Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism

Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg²⁺. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg²⁺ from Ca²⁺. Using a metal ion chelator β-thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution.     

Mononuclear and dinuclear mechanisms for catalysis of phosphodiester cleavage by alkaline earth metal ions in aqueous solution

In biological systems, enzymes often use metal ions, especially Mg²⁺, to catalyze phosphodiesterolysis, and model aqueous studies represent an important avenue of examining the contributions of these ions to catalysis. We have examined Mg²⁺ and Ca²⁺ catalyzed hydrolysis of the model phosphodiester thymidine-5′-p-nitrophenyl phosphate (T5PNP). At 25 °C, we find that, despite their different Lewis acidities, these ions have similar catalytic ability with second-order rate constants for attack of T5PNP by hydroxide (k_OH) of 4.1 × 10⁻⁴ M⁻¹s⁻¹ and 3.7 × 10⁻⁴ M⁻¹s⁻¹ in the presence of 0.30 M Mg²⁺ and Ca²⁺, respectively, compared to 8.3 × 10⁻⁷ M⁻¹s⁻¹ in the absence of divalent metal ion. Examining the dependence of k_OH on [M²⁺] at 50 °C indicates different kinetic mechanisms with Mg²⁺ utilizing a single ion mechanism and Ca²⁺ operating by parallel single and double ion mechanisms. Association of the metal ion(s) occurs prior to nucleophilic attack by hydroxide. Comparing the k_OH values reveals a single Mg²⁺ catalyzes the reaction by 1800-fold whereas a single Ca²⁺ ion catalyzes the reaction by only 90-fold. The second Ca²⁺ provides an additional 10-fold catalysis, significantly reducing the catalytic disparity between Mg²⁺ and Ca²⁺.     

Evidence for two distinct Mg2+ binding sites in G(s alpha) and G(i alpha1) proteins

The function of guanine nucleotide binding (G) proteins is Mg²⁺ dependent with guanine nucleotide exchange requiring higher metal ion concentration than guanosine 5′-triphosphate hydrolysis. It is unclear whether two Mg²⁺ binding sites are present or if one Mg²⁺ binding site exhibits different affinities for the inactive GDP-bound or the active GTP-bound conformations. We used furaptra, a Mg²⁺-specific fluorophore, to investigate Mg²⁺ binding to α subunits in both conformations of the stimulatory (G_sα) and inhibitory (G_iα1) regulators of adenylyl cyclase. Regardless of the conformation or α protein studied, we found that two distinct Mg²⁺ sites were present with dissimilar affinities. With the exception of G_sα in the active conformation, cooperativity between the two Mg²⁺ sites was also observed. Whereas the high affinity Mg²⁺ site corresponds to that observed in published X-ray structures of G proteins, the low affinity Mg²⁺ site may involve coordination to the terminal phosphate of the nucleotide.     

Mobile loop mutations in an archaeal inositol monophosphatase: Modulating three‐metal ion assisted catalysis and lithium inhibition

The inositol monophosphatase (IMPase) enzyme from the hyperthermophilic archaeon Methanocaldococcus jannaschii requires Mg²⁺ for activity and binds three to four ions tightly in the absence of ligands: K_D = 0.8 μM for one ion with a K_D of 38 μM for the other Mg²⁺ ions. However, the enzyme requires 5–10 mM Mg²⁺ for optimum catalysis, suggesting substrate alters the metal ion affinity. In crystal structures of this archaeal IMPase with products, one of the three metal ions is coordinated by only one protein contact, Asp38. The importance of this and three other acidic residues in a mobile loop that approaches the active site was probed with mutational studies. Only D38A exhibited an increased kinetic K_D for Mg²⁺; D26A, E39A, and E41A showed no significant change in the Mg²⁺ requirement for optimal activity. D38A also showed an increased K_m, but little effect on k_cat. This behavior is consistent with this side chain coordinating the third metal ion in the substrate complex, but with sufficient flexibility in the loop such that other acidic residues could position the Mg²⁺ in the active site in the absence of Asp38. While lithium ion inhibition of the archaeal IMPase is very poor (IC₅₀～250 mM), the D38A enzyme has a dramatically enhanced sensitivity to Li⁺ with an IC₅₀ of 12 mM. These results constitute additional evidence for three metal ion assisted catalysis with substrate and product binding reducing affinity of the third necessary metal ion. They also suggest a specific mode of action for lithium inhibition in the IMPase superfamily.     

Catalysis by imidazolium ion and metal ions in the reaction of a phosphate diester

The rates of reaction of catechol cyclic phosphate in water and in acetonitrile-water demonstrate that imidazolium ion and metal ions (Na⁺, Mg²⁺, Zn²⁺) cause significant accelerations. These studies provide models for the potential role of cations in catalysis of reactions of phosphate anions by enzymes. In catalysis by Zn²⁺, we find that two to three imidazoles are required for coordination to Zn²⁺ for most effective catalysis. Enough water must be present to solvate imidazole and coordinate to Zn²⁺, indicating that a coordinated H₂O is the nucleophile in Zn²⁺ catalysis. Product analysis also supports this conclusion.     

Increasing cleavage specificity and activity of restriction endonuclease KpnI

Restriction enzyme KpnI is a HNH superfamily endonuclease requiring divalent metal ions for DNA cleavage but not for binding. The active site of KpnI can accommodate metal ions of different atomic radii for DNA cleavage. Although Mg²⁺ ion higher than 500 μM mediates promiscuous activity, Ca²⁺ suppresses the promiscuity and induces high cleavage fidelity. Here, we report that a conservative mutation of the metal-coordinating residue D148 to Glu results in the elimination of the Ca²⁺-mediated cleavage but imparting high cleavage fidelity with Mg²⁺. High cleavage fidelity of the mutant D148E is achieved through better discrimination of the target site at the binding and cleavage steps. Biochemical experiments and molecular dynamics simulations suggest that the mutation inhibits Ca²⁺-mediated cleavage activity by altering the geometry of the Ca²⁺-bound HNH active site. Although the D148E mutant reduces the specific activity of the enzyme, we identified a suppressor mutation that increases the turnover rate to restore the specific activity of the high fidelity mutant to the wild-type level. Our results show that active site plasticity in coordinating different metal ions is related to KpnI promiscuous activity, and tinkering the metal ion coordination is a plausible way to reduce promiscuous activity of metalloenzymes.     

Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis

Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded(ds)-RNA-specific endonuclease with key roles in diverse RNA maturation and decay pathways. E.coli RNase III is a member of a structurally distinct superfamily that includes Dicer, a central enzyme in the mechanism of RNA interference. E.coli RNase III requires a divalent metal ion for activity, with Mg²⁺ as the preferred species. However, neither the function(s) nor the number of metal ions involved in catalysis is known. To gain information on metal ion involvement in catalysis, the rate of cleavage of the model substrate R1.1 RNA was determined as a function of Mg²⁺ concentration. Single-turnover conditions were applied, wherein phosphodiester cleavage was the rate-limiting event. The measured Hill coefficient (n_H) is 2.0 ± 0.1, indicative of the involvement of two Mg²⁺ ions in phosphodiester hydrolysis. It is also shown that 2-hydroxy-4H-isoquinoline-1,3-dione—an inhibitor of ribonucleases that employ two divalent metal ions in their catalytic sites—inhibits E.coli RNase III cleavage of R1.1 RNA. The IC₅₀ for the compound is 14 μM for the Mg²⁺-supported reaction, and 8 μM for the Mn²⁺-supported reaction. The compound exhibits noncompetitive inhibitory kinetics, indicating that it does not perturb substrate binding. Neither the O-methylated version of the compound nor the unsubstituted imide inhibit substrate cleavage, which is consistent with a specific interaction of the N-hydroxyimide with two closely positioned divalent metal ions. A preliminary model is presented for functional roles of two divalent metal ions in the RNase III catalytic mechanism.     

88 Comparison of monovalent and divalent ion distributions around a DNA duplex with molecular dynamic simulation and Poisson–Boltzmann approach

Ion interactions with nucleic acids (both DNA and RNA) are an important and evolving field of investigation. Positively charged cations may interact with highly negatively charged nucleic acids via simple electrostatic interactions to help screen the electrostatic repulsion along the nucleic acids and assist their folding and/or compaction. Cations may also bind at specific sites and become integral parts of the structures, possibly playing important enzymatic roles. Two popular methods for computationally exploring a nucleic acid’s ion atmosphere are atomistic molecular dynamics (MD) simulations and the Poisson–Boltzmann (PB) equation. In general, monovalent ion results obtained from MD simulations and the PB equation agree well with experiment. However, Bai et al. (2007) observed discrepancies between experiment and the PB equation while examining the competitive binding of monovalent and divalent ions, with more significant discrepancies for divalent ions. The goal of this project was to thoroughly investigate monovalent (Na⁺) and divalent (Mg²⁺) ion distributions formed around a DNA duplex with MD simulations and the PB equation. We simulated three different cation concentrations, and matched the equilibrated bulk ion concentration for our theoretical calculations with the PB equation. Based on previous work, our Mg²⁺ ions were fully solvated, the expected state of Mg²⁺ ions when interacting with a duplex, when the production simulations began and remained throughout the simulations (Kirmizialtin, 2010; Robbins, 2012). Na⁺ ion distributions and number of Na⁺ ions within 10?Å of the DNA obtained from our two methods agreed well. However, results differed for Mg²⁺ ions, with a lower number of ions within the cut-off distance obtained from the PB equation when compared to MD simulations. The Mg²⁺ ion distributions around the DNA obtained via the two methods also differed. Based on our results, we conclude that the PB equation will systematically underestimate Mg²⁺ ions bound to DNA, and much of this deviation is attributed to dielectric saturation associated with high valency ions.     

Conformational dynamics of stem II of the U2 snRNA

The spliceosome undergoes dramatic changes in both small nuclear RNA (snRNA) composition and structure during assembly and pre-mRNA splicing. It has been previously proposed that the U2 snRNA adopts two conformations within the stem II region: stem IIa or stem IIc. Dynamic rearrangement of stem IIa into IIc and vice versa is necessary for proper progression of the spliceosome through assembly and catalysis. How this conformational transition is regulated is unclear; although, proteins such as Cus2p and the helicase Prp5p have been implicated in this process. We have used single-molecule Förster resonance energy transfer (smFRET) to study U2 stem II toggling between stem IIa and IIc. Structural interconversion of the RNA was spontaneous and did not require the presence of a helicase; however, both Mg²⁺ and Cus2p promote formation of stem IIa. Destabilization of stem IIa by a G53A mutation in the RNA promotes stem IIc formation and inhibits conformational switching of the RNA by both Mg²⁺ and Cus2p. Transitioning to stem IIa can be restored using Cus2p mutations that suppress G53A phenotypes in vivo. We propose that during spliceosome assembly, Cus2p and Mg²⁺ may work together to promote stem IIa formation. During catalysis the spliceosome could then toggle stem II with the aid of Mg²⁺ or with the use of functionally equivalent protein interactions. As noted in previous studies, the Mg²⁺ toggling we observe parallels previous observations of U2/U6 and Prp8p RNase H domain Mg²⁺-dependent conformational changes. Together these data suggest that multiple components of the spliceosome may have evolved to switch between conformations corresponding to open or closed active sites with the aid of metal and protein cofactors.     

ATP conformations and ion binding modes in the active site of anthrax edema factor: A computational analysis

The Edema Factor (EF), one of the virulence factors of anthrax, is an adenylyl cyclase that promotes the overproduction of cyclic‐AMP (cAMP) from ATP, and therefore perturbs cell signaling. Crystallographic structures of EF bound to ATP analogs and reaction products, cyclic‐AMP, and Pyrophosphate (PPi), revealed different substrate conformations and catalytic‐cation binding modes, one or two cations being observed in the active site. To shed light into the biological significance of these crystallographic structures, the energetics, geometry, and dynamics of the active site are analyzed using molecular dynamics simulations. The ATP conformation observed in the one‐metal‐ion structure allows stronger interactions with the catalytic ion, and ATP is more restrained than in the structure containing two Mg²⁺ ions. Therefore, we propose that the conformation observed in the one‐ion crystal structure is a more probable starting point for the reaction. The simulations also suggest that a C3′‐endo sugar pucker facilitates nucleophilic attack. Additionally, the two‐cation binding mode restrains the mobility of the reaction products, and thus their tendency to dissociate. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Heterogeneous behavior of metalloproteins toward metal ion binding and selectivity: insights from molecular dynamics studies

About one-third of the existing proteins require metal ions as cofactors for their catalytic activities and structural complexities. While many of them bind only to a specific metal, others bind to multiple (different) metal ions. However, the exact mechanism of their metal preference has not been deduced to clarity. In this study, we used molecular dynamics (MD) simulations to investigate whether a cognate metal (bound to the structure) can be replaced with other similar metal ions. We have chosen seven different proteins (phospholipase A₂, sucrose phosphatase, pyrazinamidase, cysteine dioxygenase (CDO), plastocyanin, monoclonal anti-CD4 antibody Q425, and synaptotagmin 1 C₂B domain) bound to seven different divalent metal ions (Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Ba²⁺, and Sr²⁺, respectively). In total, 49 MD simulations each of 50 ns were performed and each trajectory was analyzed independently. Results demonstrate that in some cases, cognate metal ions can be exchanged with similar metal ions. On the contrary, some proteins show binding affinity specifically to their cognate metal ions. Surprisingly, two proteins CDO and plastocyanin which are known to bind Fe²⁺ and Cu²⁺, respectively, do not exhibit binding affinity to any metal ion. Furthermore, the study reveals that in some cases, the active site topology remains rigid even without cognate metals, whereas, some require them for their active site stability. Thus, it will be interesting to experimentally verify the accuracy of these observations obtained computationally. Moreover, the study can help in designing novel active sites for proteins to sequester metal ions particularly of toxic nature.     

Catalytic cleavage of cis- and trans-acting antigenomic delta ribozymes in the presence of various divalent metal ions

Catalytic activity of four structural variants of the antigenomic delta ribozyme, two cis- and two trans-acting, has been compared in the presence of selected divalent metal ions that effectively support catalysis. The ribozymes differ in regions that are not directly involved in formation of the ribozyme active site: the region immediately preceding the catalytic cleavage site, the P4 stem and a stretch of the viral RNA sequence extending the minimal ribozyme sequence at its 3′-terminus. The variants show high cleavage activity in the presence of Mg²⁺, Ca²⁺ and Mn²⁺, lower with Co²⁺ and Sr²⁺ and some variants are also active with Cd²⁺ and Zn²⁺ ions. In the presence of a particular metal ion the ribozymes cleave, however with different initial rates, according to pseudo-first or higher order kinetics and to different final cleavage extents. On the other hand, relatively small differences are observed in the reactions induced by various metal ions. The cleavage of trans-acting ribozymes induced by Mg²⁺ is partially inhibited in the presence of Na⁺, spermidine and some other divalent metal ions. The inert Co(NH₃)₆³⁺ complex is unable to support catalysis, as reported earlier for the genomic ribozyme. The results are discussed in terms of the influence of structural elements peripheral to the ribozyme active site on its cleavage rate and efficiency as well as the role of metal ions in the cleavage mechanism. Some implications concerning further studies and possible applications of delta ribozymes are also considered.     

Structure and binding of Mg(II) ions and di-metal bridge complexes with biological phosphates and phosphoranes

Divalent Mg²⁺ ions often serve as cofactors in enzyme or ribozyme-catalyzed phosphoryl transfer reactions. In this work, the interaction of Mg²⁺ ions and di-metal bridge complexes with phosphates, phosphoranes, and other biological ligands relevant to RNA catalysis are characterized with density functional methods. The effect of bulk solvent is treated with two continuum solvation methods (PCM and COSMO) for comparison. The relative binding affinity for different biological ligands to Mg²⁺ are quantified in different protonation states. The structure and stability of the single-metal and di-metal complexes are characterized, and the changes in phosphate and phosphorane geometry induced by metal ion binding are discussed. Di-metal bridge complexes are a ubiquitous motif and the key factors governing their electrostatic stabilization are outlined. The results presented here provide quantitative characterization of metal ion binding to ligands of importance to RNA catalysis, and lay the groundwork for design of new generation quantum models that can be applied to the full biological enzymatic systems.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0583-7An erratum to this article can be found at     

Aggregation of fibrinogen molecules by metal ions

The ability of metal ions to cause physical aggregation of neutral solutions of bovine fibrinogen has been studied. Three categories were found: (a) ions (such as Ca²⁺, Mg²⁺ and Mn²⁺) which did not cause aggregation even when present in 1–100 mm concentrations: (b) ions (such as Fe²⁺, Cu²⁺ and Ni²⁺) which caused aggregation in the 0–10 mm concentration range, (c) ions (such as Hg²⁺, Zn²⁺, Cr³⁺, La³⁺) which caused aggregation in the 0–1000 μm concentration range. Aggregation occurs immediately the metal ion is brought into contact with the fibrinogen, and product formation reaches a steady state within 5 min. With the exception of Zn²⁺, all the ions that caused aggregation exhibited a threshold concentration below which no observable aggregation took place. The threshold concentration for Hg²⁺, the most effective ion studied, was 6 μm. Addition of excess EDTA caused resolubilization of the aggregated fibrinogen due to removal of the metal ions. Aggregation is thus thought to be a physical process initiated by binding of metal ions to those carboxyl groups in fibrinogen responsible for keeping the monomers apart in solution. The aggregation does not involve prior proteolytic degradation of the fibrinogen.     

31P-nmr studies of the effect of various metals on substrate binding to yeast enolase

The ³¹P nuclear magnetic resonance (nmr) spectra of product (phosphoenolpyruvate) and substrate (2-phosphoglycerate) binding to 1:1 molar ratios ot yeast enolase were obtained as functions of the level of various metal ions. Levels sufficient to produce substrate and product binding but not catalysis ( 1 equivalent/subunit), produced shifts (with respect to 86% H₃PO₄) to lower shielding of ca. 30 ppm in the case of Co²⁺, 5–8 ppm in the case of Mg²⁺, and 2–3 ppm in the case ofCa²⁺, but virtual obliteration in the case of Mn²⁺. The effects of Mn²⁺ and Co²⁺ are consistent with a close approach of the metal ions to the phosphate groups. The effects of the physiological cofactor and optimum activator Mg²⁺ and the nonactivator Ca²⁺ are interpreted as indicating different degrees of distortion of the R-O-P bond angle in the two metal-enzyme-substrate complexes. Levels of Mg²⁺ sufficient for optimal or near optimal catalysis (2 equivalents/subunit) produce shifts to higher shielding in the ³¹P resonances of both substrate and product. These shifts are intermediate between those in the presence of 1 equivalent/subunit and those of the free ligands. Addition of a second equivalent of Ca²⁺ produces a slight shift to lower shielding of the phosphoenolpyruvate resonance and a small shift to higher shielding in the resonance for 2-phosphoglycerate. Similar levels of Co²⁺ eliminate the resonances for both substrate and product. These effects are interpreted as arising from direct coordination between substrate-dependent metal ion binding and the phosphate esters. Higher levels of Ca²⁺, Mg²⁺, or Co²⁺ or addition of KF, all of which inhibit enzyme activity, have only minor effects on the spectra. The spectrum of inorganic phosphate, a competitive inhibitor, was also examined. KF strongly enhances binding, as does excess Mg²⁺, and the binding is accompanied by a chemical shift to lower shielding of ca 2 ppm. This is not due to formation of a magnesium-fluorophosphate complex, consistent with the findings of other workers.     

Determining the Functions of HIV-1 Tat and a Second Magnesium Ion in the CDK9/Cyclin T1 Complex: A Molecular Dynamics Simulation Study

The current paradigm of cyclin-dependent kinase (CDK) regulation based on the well-established CDK2 has been recently expanded. The determination of CDK9 crystal structures suggests the requirement of an additional regulatory protein, such as human immunodeficiency virus type 1 (HIV-1) Tat, to exert its physiological functions. In most kinases, the exact number and roles of the cofactor metal ions remain unappreciated, and the repertoire has thus gained increasing attention recently. Here, molecular dynamics (MD) simulations were implemented on CDK9 to explore the functional roles of HIV-1 Tat and the second Mg²⁺ ion at site 1 (Mg₁ ²⁺). The simulations unveiled that binding of HIV-1 Tat to CDK9 not only stabilized hydrogen bonds (H-bonds) between ATP and hinge residues Asp104 and Cys106, as well as between ATP and invariant Lys48, but also facilitated the salt bridge network pertaining to the phosphorylated Thr186 at the activation loop. By contrast, these H-bonds cannot be formed in CDK9 owing to the absence of HIV-1 Tat. MD simulations further revealed that the Mg₁ ²⁺ ion, coupled with the Mg₂ ²⁺ ion, anchored to the triphosphate moiety of ATP in its catalytic competent conformation. This observation indicates the requirement of the Mg₁ ²⁺ ion for CDK9 to realize its function. Overall, the introduction of HIV-1 Tat and Mg₁ ²⁺ ion resulted in the active site architectural characteristics of phosphorylated CDK9. These data highlighted the functional roles of HIV-1 Tat and Mg₁ ²⁺ ion in the regulation of CDK9 activity, which contributes an important complementary understanding of CDK molecular underpinnings.     

Structure-based Mechanism of CMP-2-keto-3-deoxymanno-octulonic Acid Synthetase: CONVERGENT EVOLUTION OF A SUGAR-ACTIVATING ENZYME WITH DNA/RNA POLYMERASES*

The enzyme CMP-Kdo synthetase (KdsB) catalyzes the addition of 2-keto-3-deoxymanno-octulonic acid (Kdo) to CTP to form CMP-Kdo, a key reaction in the biosynthesis of lipopolysaccharide. The reaction catalyzed by KdsB and the related CMP-acylneuraminate synthase is unique among the sugar-activating enzymes in that the respective sugars are directly coupled to a cytosine monophosphate. Using inhibition studies, in combination with isothermal calorimetry, we show the substrate analogue 2β-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2β-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg²⁺ ion (Mg-B). Both ligands occupy conformations compatible with an S_n2-type attack on the α-phosphate by the Kdo 2-hydroxyl group. Based on strong similarity with DNA/RNA polymerases, both in terms of overall chemistry catalyzed as well as active site configuration, we postulate a second Mg²⁺ ion (Mg-A) is bound by the catalytically competent KdsB-CTP-Kdo ternary complex. Modeling of this complex reveals the Mg-A coordinated to the conserved Asp¹⁰⁰ and Asp²³⁵ in addition to the CTP α-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn²⁺-substituted ternary complex support this model. We propose the KdsB/CNS sugar-activating enzymes catalyze the formation of activated sugars, such as the abundant CMP-5-N-acetylneuraminic acid, by recruitment of two Mg²⁺ to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the α-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group.     

Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme

The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu²⁺ for optimal catalysis of thiophosphoryl transfer from GTPγS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu²⁺, indicating a specific catalytic role independent of any potential interactions with the GTPγS thiophosphoryl group. In-line probing and ATPγS competition both argue against direct Cu²⁺ binding by RNA; rather, these data establish that Cu²⁺ enters the active site within a Cu²⁺•GTPγS or Cu²⁺•GTP chelation complex, and that Cu²⁺•nucleobase interactions further enforce Cu²⁺ selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg²⁺ with [Co(NH₃)₆]³⁺ significantly reduced product yield, but not k_obs, indicating that the role of inner-sphere Mg²⁺ coordination is structural rather than catalytic. Replacing Mg²⁺ with alkaline earths of increasing ionic radii (Ca²⁺, Sr²⁺ and Ba²⁺) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step.     

Mg2+ Impacts the Twister Ribozyme through Push-Pull Stabilization of Nonsequential Phosphate Pairs

RNA molecules perform a variety of biological functions for which the correct three-dimensional structure is essential, including as ribozymes where they catalyze chemical reactions. Metal ions, especially Mg²⁺, neutralize these negatively charged nucleic acids and specifically stabilize RNA tertiary structures as well as impact the folding landscape of RNAs as they assume their tertiary structures. Specific binding sites of Mg²⁺ in folded conformations of RNA have been studied extensively; however, the full range of interactions of the ion with compact intermediates and unfolded states of RNA is challenging to investigate, and the atomic details of the mechanism by which the ion facilitates tertiary structure formation is not fully known. Here, umbrella sampling combined with oscillating chemical potential Grand Canonical Monte Carlo/molecular dynamics simulations are used to capture the energetics and atomic-level details of Mg²⁺-RNA interactions that occur along an unfolding pathway of the Twister ribozyme. The free energy profiles reveal stabilization of partially unfolded states by Mg²⁺, as observed in unfolding experiments, with this stabilization being due to increased sampling of simultaneous interactions of Mg²⁺ with two or more nonsequential phosphate groups. Notably, these results indicate a push-pull mechanism in which the Mg²⁺-RNA interactions actually lead to destabilization of specific nonsequential phosphate-phosphate interactions (i.e., pushed apart), whereas other interactions are stabilized (i.e., pulled together), a balance that stabilizes unfolded states and facilitates the folding of Twister, including the formation of hydrogen bonds associated with the tertiary structure. This study establishes a better understanding of how Mg²⁺-ion interactions contribute to RNA structural properties and stability.