Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction

Mir, Aamir; Chen, Ji; Robinson, Kyle; Lendy, Emma K.; Goodman, Jaclyn; Neau, David; Golden, Barbara L.

doi:10.1021/acs.biochem.5b00824

Cited by 61 publications

(119 citation statements)

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“…In particular, the specific mechanism whereby the nucleophile becomes activated by a general base is not yet resolved. Biochemical studies(30, 31, 36) have suggested that the guanine nucleobase in residue G12 may act as the general base in HHR, which is also supported by crystal structures(13, 15) in which the N1 site of G12 is well-positioned to interact with the 2′-hydroxyl group of C17 (Fig. 1).…”

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confidence: 67%

Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations

et al. 2017

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The hammerhead ribozyme is a well-studied nucleolytic ribozyme that catalyzes the self-cleavage of the RNA phosphodiester backbone. Despite experimental and theoretical efforts, there remain key questions about details of the mechanism with regard to the activation of the nucleophile by the putative general base guanine (G12). Straight-forward interpretation of the measured activity-pH data implies the pKa value of the G12 nucleobase is significantly shifted by the ribozyme environment. Recent crystallographic and biochemical work has identified pH-dependent divalent metal ion binding at the N7/O6 position of G12, leading to the hypothesis that this binding mode could induce a pKa shift of G12 towards neutrality. We present computational results that unify the interpretation of available structural and biochemical data, and paint a detailed mechanistic picture of the general base step of the reaction. Electronic structure calculations quantify the magnitude of predicted pKa shifts induced by Mg2+ binding in several Mg2+-guanine complexes. Molecular dynamics and free energy simulations using newly developed 12-6-4 parameters for divalent metal ion binding to nucleic acids are used to characterize the ribozyme active site environment and evaluate the pKa of G12 in HHR with and without the Mg2+ ion bound. The results suggest that Mg2+ is able to down-shift the pKa of G12 by −1.2 units in accord with the apparent pKa value determined from activity-pH measurements. In addition, ab initio quantum mechanical/molecular mechanical simulations are performed to explore the free energy profile for the general base step in the presence and absence of Mg2+. Taken together, these results are in quantitative agreement with available experimental data, and support a cooperative mechanism whereby Mg2+ binding at the N7/O6 position serves to stabilize G12 in the functional, deprotonated form that can abstract a proton from the nucleophile in the general base step of the reaction. In this scenario, site-specific Mg2+ ion binding acts as a switch to activate the general base in HHR. Finally, experimentally-testable predictions are made on the mutational and rescue effects on G12, which will give further insights into the catalytic mechanism. These results contribute to our growing knowledge of the potential roles of divalent metal ions in RNA catalysis.

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Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations

et al. 2017

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“…Interestingly, the position of G12 in this transition state analog is similar to that previously observed at high pH and in structures of the G12A mutants. 9, 17, 18 …”

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“…The importance of this divalent ion has been demonstrated using a G12A mutant of the hammerhead ribozyme. When the keto oxygen of G12 is replaced with the exo-cyclic amine of A12, a metal specificity switch is observed, with the G12A mutant exhibiting a clear preference for Zn 2+ ion 9 . This suggests that there is a direct interaction between the Zn 2+ ion and the exocyclic amine of A12 and, by analogy, an interaction between a divalent metal ion and the O6 of G12 in the wild-type ribozyme.…”

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“…A general base with a p K A of 8–9 and a Mg 2+ -bound water (p K A ~11.4) 26 would be consistent with the observed pH-rate profiles for this ribozyme. 9, 13 While there is significant experimental data available to support this model, it will clearly require further validation, both to determine if G12 is, in fact, a general base, and to learn if and how the hammerhead ribozyme stabilizes the leaving group.…”

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Two Active Site Divalent Ions in the Crystal Structure of the Hammerhead Ribozyme Bound to a Transition State Analogue

Mir

Golden

2016

Biochemistry

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The crystal structure of the hammerhead ribozyme bound to the pentavalent transition state analog vanadate reveals significant rearrangements relative to the previously solved structures. The active site contracts, bringing G10.1 closer to the cleavage site, and repositioning a divalent metal ion such that it could, ultimately, interact directly with the scissile phosphate. This ion could also position a water molecule to serve as a general acid in the cleavage reaction. A second divalent ion is observed coordinated to the O6 of G12. This metal ion is well-placed to help tune the pKA of G12. Based on this crystal structure as well as a wealth of biochemical studies, we propose a mechanism in which G12 serves as the general base and a magnesium-bound water serves as a general acid.

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“…[34][35][36][37][38][39][40][41][42][43][44][45][46] Further dedicated investigations and well-designed experiments will be tailored to fully explore and understand the mechanistic intricacies of this fast-cleaving ribozyme class. [49] Košutić et al …”

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A Mini‐Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class

Košutić¹,

Neuner²,

Ren

et al. 2015

Angewandte Chemie

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Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pK a value of 5.1 was determined for a 13 C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pK a of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg 2+ ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn 2+ or Cd 2+ accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X-ray structure of a 2′-OCH 3 -U5 modified twister ribozyme. Keywordsmetal ion rescue; nucleoside modifications; oligoribonucleotides; perturbed pK a ; solid-phase synthesis Small self-cleaving ribozymes are widely distributed in nature [1] and are essential for rolling-circle-based replication of satellite RNAs. [2,3] Among them, the hepatitis delta virus (HDV) ribozyme [4][5][6][7][8] employs a divalent cation in the active site for catalysis, while the remaining small self-cleaving ribozymes including hammerhead, [2,9,10] hairpin, [3,[11][12][13] glmS, [14][15][16] and Varkud Satellite [17] employ principles of general acid-base and electrostatics for catalysis. Very recently, a new class of nucleolytic ribozymes (termed twister) has been discovered, [18] and soon thereafter, crystal structures were published that revealed a common double-pseudoknot overall architecture for the twister ribozyme but showed clear distinctions in residue and divalent cation alignments at the cleavage site. [19][20][21] While the O. sativa twister ribozyme was off-line orthogonally aligned with a fully base-paired stem P1, [19,20] the env22 twister ribozyme was in-line oriented at the cleavage step A6-U5, with a Mg 2+ coordinated to the scissile phosphate. Furthermore, for the env22 twister ribozyme, stem P1 formed only the two central base pairs (Figure 1) while the neighboring nucleotides U1 and U4 were instead engaged in stacked base triplet interactions (U4-A49-A34 and U1-A50-U33; Figure 1B). [21] Košutić et al.Page 2 Angew Chem Int Ed Engl. Author manuscript; available in PMC 2016 January 16. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThese contrasting observations were the starting point for the present investigation. A thorough comparison of the two structures (PDB: 4OJI for O. sativa and PDB: 4RGF for env22) revealed that the conserved adenosine (A6; env22 numbering is used throughout) at the cleavage site adopts nearly identical conformations involving ex...

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Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction

Cited by 61 publications

References 68 publications

Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations

Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations

Two Active Site Divalent Ions in the Crystal Structure of the Hammerhead Ribozyme Bound to a Transition State Analogue

A Mini‐Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class

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