| 1. |
- Chiruvella, Kishore K., et al.
(författare)
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Biochemical Characterization of Kat1 : a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MAT alpha). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons.
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| 2. |
- Crona, Mikael, et al.
(författare)
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A ribonucleotide reductase inhibitor with deoxyribonucleoside-reversible cytotoxicity
- 2016
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Ingår i: Molecular Oncology. - : Wiley. - 1574-7891 .- 1878-0261. ; 10:9, s. 1375-1386
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide Reductase (RNR) is the sole enzyme that catalyzes the reduction of ribonucleotides into deoxyribonucleotides. Even though RNR is a recognized target for antiproliferative molecules, and the main target of the approved drug hydroxyurea, few new leads targeted to this enzyme have been developed. We have evaluated a recently identified set of RNR inhibitors with respect to inhibition of the human enzyme and cellular toxicity. One compound, NSC73735, is particularly interesting; it is specific for leukemia cells and is the first identified compound that hinders oligomerization of the mammalian large RNR subunit. Similar to hydroxyurea, it caused a disruption of the cell cycle distribution of cultured HL-60 cells. In contrast to hydroxyurea, the disruption was reversible, indicating higher specificity. NSC73735 thus defines a potential lead candidate for RNR-targeted anticancer drugs, as well as a chemical probe with better selectivity for RNR inhibition than hydroxyurea.
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| 3. |
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| 4. |
- Gupta, Arun A., et al.
(författare)
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Formation of a Secretion-Competent Protein Complex by a Dynamic Wrap-around Binding Mechanism
- 2018
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 430:18, Part B, s. 3157-3169
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial virulence is typically initiated by translocation of effector or toxic proteins across host cell membranes. A class of gram-negative pathogenic bacteria including Yersinia pseudotuberculosis and Yersinia pestis accomplishes this objective with a protein assembly called the type III secretion system. Yersinia effector proteins (Yop) are presented to the translocation apparatus through formation of specific complexes with their cognate chaperones (Syc). In the complexes where the structure is available, the Yops are extended and wrap around their cognate chaperone. This structural architecture enables secretion of the Yop from the bacterium in early stages of translocation. It has been shown previously that the chaperone-binding domain of YopE is disordered in its isolation but becomes substantially more ordered in its wrap-around complex with its chaperone SycE. Here, by means of NMR spectroscopy, small-angle X-ray scattering and molecular modeling, we demonstrate that while the free chaperone-binding domain of YopH (YopHCBD) adopts a fully ordered and globular fold, it populates an elongated, wrap-around conformation when it engages in a specific complex with its chaperone SycH2. Hence, in contrast to YopE that is unstructured in its free state, YopH transits from a globular free state to an elongated chaperone-bound state. We demonstrate that a sparsely populated YopHCBD state has an elevated affinity for SycH2 and represents an intermediate in the formation of the protein complex. Our results suggest that Yersinia has evolved a binding mechanism where SycH2 passively stimulates an elongated YopH conformation that is presented to the type III secretion system in a secretion-competent conformation.
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| 5. |
- Johansson, Renzo, et al.
(författare)
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Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones
- 2016
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 24:6, s. 906-917
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone.
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| 6. |
- Jonna, Venkateswara Rao, 1980-
(författare)
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Class I Ribonucleotide Reductases : overall activity regulation, oligomerization, and drug targeting
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ribonucleotide reductase (RNR) is a key enzyme in the de novo biosynthesis and homeostatic maintenance of all four DNA building blocks by being able to make deoxyribonucleotides from the corresponding ribonucleotides. It is important for the cell to control the production of a balanced supply of the dNTPs to minimize misincorporations in DNA. Because RNR is the rate-limiting enzyme in DNA synthesis, it is an important target for antimicrobial and antiproliferative molecules. The enzyme RNR has one of the most sophisticated allosteric regulations known in Nature with four allosteric effectors (ATP, dATP, dGTP, and dTTP) and two allosteric sites. One of the sites (s-site) controls the substrate specificity of the enzyme, whereas the other one (a-site) regulates the overall activity. The a-site binds either dATP, which inhibits the enzyme or ATP that activates the enzyme. In eukaryotes, ATP activation is directly through the a-site and in E. coli it is a cross-talk effect between the a and s-sites. It is important to study and get more knowledge about the overall activity regulation of RNR, both because it has an important physiological function, but also because it may provide important clues to the design of antibacterial and antiproliferative drugs, which can target RNR.Previous studies of class I RNRs, the class found in nearly all eukaryotes and many prokaryotes have revealed that the overall activity regulation is dependent on the formation of oligomeric complexes. The class I RNR consists of two subunits, a large α subunit, and a small β subunit. The oligomeric complexes vary between different species with the mammalian and yeast enzymes cycle between structurally different active and inactive α6β2 complexes, and the E. coli enzyme cycles between active α2β2 and inactive α4β4 complexes. Because RNR equilibrates between many different oligomeric forms that are not resolved by most conventional methods, we have used a technique termed gas-phase electrophoretic macromolecule analysis (GEMMA). In the present studies, our focus is on characterizing both prokaryotic and mammalian class I RNRs. In one of our projects, we have studied the class I RNR from Pseudomonas aeruginosa and found that it represents a novel mechanism of overall activity allosteric regulation, which is different from the two known overall activity allosteric regulation found in E. coli and eukaryotic RNRs, respectively. The structural differences between the bacterial and the eukaryote class I RNRs are interesting from a drug developmental viewpoint because they open up the possibility of finding inhibitors that selectively target the pathogens. The biochemical data that we have published in the above project was later supported by crystal structure and solution X-ray scattering data that we published together with Derek T. Logan`s research group.We have also studied the effect of a novel antiproliferative molecule, NSC73735, on the oligomerization of the human RNR large subunit. This collaborative research results showed that the molecule NSC73735 is the first reported non-nucleoside molecule which alters the oligomerization to inhibit human RNR and the molecule disrupts the cell cycle distribution in human leukemia cells.
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| 7. |
- Jonna, Venkateswara Rao, et al.
(författare)
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Diversity in Overall Activity Regulation of Ribonucleotide Reductase
- 2015
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; , s. 1-24
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides, which are used as building blocks for DNA replication and repair. This process is tightly regulated via two allosteric sites, the specificity site (s-site) and the overall activity site (a-site). The a-site resides in an N-terminal ATP cone domain that binds dATP or ATP and functions as an on/off switch, whereas the composite s-site binds ATP, dATP, dTTP, or dGTP and determines which substrate to reduce. There are three classes of RNRs, and class I RNRs consist of different combinations of α and β subunits. In eukaryotic and Escherichia coli canonical class I RNRs, dATP inhibits enzyme activity through the formation of inactive α6 and α4β4 complexes, respectively. Here we show that the Pseudomonas aeruginosa class I RNR has a duplicated ATP cone domain and represents a third mechanism of overall activity regulation. Each α polypeptide binds three dATP molecules, and the N-terminal ATP cone is critical for binding two of the dATPs because a truncated protein lacking this cone could only bind one dATP to its s-site. ATP activates the enzyme solely by preventing dATP from binding. The dATP-induced inactive form is an α4 complex, which can interact with β2 to form a non-productive α4β2 complex. Other allosteric effectors induce a mixture of α2 and α4 forms, with the former being able to interact with β2 to form active α2β2 complexes. The unique features of the P. aeruginosa RNR are interesting both from evolutionary and drug discovery perspectives.
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| 8. |
- Loderer, Christoph, et al.
(författare)
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A unique cysteine-rich zinc finger domain present in a majority of class II ribonucleotide reductases mediates catalytic turnover
- 2017
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Ingår i: Journal of Biological Chemistry. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 0021-9258 .- 1083-351X. ; 292:46, s. 19044-19054
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, used in DNA synthesis and repair. Two different mechanisms help deliver the required electrons to the RNR active site. Formate can be used as reductant directly in the active site, or glutaredoxins or thioredoxins reduce a C-terminal cysteine pair, which then delivers the electrons to the active site. Here, we characterized a novel cysteine-rich C-terminal domain (CRD), which is present in most class II RNRs found in microbes. The NrdJd-type RNR from the bacterium Stackebrandtia nassauensis was used as a model enzyme. We show that the CRD is involved in both higher oligomeric state formation and electron transfer to the active site. The CRD-dependent formation of high oligomers, such as tetramers and hexamers, was induced by addition of dATP or dGTP, but not of dTTP or dCTP. The electron transfer was mediated by an array of six cysteine residues at the very C-terminal end, which also coordinated a zinc atom. The electron transfer can also occur between subunits, depending on the enzyme's oligomeric state. An investigation of the native reductant of the system revealed no interaction with glutaredoxins or thioredoxins, indicating that this class II RNR uses a different electron source. Our results indicate that the CRD has a crucial role in catalytic turnover and a potentially new terminal reduction mechanism and suggest that the CRD is important for the activities of many class II RNRs.
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| 9. |
- Mangold, Stefanie, et al.
(författare)
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Response of Acidithiobacillus caldus toward suboptimal pH conditions
- 2013
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Ingår i: Extremophiles. - : Springer Science and Business Media LLC. - 1431-0651 .- 1433-4909. ; 17:4, s. 689-696
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Tidskriftsartikel (refereegranskat)abstract
- Maintenance of a circumneutral intracellular pH is important for any organism. Acidophilic microorganisms thrive at low pH while maintaining their intracellular pH around 6.5. However, the mechanisms contributing to acidophile pH homeostasis are not well characterized. The authors investigated the proteomic response and cytoplasmic membrane fatty acid profiles of Acidithiobacillus caldus toward three pH values: 1.1, 2.5, and 4.0. Major rearrangements were observed but lower pH elicited larger changes. Differentially expressed transcription factors suggested tight transcriptional control of pH induced genes. Enzymes involved in sulfur metabolism were up-regulated at pH 1.1 suggesting either that: (1) cells required more energy for maintenance or (2) increased metabolic activity was a specific acid stress response to export intracellular protons via 1A degrees electron transport proton pumps. Furthermore, glutamate decarboxylase, an important enzyme in Escherichia coli acid resistance, was uniquely expressed at pH 1.1. Other proteins previously shown to be involved in neutrophilic acid response, such as spermidine synthase, PspA, and toluene tolerance protein, were differentially expressed in At. caldus but require further investigation to show a direct link to pH homeostasis. Their roles in acidophilic organisms are discussed. Active modulation of fatty acid profiles was detected and suggested a more rigid membrane at low pH.
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| 10. |
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