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

Mir, Aamir; Golden, Barbara L.

doi:10.1021/acs.biochem.5b01139

Cited by 71 publications

(103 citation statements)

References 23 publications

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“…Starting from the recent crystal structure of the RzB hammerhead ribozyme with divalent metal ion bound at G12(15, 16) (PDB code 5DI2), the RNA was solvated in a box of TIP4P-Ew(75) water molecules with a 12 A buffer. 67 Na + and 15 Cl − ions were added to neutralize the system and solvate it with 0.14 M NaCl (roughly physiological salt conditions).…”

Section: Methodsmentioning

confidence: 99%

“…Recently, a new set of crystal structures(15, 16) of a full length hammerhead ribozyme (RzB) has been obtained that indicate a Mg 2+ ion bound directly to the Hoogsteen face of G12 at pH 8. This binding mode is similar to the position of an electron density peak found in the previous crystal structures and interpreted as a likely water molecule(43) or Na + ion(14).…”

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confidence: 99%

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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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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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Section: Methodsmentioning

confidence: 99%

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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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“…4,8 In contrast, the observation of metal ion coordination by nucleobases has been limited particularly for catalytic RNAs 13–15 ; structural and biochemical studies have been carried out for inner-sphere monovalent ions 10,16 , outer-sphere divalent ions 17–20 , and one recent observation of an inner-sphere divalent metal ion in a small, self-cleaving ribozyme. 21 A recent structural survey finds that the second largest groups of sites, besides the phosphate backbone, that form inner-sphere interactions with Mg(II) in RNA crystal structures are the more than 1300 observed interactions with O4 of U and O6 of G. 22 However, the biochemical functions of this abundant group of RNA nucleobase-Mg(II) interactions remain poorly understood.…”

Section: Introductionmentioning

confidence: 99%

Inner-Sphere Coordination of Divalent Metal Ion with Nucleobase in Catalytic RNA

2017

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Identification of the function of metal ions and the RNA moieties, particularly nucleobases, that bind metal ions are important questions in RNA catalysis. Here we combine single-atom and abasic substitutions to probe functions of conserved nucleobases in ribonuclease P (RNase P). Structural and biophysical studies of bacterial RNase P propose direct coordination of metal ions by the nucleobases of conserved uridine and guanosine in helix P4 of the RNA subunit (P RNA). To biochemically probe the function of metal ion interactions, we substituted the universally conserved bulged uridine (U51) in the P4 helix of circularly permuted Bacillus subtilis P RNA with 4-thiouridine, 4-deoxyuridine and abasic modifications and G378/379 with 2-aminopurine, N7-deazaguanosine, and 6-thioguanosine. The functional group modifications of U51 decrease RNase P-catalyzed phosphodiester bond cleavage 16- to 23-fold, as measured by the single-turnover cleavage rate constant. The activity of the 4-thiouridine RNase P is partially rescued by addition of Cd(II) or Mn(II) ions. This is the first time a metal-rescue experiment provides evidence for inner-sphere divalent metal ion coordination with a nucleobase. Modifications of G379 modestly decrease the cleavage activity of RNase P, suggesting outer-sphere coordination of O6 on G379 to a metal ion. These data provide biochemical evidence for catalytically important interactions of the P4 helix of P RNA with metal ions, demonstrating that the bulged uridine coordinates at least one catalytic metal ion through an inner-sphere interaction. The combination of single-atom and abasic nucleotide substitutions provides a powerful strategy to probe functions of conserved nucleobases in large RNAs.

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“…In other words, such interaction redundancy might serve as a sort of β catalysis buffer to overcome a deficiency in either single interaction. A survey of the predominant active site models for the hammerhead, 20 hairpin, 21 HDV, 22 VS, 23 and glmS 24 self-cleaving RNA classes reveals that none of these five natural ribozyme classes exploit simultaneous ligands to both non-bridging phosphate oxygen atoms, although there is some evidence that hairpin ribozymes might make both contacts. 25 Thus, most other self-cleaving ribozyme classes appear to attain their high speeds without using this structural feature, and so twister ribozymes would be very unusual if they did.…”

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Mechanistic Debris Generated by Twister Ribozymes

Breaker

2017

ACS Chem. Biol.

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Twister RNAs represent a recently-discovered class of natural ribozymes that promote rapid cleaving of RNA backbones. Although an abundance of theoretical, biochemical, and structural data exists for several members of the twister class, disagreements about the architecture and mechanism of its active site have emerged. Historically, such storms regarding mechanistic details typically occur soon after each new self-cleaving ribozyme class is reported, but paths forward exist to quickly reach calmer conditions.

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

Cited by 71 publications

References 23 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

Inner-Sphere Coordination of Divalent Metal Ion with Nucleobase in Catalytic RNA

Mechanistic Debris Generated by Twister Ribozymes

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