Structure and mechanism of <i>E. coli</i> RNA 2′,3′-cyclic phosphodiesterase

Remus, Barbara S.; Jacewicz, Agata; Shuman, Stewart

doi:10.1261/rna.046797.114

Cited by 22 publications

(24 citation statements)

References 34 publications

(34 reference statements)

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“…Structures of several 2H phosphoesterases have been determined, but only a few have been resolved with substrate. Besides the structures of AKAP7 with AMP and, now, the dimer of RVA VP3 CTD with 2-5A, only the catalytic domain of mouse 2=,3=cyclic nucleotide 3=-PDE with 2=-O-(sulfidophosphinato)adenine (PDB 2YOZ) and the E. coli 2=,3=-cyclic PDE ThpR with AMP (PDB 4QAK) have been published (20,40,41). Each of the 2=,3=-PDEs interacts with substrate through H⌽(S/T)⌽ histidine and threonine residues and astacking interaction between the adenine base and a phenylalanine three amino acids downstream from the first H⌽(S/T)⌽ threonine.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for 2′-5′-Oligoadenylate Binding and Enzyme Activity of a Viral RNase L Antagonist

Ogden

Jha

et al. 2015

J Virol

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Synthesis of 2=-5=-oligoadenylates (2-5A) by oligoadenylate synthetase (OAS) is an important innate cellular response that limits viral replication by activating the latent cellular RNase, RNase L, to degrade single-stranded RNA. Some rotaviruses and coronaviruses antagonize the OAS/RNase L pathway through the activity of an encoded 2H phosphoesterase domain that cleaves 2-5A. These viral 2H phosphoesterases are phylogenetically related to the cellular A kinase anchoring protein 7 (AKAP7) and share a core structure and an active site that contains two well-defined H⌽(S/T)⌽ (where ⌽ is a hydrophobic residue) motifs, but their mechanism of substrate binding is unknown. Here, we report the structures of a viral 2H phosphoesterase, the C-terminal domain (CTD) of the group A rotavirus (RVA) VP3 protein, both alone and in complex with 2-5A. The domain forms a compact fold, with a concave ␤-sheet that contains the catalytic cleft, but it lacks two ␣-helical regions and two ␤-strands observed in AKAP7 and other 2H phosphoesterases. The cocrystal structure shows significant conformational changes in the R loop upon ligand binding. Bioinformatics and biochemical analyses reveal that conserved residues and residues required for catalytic activity and substrate binding comprise the catalytic motifs and a region on one side of the binding cleft. We demonstrate that the VP3 CTD of group B rotavirus, but not that of group G, cleaves 2-5A. These findings suggest that the VP3 CTD is a streamlined version of a 2H phosphoesterase with a ligand-binding mechanism that is shared among 2H phosphodiesterases that cleave 2-5A. IMPORTANCEThe C-terminal domain (CTD) of rotavirus VP3 is a 2H phosphoesterase that cleaves 2=-5=-oligoadenylates (2-5A), potent activators of an important innate cellular antiviral pathway. 2H phosphoesterase superfamily proteins contain two conserved catalytic motifs and a proposed core structure. Here, we present structures of a viral 2H phosphoesterase, the rotavirus VP3 CTD, alone and in complex with its substrate, 2-5A. The domain lacks two ␣-helical regions and ␤-strands present in other 2H phosphoesterases. A loop of the protein undergoes significant structural changes upon substrate binding. Together with our bioinformatics and biochemical findings, the crystal structures suggest that the RVA VP3 CTD domain is a streamlined version of a cellular enzyme that shares a ligand-binding mechanism with other 2H phosphodiesterases that cleave 2-5A but differs from those of 2H phosphodiesterases that cleave other substrates. These findings may aid in the future design of antivirals targeting viral phosphodiesterases with cleavage specificity for 2-5A. O ne of the earliest steps in the battle between a virus and its host is the detection of pathogen-associated molecular patterns by cellular sensors. For RNA viruses, these patterns may be found in RNA products synthesized during viral transcription and replication. A number of cytoplasmic cellular molecules have been described that sense foreign RNA and resp...

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Section: Discussionmentioning

confidence: 99%

Structural Basis for 2′-5′-Oligoadenylate Binding and Enzyme Activity of a Viral RNase L Antagonist

Ogden

Jha

et al. 2015

J Virol

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“…We next compared the drCPDase structure with that of its E. coli homologue (ecCPDase), which has been recently solved in complex with 2 0 -AMP (Remus et al, 2014). The overall structure of drCPDase could be superimposed on that of ecCPDase with an r.m.s.d.…”

Section: Resultsmentioning

confidence: 99%

“…The structure of drCPDase was determined by molecular replacement using the E. coli homologue (PDB entry 4qak; Remus et al, 2014) as a search model. The structure was refined using PHENIX interspersed with manual model building using Coot (Emsley et al, 2010;Adams et al, 2010).…”

Section: Structure Solution and Refinementmentioning

confidence: 99%

“…These subfamilies share two conserved histidines in a pair of HXT/SX motifs (where X is a hydrophobic residue), hence the name of this protein family. Superimpositions of these 2H phosphoesterase structures, which have been solved independently by nuclear magnetic resonance or X-ray crystallography in recent years, revealed that they share similar overall structures (Remus et al, 2014;Myllykoski et al, 2013Myllykoski et al, , 2016Sakamoto et al, 2005). The active site is located between two lobes formed by antiparallel -strands with -helices wrapping around them.…”

Section: Introductionmentioning

confidence: 95%

“…CPDase (EC 6.5.1) is found in almost all organisms from bacteria to mammals and can exist as a 'standalone' protein in addition to forming part of tRNA ligase (Kato et al, 2003;Hofmann et al, 2000;Myllykoski et al, 2013;Remus et al, 2014;Mazumder et al, 2002). Based on domain arrangements and sequence alignments, CPDase is classified into the 2H phosphoesterase superfamily, which can be divided into four subfamilies: the archaeobacterial LigT-like, eukaryotic viral LigT-like, YjcG-like and mlr3352 groups (Mazumder et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

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Crystal structure of the RNA 2′,3′-cyclic phosphodiesterase fromDeinococcus radiodurans

Han¹,

Cheng²,

Zhou³

et al. 2017

Acta Cryst Sect F

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2',3'-Cyclic phosphodiesterase (CPDase) homologues have been found in all domains of life and are involved in diverse RNA and nucleotide metabolisms. The CPDase from Deinococcus radiodurans was crystallized and the crystals diffracted to 1.6 Å resolution, which is the highest resolution currently known for a CPDase structure. Structural comparisons revealed that the enzyme is in an open conformation in the absence of substrate. Nevertheless, the active site is well formed, and the representative motifs interact with sulfate ion, which suggests a conserved catalytic mechanism.

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Microwave‐Assisted One‐Step Synthesis of 2′,3′‐Cyclic Phosphates of Nucleosides

2023

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Cyclic canonical nucleotides are an important class of compounds playing broad roles in regulating biological processes and are investigated in the context of prebiotic chemistry as activated nucleotides for oligonucleotide formation. Despite their growing importance, synthetic access of 2 ,3 -cyclic nucleotides is constrained, resulting in their cost-prohibitive commercial prices. Here, we describe a microwave-assisted one-pot synthesis starting from commercially available nucleosides employing an easily available cyclophosphorylating reagent, bis(dimethyldiamino)phosphorodiamidate (BDMDAP). The corresponding 2 ,3 -cyclic nucleotides are isolated in good yields (70-91%) by a simple ion-exchange column with no further workup. The nucleosides require no protecting group as the cyclophosphorylation reaction is selective for the 2 ,3 -dihydroxyl groups. The experimental protocol is robust and can be run in parallel to provide access to gram quantities of these 2 ,3 -cyclic nucleotides within a day.

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Structure and mechanism of E. coli RNA 2′,3′-cyclic phosphodiesterase

Cited by 22 publications

References 34 publications

Structural Basis for 2′-5′-Oligoadenylate Binding and Enzyme Activity of a Viral RNase L Antagonist

Structural Basis for 2′-5′-Oligoadenylate Binding and Enzyme Activity of a Viral RNase L Antagonist

Crystal structure of the RNA 2′,3′-cyclic phosphodiesterase fromDeinococcus radiodurans

Microwave‐Assisted One‐Step Synthesis of 2′,3′‐Cyclic Phosphates of Nucleosides

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