The hepatitis delta virus (HDV) ribozyme catalyzes a self-cleavage reaction using a combination of nucleobase and metal ion catalysis. Both divalent and monovalent ions can catalyze this reaction, although the rate is slower with monovalent ions alone. Herein, we use quantum mechanical/molecular mechanical (QM/MM) free energy simulations to investigate the mechanism of this ribozyme and to elucidate the roles of the catalytic metal ion. With Mg2+ at the catalytic site, the self-cleavage mechanism is observed to be concerted with a phosphorane-like transition state and a free energy barrier of ∼13 kcal/mol, consistent with free energy barrier values extrapolated from experimental studies. With Na+ at the catalytic site, the mechanism is observed to be sequential, passing through a phosphorane intermediate, with free energy barriers of 2–4 kcal/mol for both steps; moreover, proton transfer from the exocyclic amine of protonated C75 to the nonbridging oxygen of the scissile phosphate occurs to stabilize the phosphorane intermediate in the sequential mechanism. To explain the slower rate observed experimentally with monovalent ions, we hypothesize that the activation of the O2′ nucleophile by deprotonation and orientation is less favorable with Na+ ions than with Mg2+ ions. To explore this hypothesis, we experimentally measure the pKa of O2′ by kinetic and NMR methods and find it to be lower in the presence of divalent ions rather than only monovalent ions. The combined theoretical and experimental results indicate that the catalytic Mg2+ ion may play three key roles: assisting in the activation of the O2′ nucleophile, acidifying the general acid C75, and stabilizing the nonbridging oxygen to prevent proton transfer to it.

Metal ion and nucleobase catalysis are important for ribozyme mechanism, but the extent to which they cooperate is unclear. A crystal structure of the hepatitis delta virus (HDV) ribozyme suggested that the pro-RP oxygen at the scissile phosphate directly coordinates a catalytic Mg2+ ion and is within hydrogen bonding distance of the amine of the general acid C75. Prior studies on the genomic HDV ribozyme, however, showed neither a thio effect nor metal ion rescue using Mn2+. Here, we combine experiment and theory to explore phosphorothioate substitutions at the scissile phosphate. We report significant thio effects at the scissile phosphate and metal ion rescue with Cd2+. Reaction profiles with an SP-phosphorothioate substitution are indistinguishable from those of the unmodified substrate in the presence of Mg2+ or Cd2+, supporting that the pro-SP oxygen does not coordinate metal ions. The RP-phosphorothioate substitution, however, exhibits biphasic kinetics, with the fast-reacting phase displaying a thio effect of up to 5-fold effect and the slow-reacting phase displaying a thio effect of ~1,000-fold. Moreover, the fast- and slow-reacting phases give metal ion rescues in Cd2+ of up to 10- and 330-fold, respectively. The metal ion rescues are unconventional in that they arise from Cd2+ inhibiting the oxo substrate but not the RP substrate. This metal ion rescue suggests a direct interaction of the catalytic metal ion with the pro-RP oxygen, in line with experiments on the antigenomic HDV ribozyme. Experiments without divalent ions, with mutants that interfere with Mg2+ binding, or with C75 deleted suggest that the pro-RP oxygen plays at most a redundant role in positioning C75. Quantum mechanical/molecular mechanical (QM/MM) studies indicate that the metal ion contributes to catalysis by interacting with both the pro-RP oxygen and the nucleophilic 2’- hydroxyl, supporting the experimental findings.

The hepatitis delta virus (HDV) ribozyme uses both metal ion and nucleobase catalysis in its cleavage mechanism. A reverse G•U wobble was observed in a recent crystal structure of the precleaved state. This unusual base pair positions a Mg 2+ ion to participate in catalysis. Herein, we used molecular dynamics (MD) and X-ray crystallography to characterize the conformation and metal binding characteristics of this base pair in product and precleaved forms. Beginning with a crystal structure of the product form, we observed formation of the reverse G•U wobble during MD trajectories. We also demonstrated that this base pair is compatible with the diffraction data for the product-bound state. During MD trajectories of the product form, Na + ions interacted with the reverse G•U wobble in the RNA active site, and a Mg 2+ ion, introduced in certain trajectories, remained bound at this site. Beginning with a crystal structure of the precleaved form, the reverse G•U wobble with bound Mg 2+ remained intact during MD simulations. When we removed Mg 2+ from the starting precleaved structure, Na + ions interacted with the reverse G•U wobble. In support of the computational results, we observed competition between Na + and Mg 2+ in the precleaved ribozyme crystallographically. Non-linear Poisson-Boltzmann calculations revealed a negatively charged patch near the reverse G•U wobble. This anionic pocket likely serves to bind metal ions and to help shift the pK a of the catalytic nucleobase, C75. Thus, the reverse G•U wobble motif serves to organize two catalytic elements, a metal ion and catalytic nucleobase, within the active site of the HDV ribozyme.RNA is involved in many aspects of biology, where it serves both informational and functional roles (1-3). Indeed, RNA can act as a riboswitch, binding small molecules and † This project was supported by NIH grant R01GM095923 (B.L.G and P.C.B), NIH grant GM56207 (S.H.S.), instrumentation funded by the NSF through grant OCI-0821527, the Purdue University Department of Biochemistry, the Markey Center for Structural Biology and the Purdue University Center for Cancer Research (B.L.G.). * To whom correspondence should be addressed. B.L.G.: telephone (765) 496-6165; fax (765) 494-7897; barbgolden@purdue.edu. S.H.-S. telephone (814) 865-6442; fax (814) 865-2927; shs@chem.psu.edu. P.C.B. telephone (814) 863-3812; fax (814) 865-2927. pcb@chem.psu.edu. ⊥ Present Address: Department of Biochemistry, University of Illinois, 600 S. Mathews Ave., Urbana IL 61801.Supporting Information Available Procedures for MD; protonated cytosine partial charges; additional MD trajectories; metal ion movement during MD; heavy-atom RMSD plots; additional NLPB results. Also provided are tables of crystallographic data collection statistics; crystallographic Na + coordinates for the pre-cleaved ribozyme; and average distances between the reverse G•U wobble and metal ions from MD. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBioche...

The hepatitis delta virus ribozyme catalyzes an RNA cleavage reaction using a catalytic nucleobase and a divalent metal ion. The catalytic base, C75, serves as a general acid and has a pKa shifted towards neutrality. Less is known about the role of metal ions in the mechanism. A recent crystal structure of the pre-cleavage ribozyme identified a Mg2+ ion that interacts through its partial hydration sphere with the G25•U20 reverse wobble. In addition, this Mg2+ ion is in position to directly coordinate the nucleophile, the 2’-hydroxyl of U(-1), suggesting it can serve as a Lewis acid to facilitate deprotonation of the 2’-hydroxyl. To test the role of the active site Mg2+ ion, we replaced the G25•U20 reverse wobble with an isosteric A25•C20 reverse wobble. This change was found to significantly reduce the negative potential at the active site, as supported by electrostatics calculations, suggesting that active site Mg2+ binding could be adversely affected by the mutation. Kinetic analysis and molecular dynamics of the A25•C20 double mutant suggest that this variant stably folds into an active structure. However, pH-rate profiles of the double mutant are inverted relative to the profiles for wild-type ribozyme, suggesting that the A25•C20 double mutant has lost the active site metal ion. Overall, these studies support a model wherein the partially hydrated Mg2+ positioned at the G25•U20 reverse wobble is catalytic and could serve as a Lewis acid, a Brønsted base, or both to facilitate deprotonation of the nucleophile.

The crystal structure of the precleaved form of the hepatitis delta virus (HDV) ribozyme reveals two G•U wobbles near the active site: a rare reverse G•U wobble involving a syn G base, and a standard G•U wobble at the cleavage site. The catalytic mechanism for this ribozyme has been proposed to involve a Mg2+ ion bound to the reverse G•U wobble, as well as a protonated C75 base. We carried out molecular dynamics simulations to analyze metal ion interaction with the reverse and standard G•U wobbles and to investigate the impact of C75 protonation on the structure and motions of the ribozyme. We identified two types of Mg2+ ions associated with the ribozyme, chelated and diffuse, at the reverse and standard G•U wobbles, respectively, which appear to contribute to catalysis and stability, respectively. These two metal ion sites exhibit relatively independent behavior. Protonation of C75 was observed to locally organize the active site in a manner that facilitates the catalytic mechanism, in which C75+ acts as a general acid and Mg2+ as a Lewis acid. The simulations also indicated that the overall structure and thermal motions of the ribozyme are not significantly influenced by the catalytic Mg2+ interaction or C75 protonation. This analysis suggests that the reaction pathway of the ribozyme is dominated by small local motions at the active site rather than large-scale global conformational changes. These results are consistent with a wealth of experimental data.

A predictive understanding of the mechanisms of RNA cleavage is important for the design of emerging technology built from biological and synthetic molecules that have promise for new biochemical and medicinal applications. Over the past 15 years, RNA cleavage reactions involving 2'-O-transphosphorylation have been discussed using a simplified framework introduced by Breaker that consists of four fundamental catalytic strategies (designated α, β, γ, and δ) that contribute to rate enhancement. As more detailed mechanistic data emerge, there is need for the framework to evolve and keep pace. We develop an ontology for discussion of strategies of enzymes that catalyze RNA cleavage via 2'-O-transphosphorylation that stratifies Breaker's framework into primary (1°), secondary (2°) and tertiary (3°) contributions to enable more precise interpretation of mechanism in the context of structure and bonding. Further, we point out instances where atomic-level changes give rise to changes in more than one catalytic contribution, a phenomenon we refer to as 'functional blurring'. We hope that this ontology will help clarify our conversations and pave the path forward toward a consensus view of these fundamental and fascinating mechanisms. The insight gained will deepen our understanding of RNA cleavage reactions catalyzed by natural protein and RNA enzymes, as well as aid in the design of new engineered DNA and synthetic enzymes.

Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pK a . Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions.light sensing | flavoprotein | photochemistry | redox | molecular dynamics C ryptochromes (CRYs) are flavin-binding proteins that perform a variety of sensory and catalytic functions in all kingdoms of life (1, 2). CRYs are closely related to the DNA photolyases (PLs), which catalyze light-driven redox reactions to break apart pyrimidine dimers in UV-damaged DNA (1, 2). CRYs and PLs share a conserved photolyase homology region that consists of an α-helical domain, which binds flavin adenine dinucleotide (FAD) and an α/β Rossman-fold domain, which sometimes binds a pteridine or deazaflavin antenna cofactor. CRYs also contain C-terminal extensions of variable sizes that contribute to their specific functions. The range of activities found for CRYs and PLs require that their flavin cofactors assume a broad range of redox, protonation, and excited states (1, 2).In the fruit fly Drosophila melanogaster, a type I cryptochrome (dCRY) is the primary light receptor of the circadian clock (1, 3). In response to blue light, dCRY coordinates interactions between Timeless (TIM) and the E3-ubiquitin ligase Jetlag (JET) (4). JETmediated proteolysis of TIM destabilizes its partner Period (PER). PER serves as the principal repressor of circadian gene expression and its degradation phase-shifts the clock (3). dCRY also catalyzes light-induced self-degradation that involves another E3-ligase: Brwd3 or RAMSHACKLE (5)...

The glmS ribozyme catalyzes a self-cleavage reaction at the phosphodiester bond between residues A-1 and G1. This reaction is thought to occur by an acid–base mechanism involving the glucosamine-6-phosphate cofactor and G40 residue. Herein quantum mechanical/molecular mechanical free energy simulations and pKa calculations, as well as experimental measurements of the rate constant for self-cleavage, are utilized to elucidate the mechanism, particularly the role of G40. Our calculations suggest that an external base deprotonates either G40(N1) or possibly A-1(O2′), which would be followed by proton transfer from G40(N1) to A-1(O2′). After this initial deprotonation, A-1(O2′) starts attacking the phosphate as a hydroxyl group, which is hydrogen-bonded to deprotonated G40, concurrent with G40(N1) moving closer to the hydroxyl group and directing the in-line attack. Proton transfer from A-1(O2′) to G40 is concomitant with attack of the scissile phosphate, followed by the remainder of the cleavage reaction. A mechanism in which an external base does not participate, but rather the proton transfers from A-1(O2′) to a nonbridging oxygen during nucleophilic attack, was also considered but deemed to be less likely due to its higher effective free energy barrier. The calculated rate constant for the favored mechanism is in agreement with the experimental rate constant measured at biological Mg2+ ion concentration. According to these calculations, catalysis is optimal when G40 has an elevated pKa rather than a pKa shifted toward neutrality, although a balance among the pKa’s of A-1, G40, and the nonbridging oxygen is essential. These results have general implications, as the hammerhead, hairpin, and twister ribozymes have guanines at a similar position as G40.