| 1. |
- Hall, Michael, et al.
(författare)
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Structural basis for glutathione-mediated activation of the virulence regulatory protein PrfA in Listeria
- 2016
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 113:51, s. 14733-14738
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Tidskriftsartikel (refereegranskat)abstract
- Infection by the human bacterial pathogen Listeria monocytogenes is mainly controlled by the positive regulatory factor A (PrfA), a member of the Crp/Fnr family of transcriptional activators. Published data suggest that PrfA requires the binding of a cofactor for full activity, and it was recently proposed that glutathione (GSH) could fulfill this function. Here we report the crystal structures of PrfA in complex with GSH and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. These structures reveal the structural basis for a GSH-mediated allosteric mode of activation of PrfA in the cytosol of the host cell. The crystal structure of PrfAWT in complex only with DNA confirms that PrfAWT can adopt a DNA binding-compatible structure without binding the GSH activator molecule. By binding to PrfA in the cytosol of the host cell, GSH induces the correct fold of the HTH motifs, thus priming the PrfA protein for DNA interaction.
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| 2. |
- Kovermann, Michael, et al.
(författare)
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Structural basis for catalytically restrictive dynamics of a high-energy enzyme state
- 2015
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Ingår i: Nature Communications. - : Macmillan Publishers Ltd.. - 2041-1723. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible’ states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme’s catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes’ conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions.
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| 3. |
- Kovermann, Michael, et al.
(författare)
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Structural basis for ligand binding to an enzyme by a conformational selection pathway
- 2017
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:24, s. 6298-6303
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Tidskriftsartikel (refereegranskat)abstract
- Proteins can bind target molecules through either induced fit or conformational selection pathways. In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation. In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the rate-limiting step for catalytic turnover. The transient nature of these states has precluded direct observation of their properties. Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography. Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis. A 1.9-angstrom X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic side-chains are properly aligned for catalysis. We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state. In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance. Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change.
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| 4. |
- Kulén, Martina, et al.
(författare)
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Structure-based design of inhibitors targeting PrfA, the master virulence regulator of Listeria monocytogenes
- 2018
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 61:9, s. 4165-4175
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Tidskriftsartikel (refereegranskat)abstract
- Listeria monocytogenes is a bacterial pathogen that controls much of its virulence through the transcriptional regulator PrfA. In this study, we describe structure guided design and synthesis of a set of PrfA inhibitors based on ring-fused 2-pyridone heterocycles. Our most effective compound decreased virulence factor expression, reduced bacterial uptake into eukaryotic cells, and improved survival of chicken embryos infected with L. monocytogenes compared to previously identified compounds. Crystal structures identified an intraprotein "tunnel" as the main inhibitor binding site (A1), where the compounds participate in an extensive hydrophobic network that restricts the protein's ability to form functional DNA-binding helix−turn−helix (HTH) motifs. Our studies also revealed a hitherto unsuspected structural plasticity of the HTH motif. In conclusion, we have designed 2-pyridone analogues that function as site-A1 selective PrfA inhibitors with potent antivirulence properties.
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| 5. |
- Montoliu-Gaya, Laia, et al.
(författare)
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Optimal blood tau species for the detection of Alzheimer's disease neuropathology: an immunoprecipitation mass spectrometry and autopsy study.
- 2024
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Ingår i: Acta neuropathologica. - 1432-0533. ; 147:1
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Tidskriftsartikel (refereegranskat)abstract
- Plasma-to-autopsy studies are essential for validation of blood biomarkers and understanding their relation to Alzheimer's disease (AD) pathology. Few such studies have been done on phosphorylated tau (p-tau) and those that exist have made limited or no comparison of the different p-tau variants. This study is the first to use immunoprecipitation mass spectrometry (IP-MS) to compare the accuracy of eight different plasma tau species in predicting autopsy-confirmed AD. The sample included 123 participants (AD=69, non-AD=54) from the Boston University Alzheimer's disease Research Center who had an available ante-mortem plasma sample and donated their brain. Plasma samples proximate to death were analyzed by targeted IP-MS for six different tryptic phosphorylated (p-tau-181, 199, 202, 205, 217, 231), and two non-phosphorylated tau (195-205, 212-221) peptides. NIA-Reagan Institute criteria were used for the neuropathological diagnosis of AD. Binary logistic regressions tested the association between each plasma peptide and autopsy-confirmed AD status. Area under the receiver operating curve (AUC) statistics were generated using predicted probabilities from the logistic regression models. Odds Ratio (OR) was used to study associations between the different plasma tau species and CERAD and Braak classifications. All tau species were increased in AD compared to non-AD, but p-tau217, p-tau205 and p-tau231 showed the highest fold-changes. Plasma p-tau217 (AUC=89.8), p-tau231 (AUC=83.4), and p-tau205 (AUC=81.3) all had excellent accuracy in discriminating AD from non-AD brain donors, even among those with CDR<1). Furthermore, p-tau217, p-tau205 and p-tau231 showed the highest ORs with both CERAD (ORp-tau217=15.29, ORp-tau205=5.05 and ORp-tau231=3.86) and Braak staging (ORp-tau217=14.29, ORp-tau205=5.27 and ORp-tau231=4.02) but presented increased levels at different amyloid and tau stages determined by neuropathological examination. Our findings support plasma p-tau217 as the most promising p-tau species for detecting AD brain pathology. Plasma p-tau231 and p-tau205 may additionally function as markers for different stages of the disease.
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| 6. |
- Alseekh, Saleh, et al.
(författare)
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Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices
- 2021
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 18:7, s. 747-756
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Forskningsöversikt (refereegranskat)abstract
- This Perspective, from a large group of metabolomics experts, provides best practices and simplified reporting guidelines for practitioners of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics. Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.
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| 7. |
- Babes, Alexandru, et al.
(författare)
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Photosensitization in porphyrias and photodynamic therapy involves TRPA1 and TRPV1
- 2016
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Ingår i: The Journal of Neuroscience. - 0270-6474. ; 36:19, s. 5264-5278
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Tidskriftsartikel (refereegranskat)abstract
- Photosensitization, an exaggerated sensitivity to harmless light, occurs genetically in rare diseases, such as porphyrias, and in photodynamic therapy where short-term toxicity is intended. A common feature is the experience of pain from bright light. In human subjects, skin exposure to 405 nm light induced moderate pain, which was intensified by pretreatment with aminolevulinic acid. In heterologous expression systems and cultured sensory neurons, exposure to blue light activated TRPA1 and, to a lesser extent, TRPV1 channels in the absence of additional photosensitization. Pretreatment with aminolevulinic acid or with protoporphyrin IX dramatically increased the light sensitivity of both TRPA1 and TRPV1 via generation of reactive oxygen species. Artificial lipid bilayers equipped with purified human TRPA1 showed substantial single-channel activity only in the presence of protoporphyrin IX and blue light. Photosensitivity and photosensitization could be demonstrated in freshly isolated mouse tissues and led to TRP channel-dependent release of proinflammatory neuropeptides upon illumination. With antagonists in clinical development, these findings may help to alleviate pain during photodynamic therapy and also allow for disease modification in porphyria patients.
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| 8. |
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| 9. |
- Campbell, Charles, et al.
(författare)
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Bridging model and real catalysts: general discussion
- 2016
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Ingår i: Faraday Discussions. - 1359-6640 .- 1364-5498. ; 188, s. 565-589
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Charles Campbell opened the discussion of the paper by Hans-JoachimFreund: If you have a 3D gold particle and it spreads out to be a 2D particle whenyou adsorb CO2, it must gain energy stability. Did you estimate the energy changeof the overall process to do that?
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| 10. |
- Campbell, Charles, et al.
(författare)
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Catalyst design from theory to practice: general discussion
- 2016
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Ingår i: Faraday Discussions. - 1359-6640 .- 1364-5498. ; 188, s. 279-307
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Hans-Joachim Freund opened the discussion of the paper by Alberto Roldan:How is the atomic hydrogen produced on the greigite surface? In the paper (DOI:10.1039/C5FD00186B) there is no comment whether you studied dissociatehydrogen adsorption.
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