scholarly journals Structure of a trapped radical transfer pathway within a ribonucleotide reductase holocomplex

Science ◽  
2020 ◽  
Vol 368 (6489) ◽  
pp. 424-427 ◽  
Author(s):  
Gyunghoon Kang ◽  
Alexander T. Taguchi ◽  
JoAnne Stubbe ◽  
Catherine L. Drennan
Keyword(s):  
Ribonucleotide Reductase ◽  
Active Site ◽  
De Novo ◽  
Reduction Reaction ◽  
Wild Type ◽  
Cryo Electron Microscopy ◽  
Ribonucleotide Reductases ◽  
Dna Biosynthesis ◽  
Radical Transfer ◽  
Resolution Structure

Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2′-deoxynucleotides de novo and are thus critical in DNA biosynthesis and repair. The nucleotide reduction reaction in all RNRs requires the generation of a transient active site thiyl radical, and in class I RNRs, this process involves a long-range radical transfer between two subunits, α and β. Because of the transient subunit association, an atomic resolution structure of an active α2β2 RNR complex has been elusive. We used a doubly substituted β2, E52Q/(2,3,5)-trifluorotyrosine122-β2, to trap wild-type α2 in a long-lived α2β2 complex. We report the structure of this complex by means of cryo–electron microscopy to 3.6-angstrom resolution, allowing for structural visualization of a 32-angstrom-long radical transfer pathway that affords RNR activity.

scholarly journals 3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Edward J Brignole ◽  
Kuang-Lei Tsai ◽  
Johnathan Chittuluru ◽  
Haoran Li ◽  
Yimon Aye ◽  
...  
Keyword(s):  
Active Site ◽  
Molecular Basis ◽  
Α Subunit ◽  
Cryo Electron Microscopy ◽  
Ribonucleotide Reductases ◽  
Dna Replication And Repair ◽  
Radical Transfer ◽  
Allosteric Regulators ◽  
Insight Into ◽  
Resolution Structure

Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit contains the active site, and the β subunit houses the radical cofactor. Here, we present a 3.3-Å resolution structure by cryo-electron microscopy (EM) of a dATP-inhibited state of human RNR. This structure, which was determined in the presence of substrate CDP and allosteric regulators ATP and dATP, has three α2 units arranged in an α6 ring. At near-atomic resolution, these data provide insight into the molecular basis for CDP recognition by allosteric specificity effectors dATP/ATP. Additionally, we present lower-resolution EM structures of human α6 in the presence of both the anticancer drug clofarabine triphosphate and β2. Together, these structures support a model for RNR inhibition in which β2 is excluded from binding in a radical transfer competent position when α exists as a stable hexamer.

scholarly journals Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus

2016 ◽  
Vol 90 (21) ◽  
pp. 9733-9742 ◽  
Author(s):  
Lindsey J. Organtini ◽  
Hyunwook Lee ◽  
Sho Iketani ◽  
Kai Huang ◽  
Robert E. Ashley ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Receptor Binding ◽  
Conformational Changes ◽  
De Novo ◽  
Mutational Analysis ◽  
Antibody Fragment ◽  
Canine Parvovirus ◽  
Single Chain ◽  
Cryo Electron Microscopy ◽  
Resolution Structure

ABSTRACT Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM. IMPORTANCE Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time.

scholarly journals The U28 ORF of human herpesvirus-7 does not encode a functional ribonucleotide reductase R1 subunit

1999 ◽  
Vol 80 (10) ◽  
pp. 2713-2718 ◽  
Author(s):  
Yunming Sun ◽  
Joe Conner
Keyword(s):  
Ribonucleotide Reductase ◽  
De Novo ◽  
Human Herpesvirus ◽  
Cloning And Expression ◽  
Subunit Interaction ◽  
Viral Dna ◽  
Sequence Comparisons ◽  
E Coli ◽  
Ribonucleotide Reductases ◽  
Functional Equivalent

Herpesvirus ribonucleotide reductases, essential for the de novo synthesis of viral DNA, are composed of two non-identical subunits, termed R1 and R2. The U28 ORF from human herpesvirus-7 has been classified, by sequence comparisons, as a homologue of the R1 subunit from ribonucleotide reductase but no R2 ORF is present. Detailed analysis of the U28 amino acid sequence indicated that a number of essential R1 catalytic residues are absent. Cloning and expression of the U28 protein in E. coli and its subsequent characterization in subunit interaction and enzyme activity assays confirmed that it is not a functional equivalent of a herpesvirus R1. In the absence of the R2 gene, we propose that the R1 ORF has evolved a distinct, as yet unidentified, function not only in human herpesvirus-7 but also in other human betaherpesviruses.

scholarly journals In Vivo Assay for Low-Activity Mutant Forms of Escherichia coli Ribonucleotide Reductase

2003 ◽  
Vol 185 (4) ◽  
pp. 1167-1173 ◽  
Author(s):  
Monica Ekberg ◽  
Pernilla Birgander ◽  
Britt-Marie Sjöberg
Keyword(s):  
Escherichia Coli ◽  
Ribonucleotide Reductase ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Tyrosyl Radical ◽  
In Vivo Assay ◽  
Radical Transfer

ABSTRACT Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells. In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic Escherichia coli cells. The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR. This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays. E. coli RNR is composed of two homodimeric proteins, R1 and R2. The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site. The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues. Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity. In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.

Crystallographic analysis of 1,2,3-trichloropropane biodegradation by the haloalkane dehalogenase DhaA31

2014 ◽  
Vol 70 (2) ◽  
pp. 209-217 ◽  
Author(s):  
Maryna Lahoda ◽  
Jeroen R. Mesters ◽  
Alena Stsiapanava ◽  
Radka Chaloupkova ◽  
Michal Kuty ◽  
...  
Keyword(s):  
Active Site ◽  
Rational Design ◽  
Chloride Ions ◽  
Saturation Mutagenesis ◽  
Hydrolytic Cleavage ◽  
Wild Type ◽  
Haloalkane Dehalogenase ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Resolution Structure

Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon–halogen bonds, which is a key step in the aerobic mineralization of many environmental pollutants. One important pollutant is the toxic and anthropogenic compound 1,2,3-trichloropropane (TCP). Rational design was combined with saturation mutagenesis to obtain the haloalkane dehalogenase variant DhaA31, which displays an increased catalytic activity towards TCP. Here, the 1.31 Å resolution crystal structure of substrate-free DhaA31, the 1.26 Å resolution structure of DhaA31 in complex with TCP and the 1.95 Å resolution structure of wild-type DhaA are reported. Crystals of the enzyme–substrate complex were successfully obtained by adding volatile TCP to the reservoir after crystallization at pH 6.5 and room temperature. Comparison of the substrate-free structure with that of the DhaA31 enzyme–substrate complex reveals that the nucleophilic Asp106 changes its conformation from an inactive to an active state during the catalytic cycle. The positions of three chloride ions found inside the active site of the enzyme indicate a possible pathway for halide release from the active site through the main tunnel. Comparison of the DhaA31 variant with wild-type DhaA revealed that the introduced substitutions reduce the volume and the solvent-accessibility of the active-site pocket.

scholarly journals How ATP and dATP act as molecular switches to regulate enzymatic activity in the prototypic bacterial class Ia ribonucleotide reductase

2021 ◽  
Author(s):  
Michael A Funk ◽  
Christina M Zimanyi ◽  
Gisele A. Andree ◽  
Allison E. Hamilos ◽  
Catherine L Drennan
Keyword(s):  
Molecular Switches ◽  
Binding Pocket ◽  
Allosteric Inhibitors ◽  
Α Subunit ◽  
E Coli ◽  
Ribonucleotide Reductases ◽  
Dna Biosynthesis ◽  
Radical Transfer ◽  
Conformational Rearrangements

Class Ia ribonucleotide reductases (RNRs) are subject to allosteric regulation to maintain the appropriate deoxyribonucleotide levels for accurate DNA biosynthesis and repair. RNR activity requires a precise alignment of its α2 and β2 subunits such that a catalytically-essential radical species is transferred from β2 to α2. In E. coli, when too many deoxyribonucleotides are produced, dATP binding to RNR generates an inactive α4β4 state in which α2 and β2 are separated, preventing radical transfer. ATP binding breaks the α−β interface, freeing β2 and restoring activity. Here we investigate the molecular basis for allosteric activity regulation in the prototypic E. coli class Ia RNR. Through the determination of six crystal structures we are able to establish how dATP binding creates a binding pocket for β on α that traps β2 in the inactive α4β4 state. These structural snapshots also reveal the numerous ATP-induced conformational rearrangements that are responsible for freeing β2. We further discover, and validate through binding and mutagenesis studies, a previously unknown nucleotide binding site on the α subunit that is crucial for the ability of ATP to dismantle the inactive α4β4 state. These findings have implications for the design of allosteric inhibitors for bacterial RNRs.

scholarly journals Convergent Allostery in Ribonucleotide Reductase

2018 ◽  
Author(s):  
William C. Thomas ◽  
F. Phil Brooks ◽  
Audrey A. Burnim ◽  
John-Paul Bacik ◽  
JoAnne Stubbe ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ribonucleotide Reductase ◽  
Molecular Basis ◽  
Model Organism ◽  
Activity Regulation ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Microscopy ◽  
Ribonucleotide Reductases ◽  
Class Ib

AbstractRibonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a new and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible, active tetramer and two novel, inhibited filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery.

scholarly journals Molecular basis for allosteric specificity regulation in class Ia ribonucleotide reductase from Escherichia coli

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Christina M Zimanyi ◽  
Percival Yang-Ting Chen ◽  
Gyunghoon Kang ◽  
Michael A Funk ◽  
Catherine L Drennan
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Bonds ◽  
Substrate Specificity ◽  
Ribonucleotide Reductase ◽  
Active Site ◽  
Molecular Basis ◽  
Allosteric Site ◽  
Dna Biosynthesis ◽  
Conformational Rearrangements ◽  
Proper Balance

Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, a reaction that is essential for DNA biosynthesis and repair. This enzyme is responsible for reducing all four ribonucleotide substrates, with specificity regulated by the binding of an effector to a distal allosteric site. In all characterized RNRs, the binding of effector dATP alters the active site to select for pyrimidines over purines, whereas effectors dGTP and TTP select for substrates ADP and GDP, respectively. Here, we have determined structures of Escherichia coli class Ia RNR with all four substrate/specificity effector-pairs bound (CDP/dATP, UDP/dATP, ADP/dGTP, GDP/TTP) that reveal the conformational rearrangements responsible for this remarkable allostery. These structures delineate how RNR ‘reads’ the base of each effector and communicates substrate preference to the active site by forming differential hydrogen bonds, thereby maintaining the proper balance of deoxynucleotides in the cell.

scholarly journals Coenzyme B12 Controls Transcription of the Streptomyces Class Ia Ribonucleotide Reductase nrdABS Operon via a Riboswitch Mechanism

2006 ◽  
Vol 188 (7) ◽  
pp. 2512-2520 ◽  
Author(s):  
Ilya Borovok ◽  
Batia Gorovitz ◽  
Rachel Schreiber ◽  
Yair Aharonowitz ◽  
Gerald Cohen
Keyword(s):  
Ribonucleotide Reductase ◽  
Untranslated Region ◽  
De Novo ◽  
Streptomyces Coelicolor ◽  
Class Ii ◽  
Coenzyme B12 ◽  
Gene Encoding ◽  
Inhibition Of Growth ◽  
Ribonucleotide Reductases ◽  
Genes Encoding

ABSTRACT Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomycetes contain genes coding for two RNRs. The class Ia RNR is oxygen dependent, and the class II RNR is oxygen independent and requires coenzyme B12. Either RNR is sufficient for vegetative growth. We show here that the Streptomyces coelicolor M145 nrdABS genes encoding the class Ia RNR are regulated by coenzyme B12. The 5′-untranslated region of nrdABS contains a 123-nucleotide B12 riboswitch. Similar B12 riboswitches are present in the corresponding regions of eight other S. coelicolor genes. The effect of B12 on growth and nrdABS transcription was examined in a mutant in which the nrdJ gene, encoding the class II RNR, was deleted. B12 concentrations of just 1 μg/liter completely inhibited growth of the NrdJ mutant strain. Likewise, B12 significantly reduced nrdABS transcription. To further explore the mechanism of B12 repression, we isolated in the nrdJ deletion strain mutants that are insensitive to B12 inhibition of growth. Two classes of mutations were found to map to the B12 riboswitch. Both conferred resistance to B12 inhibition of nrdABS transcription and are likely to affect B12 binding. These results establish that B12 regulates overall RNR expression in reciprocal ways, by riboswitch regulation of the class Ia RNR nrdABS genes and by serving as a cofactor for the class II RNR.

scholarly journals Direct interfacial Y731 oxidation in α2 by a photoβ2 subunit of E. coli class Ia ribonucleotide reductase

2015 ◽  
Vol 6 (8) ◽  
pp. 4519-4524 ◽  
Author(s):  
David Y. Song ◽  
Arturo A. Pizano ◽  
Patrick G. Holder ◽  
JoAnne Stubbe ◽  
Daniel G. Nocera
Keyword(s):  
Ribonucleotide Reductase ◽  
De Novo ◽  
Building Blocks ◽  
Biological Processes ◽  
E Coli ◽  
Proton Coupled Electron Transfer ◽  
Ribonucleotide Reductases ◽  
Wide Range ◽  
Dna Replication And Repair ◽  
Fundamental Mechanism

Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair.

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