ACES: Optimized Alchemically Enhanced Sampling.

We present an alchemical enhanced sampling (ACES) method implemented in the GPU-accelerated AMBER free energy MD engine. The methods hinges on the creation of an "enhanced sampling state" by reducing or eliminating selected potential energy terms and interactions that lead to kinetic traps and conformational barriers while maintaining those terms that curtail the need to otherwise sample large volumes of phase space. For example, the enhanced sampling state might involve transforming regions of a ligand and/or protein side chain into a noninteracting "dummy state" with internal electrostatics and torsion angle terms turned off. The enhanced sampling state is connected to a real-state end point through a Hamiltonian replica exchange (HREMD) framework that is facilitated by newly developed alchemical transformation pathways and smoothstep softcore potentials. This creates a counterdiffusion of real and enhanced-sampling states along the HREMD network. The effect of a differential response of the environment to the real and enhanced-sampling states is minimized by leveraging the dual topology framework in AMBER to construct a counterbalancing HREMD network in the opposite alchemical direction with the same (or similar) real and enhanced sampling states at inverted end points. The method has been demonstrated in a series of test cases of increasing complexity where traditional MD, and in several cases alternative REST2-like enhanced sampling methods, are shown to fail. The hydration free energy for acetic acid was shown to be independent of the starting conformation, and the values for four additional edge case molecules from the FreeSolv database were shown to have a significantly closer agreement with experiment using ACES. The method was further able to handle different rotamer states in a Cdk2 ligand identified as fractionally occupied in crystal structures. Finally, ACES was applied to T4-lysozyme and demonstrated that the side chain distribution of V111χ1 could be reliably reproduced for the apo state, bound to p-xylene, and in p-xylene→ benzene transformations. In these cases, the ACES method is shown to be highly robust and superior to a REST2-like enhanced sampling implementation alone.

AMBER Free Energy Tools: A New Framework for the Design of Optimized Alchemical Transformation Pathways.

We develop a framework for the design of optimized alchemical transformation pathways in free energy simulations using nonlinear mixing and a new functional form for so-called "softcore" potentials. We describe the implementation and testing of this framework in the GPU-accelerated AMBER software suite. The new optimized alchemical transformation pathways integrate a number of important features, including (1) the use of smoothstep functions to stabilize behavior near the transformation end points, (2) consistent power scaling of Coulomb and Lennard-Jones (LJ) interactions with unitless control parameters to maintain balance of electrostatic attractions and exchange repulsions, (3) pairwise form based on the LJ contact radius for the effective interaction distance with separation-shifted scaling, and (4) rigorous smoothing of the potential at the nonbonded cutoff boundary. The new softcore potential form is combined with smoothly transforming nonlinear λ weights for mixing specific potential energy terms, along with flexible λ-scheduling features, to enable robust and stable alchemical transformation pathways. The resulting pathways are demonstrated and tested, and shown to be superior to the traditional methods in terms of numerical stability and minimal variance of the free energy estimates for all cases considered. The framework presented here can be used to design new alchemical enhanced sampling methods, and leveraged in robust free energy workflows for large ligand data sets.

AMBER Drug Discovery Boost Tools: Automated Workflow for Production Free-Energy Simulation Setup and Analysis (ProFESSA).

We report an automated workflow for production free-energy simulation setup and analysis (ProFESSA) using the GPU-accelerated AMBER free-energy engine with enhanced sampling features and analysis tools, part of the AMBER Drug Discovery Boost package that has been integrated into the AMBER22 release. The workflow establishes a flexible, end-to-end pipeline for performing alchemical free-energy simulations that brings to bear technologies, including new enhanced sampling features and analysis tools, to practical drug discovery problems. ProFESSA provides the user with top-level control of large sets of free-energy calculations and offers access to the following key functionalities: (1) automated setup of file infrastructure; (2) enhanced conformational and alchemical sampling with the ACES method; and (3) network-wide free-energy analysis with the optional imposition of cycle closure and experimental constraints. The workflow is applied to perform absolute and relative solvation free-energy and relative ligand-protein binding free-energy calculations using different atom-mapping procedures. Results demonstrate that the workflow is internally consistent and highly robust. Further, the application of a new network-wide Lagrange multiplier constraint analysis that imposes key experimental constraints substantially improves binding free-energy predictions.

Room-temperature serial synchrotron crystallography of Drosophila cryptochrome.

Fixed-target serial crystallography allows the high-throughput collection of diffraction data from small crystals at room temperature. This methodology is particularly useful for difficult samples that have sensitivity to radiation damage or intolerance to cryoprotection measures; fixed-target methods also have the added benefit of low sample consumption. Here, this method is applied to the structure determination of the circadian photoreceptor cryptochrome (CRY), previous structures of which have been determined at cryogenic temperature. In determining the structure, several data-filtering strategies were tested for combining observations from the hundreds of crystals that contributed to the final data set. Removing data sets based on the average correlation coefficient among equivalent reflection intensities between a given data set and all others was most effective at improving the data quality and maintaining overall completeness. CRYs are light sensors that undergo conformational photoactivation. Comparisons between the cryogenic and room-temperature CRY structures reveal regions of enhanced mobility at room temperature in loops that have functional importance within the CRY family of proteins. The B factors of the room-temperature structure correlate well with those predicted from molecular-dynamics simulations.

Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign.

DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, internucleotidyl O-P linkages. Can this linkage selectivity be overcome by design to produce xenonucleic acids? Here, we report that the structure-guided redesign of an archaeal DNA polymerase, 9°N, exhibits a new activity undetectable in the wild-type enzyme: catalyzing the formation of internucleotidyl N-P linkages using 3'-NH2-ddNTPs. Replacing a metal-binding aspartate in the 9°N active site with asparagine was key to the emergence of this unnatural enzyme activity. MD simulations provided insights into how a single substitution enhances the productive positioning of a 3'-amino nucleophile in the active site. Further remodeling of the protein-nucleic acid interface in the finger subdomain yielded a quadruple-mutant variant (9°N-NRQS) displaying DNA-dependent NP-DNA polymerase activity. In addition, the engineered promiscuity of 9°N-NRQS was leveraged for one-pot synthesis of DNA─NP-DNA copolymers. This work sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.

Mechanistic insight into light-dependent recognition of Timeless by Drosophila Cryptochrome.

Cryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM recognition. We present a generally applicable select western-blot-free tagged-protein interaction (SWFTI) assay that allowed the quantification of CRY binding to TIM in dark and light. The assay was used to study CRY variants with residue substitutions in the flavin pocket and correlate their TIM affinities with CTT undocking, as measured by pulse-dipolar ESR spectroscopy and evaluated by molecular dynamics simulations. CRY variants with the CTT removed or undocked bound TIM constitutively, whereas those incapable of photoreduction bound TIM weakly. In response to the flavin redox state, two conserved histidine residues contributed to a robust on/off switch by mediating CTT interactions with the flavin pocket and TIM. Our approach provides an expeditious means to quantify the interactions of difficult-to-produce proteins.

Peripheral Methionine Residues Impact Flavin Photoreduction and Protonation in an Engineered LOV Domain Light Sensor.

Proton-coupled electron transfer reactions play critical roles in many aspects of sensory phototransduction. In the case of flavoprotein light sensors, reductive quenching of flavin excited states initiates chemical and conformational changes that ultimately transmit light signals to downstream targets. These reactions generally require neighboring aromatic residues and proton-donating side chains for rapid and coordinated electron and proton transfer to flavin. Although photoreduction of flavoproteins can produce either the anionic (ASQ) or neutral semiquinone (NSQ), the factors that favor one over the other are not well understood. Here we employ a biologically active variant of the light-oxygen-voltage (LOV) domain protein VVD devoid of the adduct-forming Cys residue (VVD-III) to probe the mechanism of flavin photoreduction and protonation. A series of isosteric and conservative residue replacements studied by rate measurements, fluorescence quantum yields, FTIR difference spectroscopy, and molecular dynamics simulations indicate that tyrosine residues facilitate charge recombination reactions that limit sustained flavin reduction, whereas methionine residues facilitate radical propagation and quenching and also gate solvent access for flavin protonation. Replacement of a single surface Met residue with Leu favors formation of the ASQ over the NSQ and desensitizes photoreduction to oxidants. In contrast, increasing site hydrophilicity by Gln substitution promotes rapid NSQ formation and weakens the influence of the redox environment. Overall, the photoreactivity of VVD-III can be understood in terms of redundant electron donors, internal hole quenching, and coupled proton transfer reactions that all depend upon protein conformation, dynamics, and solvent penetration.

Tuning flavin environment to detect and control light-induced conformational switching in Drosophila cryptochrome.

Light-induction of an anionic semiquinone (SQ) flavin radical in Drosophila cryptochrome (dCRY) alters the dCRY conformation to promote binding and degradation of the circadian clock protein Timeless (TIM). Specific peptide ligation with sortase A attaches a nitroxide spin-probe to the dCRY C-terminal tail (CTT) while avoiding deleterious side reactions. Pulse dipolar electron-spin resonance spectroscopy from the CTT nitroxide to the SQ shows that flavin photoreduction shifts the CTT ~1 nm and increases its motion, without causing full displacement from the protein. dCRY engineered to form the neutral SQ serves as a dark-state proxy to reveal that the CTT remains docked when the flavin ring is reduced but uncharged. Substitutions of flavin-proximal His378 promote CTT undocking in the dark or diminish undocking in the light, consistent with molecular dynamics simulations and TIM degradation activity. The His378 variants inform on recognition motifs for dCRY cellular turnover and strategies for developing optogenetic tools.

Confluence of theory and experiment reveals the catalytic mechanism of the Varkud satellite ribozyme.

The Varkud satellite ribozyme catalyses site-specific RNA cleavage and ligation, and serves as an important model system to understand RNA catalysis. Here, we combine stereospecific phosphorothioate substitution, precision nucleobase mutation and linear free-energy relationship measurements with molecular dynamics, molecular solvation theory and ab initio quantum mechanical/molecular mechanical free-energy simulations to gain insight into the catalysis. Through this confluence of theory and experiment, we unify the existing body of structural and functional data to unveil the catalytic mechanism in unprecedented detail, including the degree of proton transfer in the transition state. Further, we provide evidence for a critical Mg2+ in the active site that interacts with the scissile phosphate and anchors the general base guanine in position for nucleophile activation. This novel role for Mg2+ adds to the diversity of known catalytic RNA strategies and unifies functional features observed in the Varkud satellite, hairpin and hammerhead ribozyme classes.

Evidence for a Catalytic Strategy to Promote Nucleophile Activation in Metal-Dependent RNA-Cleaving Ribozymes and 8-17 DNAzyme.

An unique catalytic strategy was recently reported for the glmS ribozyme [Bingaman et al., Nat. Chem. Biol.2017, 13, 439-445] that involves promotion of productive hydrogen bonding of the O2' nucleophile to facilitate its activation. We provide broad evidence of this strategy in the hammerhead, pistol, and VS ribozymes and 8-17 DNAzyme, enabled by a functionally important divalent metal ion that interacts with the scissile phosphate and disrupts nonproductive competitive hydrogen bonding with the O2' nucleophile. This strategy, designated tertiary gamma (3°Î³) catalysis, illustrates an additional role for divalent ions in ribozyme catalysis.

An Ontology for Facilitating Discussion of Catalytic Strategies of RNA-Cleaving Enzymes.

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, ß, 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.

Elucidation of the Catalytic Mechanism of a Miniature Zinc Finger Hydrolase.

To improve our mechanistic understanding of zinc metalloenzymes, we report a joint computational and experimental study of a minimal carbonic anhydrase (CA) mimic, a 22-residue Zn-finger hydrolase. We combine classical molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) geometry optimizations, and QM/MM free energy simulations with ambient and high-pressure kinetic measurements to investigate the mechanism of the hydrolysis of the substrate p-nitrophenylacetate (pNPA). The zinc center of the hydrolase prefers a pentacoordinated geometry, as found in most naturally occurring CAs and CA-like enzymes. Two possible mechanisms for the catalytic reaction are investigated. The first one is analogous to the commonly accepted mechanism for CA-like enzymes: a sequential pathway, in which a Zn2+-bound hydroxide acts as a nucleophile and the hydrolysis proceeds through a tetrahedral intermediate. The initial rate-limiting step of this reaction is the nucleophilic attack of the hydroxide on pNPA to form the tetrahedral intermediate. The computed free energy barrier of 18.5 kcal/mol is consistent with the experimental value of 20.5 kcal/mol obtained from our kinetics experiments. We also explore an alternative reverse protonation pathway for the hydrolase, in which a nearby hydroxide ion from the bulk acts as the nucleophile (instead of a zinc-bound hydroxide). According to QM/MM MD simulations, hydrolysis occurs spontaneously along this pathway. However, this second scenario is not viable in our system, as the tertiary structure of the hydrolase lacks a suitably positioned residue that would act as a general base and generate a hydroxide ion from a nearby bulk water molecule. Hence, our combined theoretical and experimental study indicates that the investigated minimal CA mimic retains the essential mechanistic features of CA-like enzyme catalysis. The high-pressure experiments show that its catalytic efficiency can be enhanced by applying hydrostatic pressure. According to the simulations, more drastic improvements might be afforded by mutations that make the reverse protonation pathway accessible.

Importance of MM Polarization in QM/MM Studies of Enzymatic Reactions: Assessment of the QM/MM Drude Oscillator Model.

For accurate quantum mechanics/molecular mechanics (QM/MM) studies of enzymatic reactions, it is desirable to include MM polarization, for example by using the Drude oscillator (DO) model. For a long time, such studies were hampered by the lack of well-tested polarizable force fields for proteins. Following up on a recent preliminary QM/MM-DO assessment (J. Chem. THEORY: Comput. 2014, 10, 1795-1809), we now report a comprehensive investigation of the effects of MM polarization on two enzymatic reactions, namely the Claisen rearrangement in chorismate mutase and the hydroxylation reaction in p-hydroxybenzoate hydroxylase, using the QM/CHARMM-DO model and two QM methods (B3LYP, OM2). We compare the results from extensive geometry optimizations and free energy simulations at the QM/MM-DO level to those obtained from analogous calculations at the conventional QM/MM level.

Glutamine Amide Flip Elicits Long Distance Allosteric Responses in the LOV Protein Vivid.

Light-oxygen-voltage (LOV) domains sense blue light through the photochemical formation of a cysteinyl-flavin covalent adduct. Concurrent protonation at the flavin N5 position alters the hydrogen bonding interactions of an invariant Gln residue that has been proposed to flip its amide side chain as a critical step in the propagation of conformational change. Traditional molecular dynamics (MD) and replica-exchange MD (REMD) simulations of the well-characterized LOV protein Vivid (VVD) demonstrate that the Gln182 amide indeed reorients by ∼180° in response to either adduct formation or reduction of the isoalloxazine ring to the neutral semiquinone, both of which involve N5 protonation. Free energy simulations reveal that the relative free energies of the flipped Gln conformation and the flipping barrier are significantly lower in the light-adapted state. The Gln182 flip stabilizes an important hinge-bß region between the PAS ß-sheet and the N-terminal cap helix that in turn destabilizes an N-terminal latch region against the PAS core. Release of the latch, observed both experimentally and in the simulations, is known to mediate light-induced VVD dimerization. This computational study of a LOV protein, unprecedented in its agreement with experiment, provides an atomistic view of long-range allosteric coupling in a photoreceptor.

Changes in active site histidine hydrogen bonding trigger cryptochrome activation.

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 pKa 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.

Inverse thio effects in the hepatitis delta virus ribozyme reveal that the reaction pathway is controlled by metal ion charge density.

The hepatitis delta virus (HDV) ribozyme self-cleaves in the presence of a wide range of monovalent and divalent ions. Prior theoretical studies provided evidence that self-cleavage proceeds via a concerted or stepwise pathway, with the outcome dictated by the valency of the metal ion. In the present study, we measure stereospecific thio effects at the nonbridging oxygens of the scissile phosphate under a wide range of experimental conditions, including varying concentrations of diverse monovalent and divalent ions, and combine these with quantum mechanical/molecular mechanical (QM/MM) free energy simulations on the stereospecific thio substrates. The RP substrate gives large normal thio effects in the presence of all monovalent ions. The SP substrate also gives normal or no thio effects, but only for smaller monovalent and divalent cations, such as Li(+), Mg(2+), Ca(2+), and Sr(2+); in contrast, sizable inverse thio effects are found for larger monovalent and divalent cations, including Na(+), K(+), NH4(+), and Ba(2+). Proton inventories are found to be unity in the presence of the larger monovalent and divalent ions, but two in the presence of Mg(2+). Additionally, rate-pH profiles are inverted for the low charge density ions, and only imidazole plus ammonium ions rescue an inactive C75Δ variant in the absence of Mg(2+). Results from the thio effect experiments, rate-pH profiles, proton inventories, and ammonium/imidazole rescue experiments, combined with QM/MM free energy simulations, support a change in the mechanism of HDV ribozyme self-cleavage from concerted and metal ion-stabilized to stepwise and proton transfer-stabilized as the charge density of the metal ion decreases.

Role of the active site guanine in the glmS ribozyme self-cleavage mechanism: quantum mechanical/molecular mechanical free energy simulations.

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 Mg(2+) 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.

Quantum mechanical/molecular mechanical free energy simulations of the self-cleavage reaction in the hepatitis delta virus ribozyme.

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 Mg(2+) 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 Mg(2+) 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 Mg(2+) 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.

Thio effects and an unconventional metal ion rescue in the genomic hepatitis delta virus ribozyme.

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 Mg(2+) ion and is within hydrogen bonding distance of the amine of the general acid C75. Prior studies of the genomic HDV ribozyme, however, showed neither a thio effect nor metal ion rescue using Mn(2+). 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 Cd(2+). Reaction profiles with an SP-phosphorothioate substitution are indistinguishable from those of the unmodified substrate in the presence of Mg(2+) or Cd(2+), supporting the idea 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 and the slow-reacting phase displaying a thio effect of ~1000-fold. Moreover, the fast- and slow-reacting phases give metal ion rescues in Cd(2+) of up to 10- and 330-fold, respectively. The metal ion rescues are unconventional in that they arise from Cd(2+) 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 with the antigenomic HDV ribozyme. Experiments without divalent ions, with a double mutant that interferes with Mg(2+) 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.

Identification of the catalytic Mg²âº ion in the hepatitis delta virus ribozyme.

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 pK(a) shifted toward neutrality. Less is known about the role of metal ions in the mechanism. A recent crystal structure of the precleavage ribozyme identified a Mg²âº ion that interacts through its partial hydration sphere with the G25·U20 reverse wobble. In addition, this Mg²âº 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 Mg²âº 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 Mg²âº binding could be adversely affected by the mutation. The 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 in the presence of Mg²âº are inverted relative to the profiles for the wild-type ribozyme, suggesting that the A25·C20 double mutant has lost the active site metal ion. Overall, these studies support a model in which the partially hydrated Mg²âº 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.