| 1. |
- Benlloch, Reyes, et al.
(författare)
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Crystal structure and functional characterization of Photosystem II-associated carbonic anhydrase CAH3 in Chlamydomonas reinhardtii
- 2015
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Ingår i: Plant Physiology. - : American Society of Plant Biologists. - 0032-0889 .- 1532-2548. ; 167:3, s. 950-962
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Tidskriftsartikel (refereegranskat)abstract
- In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO2 and water into carbohydrates.The light-driven oxidation of water that provides the electrons and protons for the subsequent CO2 fixation takes place inphotosystem II (PSII). Recent studies show that in higher plants, HCO3– increases PSII activity by acting as a mobile acceptor ofthe protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested toimprove proton removal from PSII, possibly by rapid reformation of HCO3– from CO2. In this study, we investigated the interplaybetween PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometrymeasurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen underillumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature notpreviously observed in a-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 functionwith dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO2/HCO3– on PSIIactivity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSIIpreparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at lowpH and CO2 concentration.
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| 2. |
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| 3. |
- Edwin, Aaron, et al.
(författare)
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Domain isolation, expression, purification and proteolytic activity of the metalloprotease PrtV from Vibrio cholerae
- 2014
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Ingår i: Protein Expression and Purification. - : Elsevier. - 1046-5928 .- 1096-0279. ; 96, s. 39-47
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Tidskriftsartikel (refereegranskat)abstract
- The metalloprotease PrtV from Vibrio cholerae serves an important function for the bacteria's ability to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102kDa) multidomain pre-pro-protein that so far has only been expressed in V. cholerae. Structural studies require high amounts of soluble protein with high purity. Previous attempts for recombinant expression have been hampered by low expression and solubility of protein fragments. Here, we describe results from parallel cloning experiments in Escherichia coli where fusion tagged constructs of PrtV fragments were designed, and protein products tested for expression and solubility. Of more than 100 designed constructs, three produced protein products that expressed well. These include the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25kDa fragment (residues 581-839). The soluble fusion proteins were captured with Ni(2+) affinity chromatography, and subsequently cleaved with tobacco etch virus protease. Purification protocols yielded ∼10-15mg of pure protein from 1L of culture. Proper folding of the shorter domains was confirmed by heteronuclear NMR spectra recorded on (15)N-labeled samples. A modified protocol for the native purification of the secreted 81kDa pro-protein of PrtV is provided. Proteolytic activity measurements suggest that the 37kDa catalytic metalloprotease domain alone is sufficient for activity.
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| 4. |
- Eriksson, Jonas, et al.
(författare)
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Small Molecule Screening for Inhibitors of the YopH Phosphatase of Yersinia pseudotuberculosis
- 2012
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Ingår i: Advances in Yersinia Research. - New York : Springer. - 9781461435600 - 9781461435617 ; , s. 357-363
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Bokkapitel (refereegranskat)abstract
- Bacterial virulence systems are attractive targets for development of new antibacterial agents. Yersinia spp. utilize the type III secretion (T3S) system to secrete and translocate Yersinia outer proteins (Yop effectors) into the cytosol of the target cell and thereby overcome host defenses to successfully establish an infection. Thus, the Yop effectors constitute attractive targets for drug development. In the present study we apply small molecule screening to identify inhibitors of one of the secreted proteins YopH, a tyrosine phosphatase required for virulence. Characterization of seven inhibitors indicated that both competitive and noncompetitive inhibitors were identified with IC50 values of 6–20 μM.
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| 5. |
- Good, James A. D., 1985-, et al.
(författare)
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Attenuating Listeria monocytogenes virulence by targeting the regulatory protein PrfA
- 2016
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Ingår i: Cell chemical biology. - : Elsevier BV. - 2451-9448 .- 2451-9456. ; 23:3, s. 404-414
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Tidskriftsartikel (refereegranskat)abstract
- The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes infectivity by reducing the expression of virulence genes, without compromising bacterial growth. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds within a hydrophobic pocket, located between the C- and N-terminal domains of PrfA, and interacts with residues important for PrfA activation. This indicates that these inhibitors maintain the DNA-binding helix-turn-helix motif of PrfA in a disordered state, thereby preventing a PrfA:DNA interaction. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.
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| 6. |
- 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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| 7. |
- Hansen, Sabine, et al.
(författare)
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A Novel Growth-Based Selection Strategy Identifies New Constitutively Active Variants of the Major Virulence Regulator PrfA in Listeria monocytogenes
- 2020
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Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 202:11
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Tidskriftsartikel (refereegranskat)abstract
- Listeria monocytogenes is a Gram-positive pathogen able to cause severe human infections. Its major virulence regulator is the transcriptional activator PrfA, a member of the Crp/Fnr family of transcriptional regulators. To establish a successful L. monocytogenes infection, the PrfA protein needs to be in an active conformation, either by binding the cognate inducer glutathione (GSH) or by possessing amino acid substitutions rendering the protein constitutively active (PrfA*). By a yet unknown mechanism, phosphotransferase system (PTS) sugars repress the activity of PrfA. We therefore took a transposon-based approach to identify the mechanism by which PTS sugars repress PrfA activity. For this, we screened a transposon mutant bank to identify clones able to grow in the presence of glucose-6-phosphate as the sole carbon source. Surprisingly, most of the isolated transposon mutants also carried amino acid substitutions in PrfA. In transposon-free strains, the PrfA amino acid substitution mutants displayed growth, virulence factor expression, infectivity, and DNA binding, agreeing with previously identified PrIA* mutants. Hence, the initial growth phenotype observed in the isolated clone was due to the amino acid substitution in PrfA and unrelated to the loci inactivated by the transposon mutant. Finally, we provide structural evidence for the existence of an intermediately activated PrfA state, which gives new insights into PrfA protein activation. IMPORTANCE The Gram-positive bacterium Listeria monocytogenes is a human pathogen affecting mainly the elderly, immunocompromised people, and pregnant women. It can lead to meningoencephalitis, septicemia, and abortion. The major virulence regulator in L. monocytogenes is the PrfA protein, a transcriptional activator. Using a growth-based selection strategy, we identified mutations in the PrfA protein leading to constitutively active virulence factor expression. We provide structural evidence for the existence of an intermediately activated PrfA state, which gives new insights into PrfA protein activation.
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| 8. |
- Huang, Shenghua, et al.
(författare)
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Structural studies of β-Carbonic Anhydrase from the Green Alga Coccomyxa : Inhibitor complexes with Anions and Acetazolamide
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:12, s. e28458-
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Tidskriftsartikel (refereegranskat)abstract
- The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion - a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO(2)-HCO(3) (-) interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules.
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| 9. |
- 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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| 10. |
- 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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