Oligomerization Status Directs Overall Activity Regulation of the Escherichia coli Class Ia Ribonucleotide Reductase

Rofougaran, Reza; Crona, Mikael; Vodnala, Munender; Sjöberg, Britt‐Marie; Hofer, Anders

doi:10.1074/jbc.m806738200

Cited by 83 publications

(150 citation statements)

References 41 publications

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“…ES-DMA has also been used to characterize a variety of bimolecular complexes, such as oligomerization of subunits of ribonucleotide reductase in the presence of different functional groups [68] and triphosphates [69], PEGylated-Von Willebrand factor (VWF) protein [70], quantification of coverage of antibodies (8F5) on human common cold virus [42], and quantum dots (QDs) on bacteriophages [71,72].…”

Section: Nanoparticle-biomolecule Conjugatesmentioning

confidence: 99%

Electrospray–differential mobility analysis of bionanoparticles

Guha

Tarlov

et al. 2012

Trends in Biotechnology

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The growth of the multibillion dollar bionanoparticle industry has spurred the development of new physical characterization methods. One such method, electrospraydifferential mobility analysis (ES-DMA) constitutes an electrospray for aerosolization of bionanoparticles (such as viruses, gold-nanoparticles, proteins, nanoparticle-protein complexes) and an ion mobility method that operates at atmospheric conditions, and separates bionanoparticles spatially. This dissertation identifies some relevant "problem" areas for ES-DMA by reviewing selected applications.Some such problems are: proteins while passing through ES capillaries are found to interact with it and thus produce time dependent size distributions. Further, it is thought that adsorbed proteins may subsequently desorb and influence size distributions with the ES-DMA which may concomitantly affect quantification of aggregates. These artifacts are studied systematically and it is demonstrated that ES-DMA can quantify adsorption-desorption of complex protein mixtures at high shear rates. Further, it is shown that desorbing proteins do not have a significant effect on size distributions. Another artifact of the ES takes place during the aersolization process. Two units (called monomers) of a bionanoparticle may get encapsulated in the same ES droplet and upon drying of the droplet create artificial dimers thus affecting quantification with ES-DMA. Assuming Poisson distribution, this thesis provides a systematic approach that can be undertaken to eliminate this artifact. A third artifact arises from the low sensitivity of the DMA to size increase. When a ligand (for e.g. protein) adsorbs to a bionanoparticle it creates an increase in the size of the later, which can be used to quantify the amount of ligand adsorbed per bionanoparticle. As ligands can change conformations upon adsorption, using ES-DMA for such applications may be flawed. This issue has been identified and a solution has been provided by integrating a mass analyzer after the ES-DMA.After correcting for these artifacts, this dissertation delves into characterization of different types of bionanoparticles and demonstrates that ES-DMA has several advantages over other traditional techniques such as transmission electron microscopy, size exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and plaque assay and thus has immense potential to become a process analytical technique in biomanufacturing environments. ELECTROSPRAY-DIFFERENTIAL MOBILITY ANALYSIS OF BIONANOPARTICLES

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Section: Nanoparticle-biomolecule Conjugatesmentioning

confidence: 99%

Electrospray–differential mobility analysis of bionanoparticles

Guha

Tarlov

et al. 2012

Trends in Biotechnology

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“…Thus in E. coli both subunits are needed for oligomerization, whereas the eukaryotic ␣ subunit can form hexamers on its own. Furthermore, the E. coli enzyme can be inhibited by dATP as well as by combinations of ATP and high concentrations of dTTP or dGTP (7). The overall activity regulation of the E. coli enzyme thus appears to be governed by cross-talk between the a-site and s-site, whereas the overall activity regulation of the eukaryotic enzyme seems to be entirely driven by the a-site.…”

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confidence: 99%

“…In this case, the enzyme activity is turned off via dimerization of the active ␣ 2 ␤ 2 form into an inactive ring-shaped ␣ 4 ␤ 4 complex that cannot support electron transport between the subunits (6,7,13). Thus in E. coli both subunits are needed for oligomerization, whereas the eukaryotic ␣ subunit can form hexamers on its own.…”

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confidence: 99%

“…In the previous analysis of the P. aeruginosa class I RNR, the size determination of the ATP-induced oligomers was ambiguous (14), which is a common problem with gel filtration analysis of protein complexes in rapid equilibrium. In this study we have used gas-phase electrophoretic mobility macromolecule analysis (GEMMA), which is an alternative method better suited for equilibrating complexes that has successfully been used for studies of other RNRs (7,8,10).…”

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confidence: 99%

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Diversity in Overall Activity Regulation of Ribonucleotide Reductase

Jonna

Crona

Rofougaran

et al. 2015

Journal of Biological Chemistry

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Background: Ribonucleotide reductase (RNR) makes DNA building blocks. Results: Binding of three dATP molecules to the Pseudomonas aeruginosa class I RNR ␣ subunit inactivates the enzyme by inducing an inert ␣ 4 complex. Conclusion: The number of bound dATP molecules and the tetrameric complex are unique among RNRs. Significance: The novel inhibition mechanism of P. aeruginosa RNR is a potential drug target.

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“…Allosteric regulation of this activity is key to cell survival and involves conformational changes as well as oligomeric state changes in both prokaryotic (12,14,18) and eukaryotic systems (19)(20)(21)(22)(23). For E. coli (12,14,18), mouse (20)(21)(22), yeast (19), and human (19,23), the negative effector dATP has been linked to increases in oligomeric state with a recent gas-phase electrophoretic molecular mobility analysis (GEMMA) study estimating a molecular mass of 510 kDa for the dATP-inhibited E. coli RNR (most consistent with an α 4 β 4 state) (18), whereas for human and yeast RNR, dATP has been linked to α-hexamerization (19,22,23).…”

mentioning

confidence: 99%

Structural interconversions modulate activity of Escherichia coli ribonucleotide reductase

Ando

Brignole

Zimanyi

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

102

262

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Essential for DNA biosynthesis and repair, ribonucleotide reductases (RNRs) convert ribonucleotides to deoxyribonucleotides via radical-based chemistry. Although long known that allosteric regulation of RNR activity is vital for cell health, the molecular basis of this regulation has been enigmatic, largely due to a lack of structural information about how the catalytic subunit (α 2 ) and the radical-generation subunit (β 2 ) interact. Here we present the first structure of a complex between α 2 and β 2 subunits for the prototypic RNR from Escherichia coli. Using four techniques (small-angle X-ray scattering, X-ray crystallography, electron microscopy, and analytical ultracentrifugation), we describe an unprecedented α 4 β 4 ring-like structure in the presence of the negative activity effector dATP and provide structural support for an active α 2 β 2 configuration. We demonstrate that, under physiological conditions, E. coli RNR exists as a mixture of transient α 2 β 2 and α 4 β 4 species whose distributions are modulated by allosteric effectors. We further show that this interconversion between α 2 β 2 and α 4 β 4 entails dramatic subunit rearrangements, providing a stunning molecular explanation for the allosteric regulation of RNR activity in E. coli.allostery | protein-protein interactions | conformational equilibria | nucleotide metabolism I mportant targets of anticancer and antiviral drugs, ribonucleotide reductases (RNRs) are classified by the metallocofactor used to generate a thiyl radical (1) that initiates reduction of ribonucleotides to deoxyribonucleotides (2, 3). Class Ia RNRs are found in all eukaryotes and many aerobic bacteria, with the Escherichia coli enzyme serving as the prototype. These RNRs reduce ribonucleoside 5′-diphosphates and are composed of two homodimeric subunits: α 2 and β 2 (Fig. 1A). The α 2 subunit contains the active site, where ribonucleotide reduction occurs, and two types of allosteric effector binding sites (4, 5). One effector site tunes the specificity for all four ribonucleotide substrates in response to intracellular levels of deoxyribonucleoside 5′-triphosphates (dATP, dTTP, dGTP) and ATP (6, 7) such that balanced pools of deoxyribonucleotides are maintained (8). The second effector site controls the rate of reduction, binding ATP to turn the enzyme on or dATP to turn it off (4, 6). This activity site is housed in an N-terminal cone domain (5, 9) and provides a means for negative feedback regulation to safeguard against cytotoxic elevation of deoxyribonucleotide levels (2, 3, 10). The β 2 subunit harbors the essential diferric-tyrosyl radical (Y122• in E. coli) cofactor (11) that initiates radical chemistry.Active RNR has long been proposed to be a transient α 2 β 2 complex (Fig. 1B) with enhanced subunit affinity upon binding of substrates and effectors (12-15). For each turnover, α 2 , β 2 , substrate and effector must interact, triggering long-range proton-coupled electron transfer (PCET) reducing Y122• in β 2 and oxidizing C439 to a thiyl radical in the active s...

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Oligomerization Status Directs Overall Activity Regulation of the Escherichia coli Class Ia Ribonucleotide Reductase

Cited by 83 publications

References 41 publications

Electrospray–differential mobility analysis of bionanoparticles

Electrospray–differential mobility analysis of bionanoparticles

Diversity in Overall Activity Regulation of Ribonucleotide Reductase

Structural interconversions modulate activity of Escherichia coli ribonucleotide reductase

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