2′-Fluoro Substituents Can Mimic Native 2′-Hydroxyls within Structured RNA

Forconi, Marcello; Schwans, Jason P.; Porecha, Rishi H.; Sengupta, Raghuvir N.; Piccirilli, Joseph A.; Herschlag, Daniel

doi:10.1016/j.chembiol.2011.07.014

Cited by 13 publications

(11 citation statements)

References 53 publications

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“…Furthermore, the tri-fluoro-methyl moiety of BAY 60-2770 is flanked by residues Y2 and F112 and possibly makes a hydrogen bond acceptor interaction with the hydroxyl moiety of Y83 (3.2Å; Y83 is conserved in sGC). Such a fluoro-mediated interaction has been found previously also in RNA 12 . The protein region near the tri-fluoro-methyl moiety of BAY 60-2770, encompassing residues 109-113, is well defined including side chains of residues S111 and F112.…”

Section: Resultssupporting

confidence: 78%

Insights into BAY 60-2770 Activation and S-Nitrosylation-Dependent Desensitization of Soluble Guanylyl Cyclase via Crystal Structures of Homologous Nostoc H-NOX Domain Complexes

et al. 2013

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The soluble guanylyl cyclase (sGC) is an important receptor for nitric oxide (NO). Nitric oxide activates sGC several hundred fold to generate cGMP from GTP. Because of sGC’s salutary roles in cardiovascular physiology, it has received substantial attention as a drug target. The heme domain of sGC is key to its regulation as it not only contains the NO activation site but also harbors sites for NO-independent sGC activators as well an S-nitrosylation site (β1 C122) involved in desensitization. Here we report the crystal structure of the activator BAY 60-2770 bound to the Nostoc H-NOX domain that is homologous to sGC. The structure reveals that BAY 60-2770 has displaced the heme and acts as a heme mimetic via carboxylate-mediated interactions with the conserved YxSxR motif as well as hydrophobic interactions. Comparisons with the previously determined BAY 58-2667 bound structure reveals that BAY 60-2770 is more ordered in its hydrophobic tail region. sGC activity assays demonstrate that BAY 60-2770 has about 10% higher fold maximal stimulation compared to BAY 58-2667. S-nitrosylation of the BAY 60-2770 substituted Nostoc H-NOX domain causes subtle changes in the vicinity of the S-nitrosylated C122 residue. These shifts could impact the adjacent YxSxR motif and αF helix and as such potentially inhibit either heme incorporation or NO-activation of sGC and thus provide a structural basis for desensitization.

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Section: Resultssupporting

confidence: 78%

Insights into BAY 60-2770 Activation and S-Nitrosylation-Dependent Desensitization of Soluble Guanylyl Cyclase via Crystal Structures of Homologous Nostoc H-NOX Domain Complexes

et al. 2013

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“…However the loss of product formation at acidic pH by GTP analogs suggests that the hydrogen bond acceptor ability of 29 group is critical for the synthesis reaction. Interestingly, the H-bond acceptor characteristic of organic F is debated widely, and it has been noted that it can take part in H-bonding (27,28). Hence, we predict a possibility that the product formation observed for 29-F dGTP in the alkaline condition could be a result of the H-bond acceptor characteristic of the organic fluorine.…”

Section: Examining Synthesis Activity Using 29 Modified Gtp Analogsmentioning

confidence: 81%

A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 2′-OH of GTP

Patil

Vithani

Singh

et al. 2020

Journal of Biological Chemistry

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Bacterial Rel proteins synthesize hyperphosphorylated guanosine nucleotides, denoted as (p)ppGpp, which by inhibiting energy requiring molecular pathways help bacteria to overcome the depletion of nutrients in its surroundings. (p)ppGpp synthesis by Rel involves transferring a pyrophosphate from ATP to the oxygen of 3’-OH of GTP/GDP. Initially, a conserved glutamate at the active site was believed to generate the nucleophile necessary to accomplish the reaction. Later this role was alluded to a Mg2+ ion. However, no study has unequivocally established a catalytic mechanism for (p)ppGpp synthesis. Here we present a revised mechanism, wherein for the first time we explore a role for 2’-OH of GTP and show how it is important in generating the nucleophile. Through a careful comparison of substrate-bound structures of Rel, we illustrate that the active site does not discriminate GTP from dGTP, for a substrate. Using biochemical studies, we demonstrate that both GTP and dGTP bind to Rel, but only GTP (but not dGTP) can form the product. Reactions performed using GTP analogues substituted with different chemical moieties at the 2’ position, suggest a clear role for 2’-OH in catalysis by providing an indispensable hydrogen bond; preliminary computational analysis further supports this view. This study elucidating a catalytic role for 2’-OH of GTP in (p)ppGpp synthesis allows us to propose different mechanistic possibilities by which it generates the nucleophile for the synthesis reaction. This study underscores the selection of ribose nucleotides as second messengers and finds its roots in the old RNA world hypothesis.

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“…Studies have shown that 2'-fluoro substitutions are less disruptive than 2'-deoxynucleotide substitutions in structured RNA 22 . The 2’-fluoro substitution prefers the c3’- endo sugar pucker found in RNA 23; 24 and the fluoro atom may participate as a weak hydrogen bond acceptor 22; 25; 26; 27; 28; 29;30 . In the decoding center, the 2’-hydroxyl groups of the A site codon participate mainly as hydrogen bond donors (Figure 1a).…”

Section: Resultsmentioning

confidence: 99%

Steric Complementarity in the Decoding Center Is Important for tRNA Selection by the Ribosome

Khade

Shi

Joseph

2013

Journal of Molecular Biology

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Accurate tRNA selection by the ribosome is essential for the synthesis of functional proteins. Previous structural studies indicated that the ribosome distinguishes between cognate and near-cognate tRNAs by monitoring the geometry of the codon-anticodon helix in the decoding center using the universally conserved 16S rRNA bases G530, A1492 and A1493. These bases form hydrogen bonds with the 2’-hydroxyl groups of the codon-anticodon helix, which are expected to be disrupted with a near-cognate codon-anticodon helix. However, a recent structural study showed that G530, A1492 and A1493 form hydrogen bonds in an identical manner with both cognate and near-cognate codon-anticodon helices. To understand how the ribosome discriminates between cognate and near-cognate tRNAs, we made 2’-deoxynucleotide and 2’-fluoro substituted mRNAs, which disrupt the hydrogen bonds between the A site codon and G530, A1492 and A1493. Our results show that multiple 2’-deoxynucleotide substitutions in the mRNA substantially inhibit tRNA selection, whereas multiple 2’-fluoro substitutions in the mRNA have only modest effects on tRNA selection. Furthermore, the miscoding antibiotics paromomycin and streptomycin rescue the defects in tRNA selection with the multiple 2’-deoxynucleotide substituted mRNA. These results suggest that steric complementarity in the decoding center is more important than the hydrogen bonds between the A site codon and G530, A1492 and A1493 for tRNA selection.

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2′-Fluoro Substituents Can Mimic Native 2′-Hydroxyls within Structured RNA

Cited by 13 publications

References 53 publications

Insights into BAY 60-2770 Activation and S-Nitrosylation-Dependent Desensitization of Soluble Guanylyl Cyclase via Crystal Structures of Homologous Nostoc H-NOX Domain Complexes

Insights into BAY 60-2770 Activation and S-Nitrosylation-Dependent Desensitization of Soluble Guanylyl Cyclase via Crystal Structures of Homologous Nostoc H-NOX Domain Complexes

A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 2′-OH of GTP

Steric Complementarity in the Decoding Center Is Important for tRNA Selection by the Ribosome

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