, whereas specific adaptive substitutions and interactions with the C-terminal region enhance the stability of the tetrameric state characteristic of the C4-adapted isoform. We propose that differences in the N-terminal domain between the C4 and nonC4 isoforms reflect distinct selective pressures, which have driven their evolutionary divergence to fulfill specialized cellular functions.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
The C4 carbon concentrating mechanism relies on specialized enzymes that have evolved unique expression patterns and biochemical properties distinct to their ancestral housekeeping forms. In maize and sorghum, the evolution of C4-NADP-malic enzyme (C4-NADP-ME) involved gene duplication and neofunctionalization, leading to the emergence of two plastidic isoforms: C4-NADP-ME and nonC4-NADP-ME, each with distinct kinetic and structural features. While C4-NADP-ME functions primarily as a tetramer, nonC4-NADP-ME exists in an equilibrium between dimeric and tetrameric forms, favoring the dimer in solution. This study shows which evolutionary changes in amino acid sequences influence the structure and function of these isoforms. By integrating X-ray crystallography, cryo-electron microscopy, computational molecular modeling and targeted biochemical analysis of mutant and truncated protein variants, we identify crucial roles for the N- and C-terminal regions and specific amino acid residues in governing isoform oligomerization. Our results reveal that the N-terminal region is essential for stabilizing the dimeric form of nonC4-NADP-ME, whereas specific adaptive substitutions and interactions with the C-terminal region enhance the stability of the tetrameric state characteristic of the C4-adapted isoform. We propose that differences in the N-terminal domain between the C4 and nonC4 isoforms reflect distinct selective pressures, which have driven their evolutionary divergence to fulfill specialized cellular functions. |
Kaundal, Soni RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as viral replication factories Journal Article In: 2024. @article{noKey,
title = {RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as viral replication factories},
author = {Kaundal, Soni},
url = {https://www.science.org/doi/full/10.1126/sciadv.adp9333},
doi = {https://doi.org/10.1126/sciadv.adp9333},
year = {2024},
date = {2024-12-20},
abstract = {Many viral proteins form biomolecular condensates via liquid-liquid phase separation (LLPS) to support viral replication and evade host antiviral responses, and thus, they are potential targets for designing antivirals. In the case of nonenveloped positive-sense RNA viruses, forming such condensates for viral replication is unclear and less understood. Human noroviruses (HuNoVs) are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide. Here, we show that the RNA-dependent RNA polymerase (RdRp) of pandemic GII.4 HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication. We show that such condensates are formed in HuNoV-infected human intestinal enteroid cultures and are the sites for genome replication. Our studies demonstrate the formation of phase-separated condensates as replication factories in a positive-sense RNA virus, which plausibly is an effective mechanism to dynamically isolate RdRp replicating the genomic RNA from interfering with the ribosomal translation of the same RNA.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Many viral proteins form biomolecular condensates via liquid-liquid phase separation (LLPS) to support viral replication and evade host antiviral responses, and thus, they are potential targets for designing antivirals. In the case of nonenveloped positive-sense RNA viruses, forming such condensates for viral replication is unclear and less understood. Human noroviruses (HuNoVs) are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide. Here, we show that the RNA-dependent RNA polymerase (RdRp) of pandemic GII.4 HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication. We show that such condensates are formed in HuNoV-infected human intestinal enteroid cultures and are the sites for genome replication. Our studies demonstrate the formation of phase-separated condensates as replication factories in a positive-sense RNA virus, which plausibly is an effective mechanism to dynamically isolate RdRp replicating the genomic RNA from interfering with the ribosomal translation of the same RNA. |
Dunnett, Louise Enhanced identification of small molecules binding to hnRNPA1 via cryptic pockets mapping coupled with X-Ray fragment screening Journal Article In: 2024. @article{noKey,
title = {Enhanced identification of small molecules binding to hnRNPA1 via cryptic pockets mapping coupled with X-Ray fragment screening},
author = {Dunnett, Louise},
url = {https://www.biorxiv.org/content/10.1101/2024.12.17.628909v1.abstract},
doi = {https://doi.org/10.1101/2024.12.17.628909},
year = {2024},
date = {2024-12-18},
abstract = {The human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a prototypical RNA-binding protein essential in regulating a wide range of post-transcriptional events in cells. As a multifunctional protein with a key role in RNA metabolism, deregulation of its functions has been linked to neurodegenerative diseases, tumour aggressiveness and chemoresistance, which has fuelled efforts to develop novel therapeutics that modulates its RNA binding activities. Here, using a combination of Molecular Dynamics (MD) simulations and graph neural network pockets predictions, we showed that hnRNPA1 N-terminal RNA binding domain (UP1) contains several cryptic pockets capable of binding small molecules. To identify chemical entities for development of potent drug candidates and experimentally validate identified druggable hotspots, we carried out a large fragment screening on UP1 protein crystals. Our screen identified 36 hits which extensively samples UP1 functional regions involved in RNA recognition and binding, as well as mapping hotspots onto novel protein interaction surfaces. We observed a wide range of ligand-induced conformational variation, by stabilisation of dynamic protein regions. Our high-resolution structures, the first of an hnRNP in complex with a fragment or small molecule, provides rapid routes for the rational development of a range of different inhibitors and chemical tools for studying molecular mechanisms of hnRNPA1 mediated splicing regulation.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
The human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a prototypical RNA-binding protein essential in regulating a wide range of post-transcriptional events in cells. As a multifunctional protein with a key role in RNA metabolism, deregulation of its functions has been linked to neurodegenerative diseases, tumour aggressiveness and chemoresistance, which has fuelled efforts to develop novel therapeutics that modulates its RNA binding activities. Here, using a combination of Molecular Dynamics (MD) simulations and graph neural network pockets predictions, we showed that hnRNPA1 N-terminal RNA binding domain (UP1) contains several cryptic pockets capable of binding small molecules. To identify chemical entities for development of potent drug candidates and experimentally validate identified druggable hotspots, we carried out a large fragment screening on UP1 protein crystals. Our screen identified 36 hits which extensively samples UP1 functional regions involved in RNA recognition and binding, as well as mapping hotspots onto novel protein interaction surfaces. We observed a wide range of ligand-induced conformational variation, by stabilisation of dynamic protein regions. Our high-resolution structures, the first of an hnRNP in complex with a fragment or small molecule, provides rapid routes for the rational development of a range of different inhibitors and chemical tools for studying molecular mechanisms of hnRNPA1 mediated splicing regulation. |
Naz, Zahra Biophysical Characterization of a Novel Phosphopentomutase from the Hyperthermophilic Archaeon Thermococcus kodakarensis Journal Article In: 2024. @article{noKey,
title = {Biophysical Characterization of a Novel Phosphopentomutase from the Hyperthermophilic Archaeon Thermococcus kodakarensis},
author = {Naz, Zahra},
url = {https://www.mdpi.com/1422-0067/25/23/12893},
doi = {https://doi.org/10.3390/ijms252312893},
year = {2024},
date = {2024-11-30},
abstract = {Phosphopentomutases catalyze the isomerization of ribose 1-phosphate and ribose 5-phosphate. Thermococcus kodakarensis, a hyperthermophilic archaeon, harbors a novel enzyme (PPMTk) that exhibits high homology with phosphohexomutases but has no significant phosphohexomutase activity. Instead, PPMTk catalyzes the interconversion of ribose 1-phosphate and ribose 5-phosphate. Here, we report biophysical analysis, crystallization, and three-dimensional structure determination of PPMTk by X-ray diffraction at 2.39 Å resolution. The solved structure revealed a novel catalytic motif, unique to PPMTk, which makes this enzyme distinct from the homologous counterparts. We postulate that this novel catalytic motif may enable PPMTk to isomerize phosphopentose instead of phosphohexose. To the best of our knowledge, this is the first biophysical and structural analysis of a phosphopentomutase from hyperthermophilic archaea.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Phosphopentomutases catalyze the isomerization of ribose 1-phosphate and ribose 5-phosphate. Thermococcus kodakarensis, a hyperthermophilic archaeon, harbors a novel enzyme (PPMTk) that exhibits high homology with phosphohexomutases but has no significant phosphohexomutase activity. Instead, PPMTk catalyzes the interconversion of ribose 1-phosphate and ribose 5-phosphate. Here, we report biophysical analysis, crystallization, and three-dimensional structure determination of PPMTk by X-ray diffraction at 2.39 Å resolution. The solved structure revealed a novel catalytic motif, unique to PPMTk, which makes this enzyme distinct from the homologous counterparts. We postulate that this novel catalytic motif may enable PPMTk to isomerize phosphopentose instead of phosphohexose. To the best of our knowledge, this is the first biophysical and structural analysis of a phosphopentomutase from hyperthermophilic archaea. |
Schneberger, Niels Allosteric substrate release by a sialic acid TRAP transporter substrate binding protein Journal Article In: 2024. @article{noKey,
title = {Allosteric substrate release by a sialic acid TRAP transporter substrate binding protein},
author = {Schneberger, Niels},
url = {https://www.nature.com/articles/s42003-024-07263-6},
doi = {https://doi.org/10.1038/s42003-024-07263-6},
year = {2024},
date = {2024-11-23},
abstract = {The tripartite ATP-independent periplasmic (TRAP) transporters enable Vibrio cholerae and Haemophilus influenzae to acquire sialic acid, aiding their colonization of human hosts. This process depends on SiaP, a substrate-binding protein (SBP) that captures and delivers sialic acid to the transporter. We identified 11 nanobodies that bind specifically to the SiaP proteins from H. influenzae (HiSiaP) and V. cholerae (VcSiaP). Two nanobodies inhibited sialic acid binding. Detailed structural and biophysical studies of one nanobody-SBP complex revealed an allosteric inhibition mechanism, preventing ligand binding and releasing pre-bound sialic acid. A hydrophobic surface pocket of the SBP is crucial for the allosteric mechanism and for the conformational rearrangement that occurs upon binding of sialic acid to the SBP. Our findings provide new clues regarding the mechanism of TRAP transporters, as well as potential starting points for novel drug design approaches to starve these human pathogens of important host-derived molecules.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
The tripartite ATP-independent periplasmic (TRAP) transporters enable Vibrio cholerae and Haemophilus influenzae to acquire sialic acid, aiding their colonization of human hosts. This process depends on SiaP, a substrate-binding protein (SBP) that captures and delivers sialic acid to the transporter. We identified 11 nanobodies that bind specifically to the SiaP proteins from H. influenzae (HiSiaP) and V. cholerae (VcSiaP). Two nanobodies inhibited sialic acid binding. Detailed structural and biophysical studies of one nanobody-SBP complex revealed an allosteric inhibition mechanism, preventing ligand binding and releasing pre-bound sialic acid. A hydrophobic surface pocket of the SBP is crucial for the allosteric mechanism and for the conformational rearrangement that occurs upon binding of sialic acid to the SBP. Our findings provide new clues regarding the mechanism of TRAP transporters, as well as potential starting points for novel drug design approaches to starve these human pathogens of important host-derived molecules. |
Murugesan, Gavuthami Viral sequence determines HLA-E-restricted T cell recognition of hepatitis B surface antigen Journal Article In: 2024. @article{noKey,
title = {Viral sequence determines HLA-E-restricted T cell recognition of hepatitis B surface antigen},
author = {Murugesan, Gavuthami},
url = {https://www.nature.com/articles/s41467-024-54378-9},
doi = {https://doi.org/10.1038/s41467-024-54378-9},
year = {2024},
date = {2024-11-22},
abstract = {The non-polymorphic HLA-E molecule offers opportunities for new universal immunotherapeutic approaches to chronic infectious diseases. Chronic Hepatitis B virus (HBV) infection is driven in part by T cell dysfunction due to elevated levels of the HBV envelope (Env) protein hepatitis B surface antigen (HBsAg). Here we report the characterization of three genotypic variants of an HLA-E-binding HBsAg peptide, Env371-379, identified through bioinformatic predictions and verified by biochemical and cellular assays. Using a soluble affinity-enhanced T cell receptor (TCR) (a09b08)-anti-CD3 bispecific molecule to probe HLA-E presentation of the Env371-379 peptides, we demonstrate that only the most stable Env371-379 variant, L6I, elicits functional responses to a09b08-anti-CD3-redirected polyclonal T cells co-cultured with targets expressing endogenous HBsAg. Furthermore, HLA-E-Env371-379 L6I-specific CD8+ T cells are detectable in HBV-naïve donors and people with chronic HBV after in vitro priming. In conclusion, we provide evidence for HLA-E-mediated HBV Env peptide presentation, and highlight the effect of viral mutations on the stability and targetability of pHLA-E molecules.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
The non-polymorphic HLA-E molecule offers opportunities for new universal immunotherapeutic approaches to chronic infectious diseases. Chronic Hepatitis B virus (HBV) infection is driven in part by T cell dysfunction due to elevated levels of the HBV envelope (Env) protein hepatitis B surface antigen (HBsAg). Here we report the characterization of three genotypic variants of an HLA-E-binding HBsAg peptide, Env371-379, identified through bioinformatic predictions and verified by biochemical and cellular assays. Using a soluble affinity-enhanced T cell receptor (TCR) (a09b08)-anti-CD3 bispecific molecule to probe HLA-E presentation of the Env371-379 peptides, we demonstrate that only the most stable Env371-379 variant, L6I, elicits functional responses to a09b08-anti-CD3-redirected polyclonal T cells co-cultured with targets expressing endogenous HBsAg. Furthermore, HLA-E-Env371-379 L6I-specific CD8+ T cells are detectable in HBV-naïve donors and people with chronic HBV after in vitro priming. In conclusion, we provide evidence for HLA-E-mediated HBV Env peptide presentation, and highlight the effect of viral mutations on the stability and targetability of pHLA-E molecules. |
Karuppiah, Vijaykumar Broadening alloselectivity of T cell receptors by structure guided engineering Journal Article In: 2024. @article{noKey,
title = {Broadening alloselectivity of T cell receptors by structure guided engineering},
author = {Karuppiah, Vijaykumar},
url = {https://www.nature.com/articles/s41598-024-75140-7#Abs1},
doi = {https://doi.org/10.1038/s41598-024-75140-7},
year = {2024},
date = {2024-11-06},
abstract = {Specificity of a T cell receptor (TCR) is determined by the combination of its interactions to the peptide and human leukocyte antigen (HLA). TCR-based therapeutic molecules have to date targeted a single peptide in the context of a single HLA allele. Some peptides are presented on multiple HLA alleles, and by engineering TCRs for specific recognition of more than one allele, there is potential to expand the targetable patient population. Here, as a proof of concept, we studied two TCRs, S2 and S8, binding to the PRAME peptide antigen (ELFSYLIEK) presented by HLA alleles HLA-A*03:01 and HLA-A*11:01. By structure-guided affinity maturation targeting a specific residue on the HLA surface, we show that the affinity of the TCR can be modulated for different alleles. Using a combination of affinity maturation and functional T cell assay, we demonstrate that an engineered TCR can target the same peptide on two different HLA alleles with similar affinity and potency. This work highlights the importance of engineering alloselectivity for designing TCR based therapeutics suitable for differing global populations.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Specificity of a T cell receptor (TCR) is determined by the combination of its interactions to the peptide and human leukocyte antigen (HLA). TCR-based therapeutic molecules have to date targeted a single peptide in the context of a single HLA allele. Some peptides are presented on multiple HLA alleles, and by engineering TCRs for specific recognition of more than one allele, there is potential to expand the targetable patient population. Here, as a proof of concept, we studied two TCRs, S2 and S8, binding to the PRAME peptide antigen (ELFSYLIEK) presented by HLA alleles HLA-A*03:01 and HLA-A*11:01. By structure-guided affinity maturation targeting a specific residue on the HLA surface, we show that the affinity of the TCR can be modulated for different alleles. Using a combination of affinity maturation and functional T cell assay, we demonstrate that an engineered TCR can target the same peptide on two different HLA alleles with similar affinity and potency. This work highlights the importance of engineering alloselectivity for designing TCR based therapeutics suitable for differing global populations. |
I. Sotiropoulou, Anastasia Structural studies of β-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus Journal Article In: 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 = {2024-10-01},
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 = {ROCKIMAGER},
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. |
Pinto, Miriam The toxicity of alpha-syn condensates in the presence and absence of metal ions Journal Article In: 2024. @article{noKey,
title = {The toxicity of alpha-syn condensates in the presence and absence of metal ions},
author = {Pinto, Miriam},
url = {https://repositorio.ulisboa.pt/handle/10400.5/95870},
doi = {http://hdl.handle.net/10400.5/95870},
year = {2024},
date = {2024-09-08},
abstract = {Neurodegenerative diseases (NDDs), characterized by progressive neuronal death and misfolded protein aggregation, pose significant clinical, social, and personal challenges. Parkinson's Disease (PD), the second most common neurological disorder, is notably associated with the aggregation of alpha-synuclein (aSyn). Despite its prominence, the transition of monomeric aSyn to aggregates remains inadequately understood. Recent studies suggest that Liquid-Liquid Phase Separation (LLPS) and disease related metal ions involve the transition in the molecular pathogenesis of PD. LLPS involves the separation of biomolecules into distinct phases without a membrane, potentially facilitating aSyn aggregation through dynamic condensates that eventually form solid deposits. I aim to investigate LLPS of aSyn and macroscopic dynamics of its formed droplets over time, and examine how PD related metal ions, affect the dynamic process of LLPS and modulate its toxicity to neuroblastoma cells. These metal ions, prevalent in the brain and specifically interacting with aSyn are presumably modulating LLPS, toxicity and aggregation of aSyn, making it crucial to understand their roles in the molecular pathogenesis of PD I expressed α-synuclein (aSyn) proteins in E. coli and studied the biophysical properties and toxicities of aSyn-metal ion coacervates using various techniques, including a protein crystallization robotic dispenser and confocal microscopy. In the presence of metal ions such as CuCl₂, MnCl₂, ZnCl₂, and FeCl₃, the number of droplets significantly decreased. I found that CuCl₂ ions immobilize aSyn condensates and increase their toxicity. In contrast, MnCl₂, ZnCl₂, and FeCl₃ help maintain a longer metastable state of the condensates, reducing their toxicity. This project highlights the crucial role of metal ions in modulating aSyn phase behavior, condensate toxicity and their potential involvement in the progression of PD.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
Neurodegenerative diseases (NDDs), characterized by progressive neuronal death and misfolded protein aggregation, pose significant clinical, social, and personal challenges. Parkinson's Disease (PD), the second most common neurological disorder, is notably associated with the aggregation of alpha-synuclein (aSyn). Despite its prominence, the transition of monomeric aSyn to aggregates remains inadequately understood. Recent studies suggest that Liquid-Liquid Phase Separation (LLPS) and disease related metal ions involve the transition in the molecular pathogenesis of PD. LLPS involves the separation of biomolecules into distinct phases without a membrane, potentially facilitating aSyn aggregation through dynamic condensates that eventually form solid deposits. I aim to investigate LLPS of aSyn and macroscopic dynamics of its formed droplets over time, and examine how PD related metal ions, affect the dynamic process of LLPS and modulate its toxicity to neuroblastoma cells. These metal ions, prevalent in the brain and specifically interacting with aSyn are presumably modulating LLPS, toxicity and aggregation of aSyn, making it crucial to understand their roles in the molecular pathogenesis of PD I expressed α-synuclein (aSyn) proteins in E. coli and studied the biophysical properties and toxicities of aSyn-metal ion coacervates using various techniques, including a protein crystallization robotic dispenser and confocal microscopy. In the presence of metal ions such as CuCl₂, MnCl₂, ZnCl₂, and FeCl₃, the number of droplets significantly decreased. I found that CuCl₂ ions immobilize aSyn condensates and increase their toxicity. In contrast, MnCl₂, ZnCl₂, and FeCl₃ help maintain a longer metastable state of the condensates, reducing their toxicity. This project highlights the crucial role of metal ions in modulating aSyn phase behavior, condensate toxicity and their potential involvement in the progression of PD. |
A. Lee, Alpha Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response Journal Article In: 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 = {2024-08-21},
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 = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
Baumgarten, Janosch Substrate‐Based Ligand Design for Phenazine Biosynthesis Enzyme PhzF Journal Article In: 2024. @article{noKey,
title = {Substrate‐Based Ligand Design for Phenazine Biosynthesis Enzyme PhzF},
author = {Baumgarten, Janosch},
url = {https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/cmdc.202400466},
doi = {https://doi.org/10.1002/cmdc.202400466},
year = {2024},
date = {2024-08-20},
abstract = {The phenazine pyocyanin is an important virulence factor of the pathogen Pseudomonas aeruginosa, which is on the WHO list of antibiotic resistant “priority pathogens”. In this study the isomerase PhzF, a key bacterial enzyme of the pyocyanin biosynthetic pathway, was investigated as a pathoblocker target. The aim of the pathoblocker strategy is to reduce the virulence of the pathogen without killing it, thus preventing the rapid development of resistance. Based on crystal structures of PhzF, derivatives of the inhibitor 3–hydroxyanthranilic acid were designed. Co-crystal structures of the synthesized derivatives with PhzF revealed spacial limitations of the binding pocket of PhzF in the closed conformation. In contrast, ligands aligned to the open conformation of PhzF provided more room for structural modifications. The intrinsic fluorescence of small 3–hydroxyanthranilic acid derivatives enabled direct affinity determinations using FRET assays. The analysis of structure-activity relationships showed that the carboxylic acid moiety is essential for binding to the target enzyme. The results of this study provide fundamental structural insights that will be useful for the design of PhzF-inhibitors.},
keywords = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
The phenazine pyocyanin is an important virulence factor of the pathogen Pseudomonas aeruginosa, which is on the WHO list of antibiotic resistant “priority pathogens”. In this study the isomerase PhzF, a key bacterial enzyme of the pyocyanin biosynthetic pathway, was investigated as a pathoblocker target. The aim of the pathoblocker strategy is to reduce the virulence of the pathogen without killing it, thus preventing the rapid development of resistance. Based on crystal structures of PhzF, derivatives of the inhibitor 3–hydroxyanthranilic acid were designed. Co-crystal structures of the synthesized derivatives with PhzF revealed spacial limitations of the binding pocket of PhzF in the closed conformation. In contrast, ligands aligned to the open conformation of PhzF provided more room for structural modifications. The intrinsic fluorescence of small 3–hydroxyanthranilic acid derivatives enabled direct affinity determinations using FRET assays. The analysis of structure-activity relationships showed that the carboxylic acid moiety is essential for binding to the target enzyme. The results of this study provide fundamental structural insights that will be useful for the design of PhzF-inhibitors. |
Aschenbrenner, Jasmin Crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P1 21 1 Journal Article In: 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 = {2024-08-19},
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 = {ROCKIMAGER},
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 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: 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 = {2024-08-19},
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 = {ROCKIMAGER},
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: 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 = {2024-07-02},
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 = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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: 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 = {2024-06-24},
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 = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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: 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 = {2024-06-21},
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 = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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: 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 = {2024-05-23},
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 = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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: 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 = {2024-05-15},
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 = {ROCKIMAGER},
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: 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 = {2024-05-05},
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 = {ROCKIMAGER},
pubstate = {published},
tppubtype = {article}
}
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. |
