Catalytic mechanism and pH dependence of a methyltransferase ribozyme (MTR1) from computational enzymology

Abstract A methyltransferase ribozyme (MTR1) was selected in vitro to catalyze alkyl transfer from exogenous O6-methylguanine (O6mG) to a target adenine N1, and recently, high-resolution crystal structures have become available. We use a combination of classical molecular dynamics, ab initio quantum mechanical/molecular mechanical (QM/MM) and alchemical free energy (AFE) simulations to elucidate the atomic-level solution mechanism of MTR1. Simulations identify an active reactant state involving protonation of C10 that hydrogen bonds with O6mG:N1. The deduced mechanism involves a stepwise mechanism with two transition states corresponding to proton transfer from C10:N3 to O6mG:N1 and rate-controlling methyl transfer (19.4  kcal·mol−1 barrier). AFE simulations predict the pKa for C10 to be 6.3, close to the experimental apparent pKa of 6.2, further implicating it as a critical general acid. The intrinsic rate derived from QM/MM simulations, together with pKa calculations, enables us to predict an activity–pH profile that agrees well with experiment. The insights gained provide further support for a putative RNA world and establish new design principles for RNA-based biochemical tools.

[1]  D. York,et al.  ACES: Optimized Alchemically Enhanced Sampling. , 2023, Journal of chemical theory and computation.

[2]  Timothy J. Giese,et al.  AMBER Free Energy Tools: A New Framework for the Design of Optimized Alchemical Transformation Pathways. , 2023, Journal of chemical theory and computation.

[3]  D. Case,et al.  RNA Electrostatics: How Ribozymes Engineer Active Sites to Enable Catalysis. , 2022, The journal of physical chemistry. B.

[4]  D. Lilley,et al.  Structure and mechanism of a methyltransferase ribozyme , 2022, Nature Chemical Biology.

[5]  J. Piccirilli,et al.  Structural basis for substrate binding and catalysis by a self-alkylating ribozyme , 2022, Nature Chemical Biology.

[6]  H. Schindelin,et al.  Structure and mechanism of the methyltransferase ribozyme MTR1 , 2022, Nature Chemical Biology.

[7]  Dongrong Chen,et al.  The identification and characterization of a selected SAM-dependent methyltransferase ribozyme that is present in natural sequences , 2021, Nature Catalysis.

[8]  R. Micura,et al.  A natural riboswitch scaffold with self-methylation activity , 2021, Nature Communications.

[9]  D. York,et al.  Robust, Efficient and Automated Methods for Accurate Prediction of Protein-Ligand Binding Affinities in AMBER Drug Discovery Boost , 2021 .

[10]  D. Lilley,et al.  The potential versatility of RNA catalysis , 2021, Wiley interdisciplinary reviews. RNA.

[11]  D. York,et al.  Extension of the Variational Free Energy Profile and Multistate Bennett Acceptance Ratio Methods for High-Dimensional Potential of Mean Force Profile Analysis. , 2021, The journal of physical chemistry. A.

[12]  J. Šponer,et al.  UUCG RNA Tetraloop as a Formidable Force-Field Challenge for MD Simulations. , 2020, Journal of chemical theory and computation.

[13]  Y. Yokobayashi.  High-Throughput Analysis and Engineering of Ribozymes and Deoxyribozymes by Sequencing. , 2020, Accounts of chemical research.

[14]  C. Höbartner,et al.  Site-specific RNA methylation by a methyltransferase ribozyme , 2020, Nature.

[15]  Pengfei Li,et al.  Alchemical Binding Free Energy Calculations in AMBER20: Advances and Best Practices for Drug Discovery , 2020, J. Chem. Inf. Model..

[16]  T. Katoh,et al.  An aminoacylation ribozyme evolved from a natural tRNA-sensing T-box riboswitch , 2020, Nature Chemical Biology.

[17]  D. York,et al.  The L-platform/L-scaffold framework: a blueprint for RNA-cleaving nucleic acid enzyme design , 2019, RNA.

[18]  Timothy J. Giese,et al.  Confluence of Theory and Experiment Reveal the Catalytic Mechanism of the Varkud Satellite Ribozyme , 2019, Nature Chemistry.

[19]  D. York,et al.  Dynamical ensemble of the active state and transition state mimic for the RNA-cleaving 8–17 DNAzyme in solution , 2019, Nucleic acids research.

[20]  Z. Weinberg,et al.  Novel ribozymes: discovery, catalytic mechanisms, and the quest to understand biological function , 2019, Nucleic acids research.

[21]  D. Lilley.  Classification of the nucleolytic ribozymes based upon catalytic mechanism , 2019, F1000Research.

[22]  Timothy J. Giese,et al.  Cleaning Up Mechanistic Debris Generated by Twister Ribozymes Using Computational RNA Enzymology. , 2019, ACS catalysis.

[23]  D. York,et al.  An Ontology for Facilitating Discussion of Catalytic Strategies of RNA-Cleaving Enzymes. , 2019, ACS chemical biology.

[24]  D. York,et al.  Framework for Conducting and Analyzing Crystal Simulations of Nucleic Acids to Aid in Modern Force Field Evaluation. , 2019, The journal of physical chemistry. B.

[25]  Chi Zhang,et al.  Reversible RNA Modification N1-methyladenosine (m1A) in mRNA and tRNA , 2018, Genom. Proteom. Bioinform..

[26]  Daniel D. Seith,et al.  Elucidation of Catalytic Strategies of Small Nucleolytic Ribozymes From Comparative Analysis of Active Sites. , 2018, ACS catalysis.

[27]  P. Bevilacqua,et al.  Complexity in pH-Dependent Ribozyme Kinetics: Dark pKa Shifts and Wavy Rate-pH Profiles. , 2017, Biochemistry.

[28]  Timothy J. Giese,et al.  Ambient-Potential Composite Ewald Method for ab Initio Quantum Mechanical/Molecular Mechanical Molecular Dynamics Simulation. , 2016, Journal of chemical theory and computation.

[29]  Niel M. Henriksen,et al.  Highly sampled tetranucleotide and tetraloop motifs enable evaluation of common RNA force fields , 2015, RNA.

[30]  Darrin M York,et al.  Structural fidelity and NMR relaxation analysis in a prototype RNA hairpin , 2015, RNA.

[31]  Darrin M York,et al.  Interpretation of pH-activity profiles for acid-base catalysis from molecular simulations. , 2015, Biochemistry.

[32]  David R. Liu,et al.  Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling , 2014, Nature chemical biology.

[33]  Karl-Dieter Entian,et al.  Identification of a novel methyltransferase, Bmt2, responsible for the N-1-methyl-adenosine base modification of 25S rRNA in Saccharomyces cerevisiae , 2013, Nucleic acids research.

[34]  M. Elstner,et al.  Parametrization and Benchmark of DFTB3 for Organic Molecules. , 2013, Journal of chemical theory and computation.

[35]  V. de Crécy-Lagard,et al.  Biosynthesis and function of posttranscriptional modifications of transfer RNAs. , 2012, Annual review of genetics.

[36]  Jun Lu,et al.  General acid-base catalysis mediated by nucleobases in the hairpin ribozyme. , 2012, Journal of the American Chemical Society.

[37]  Zasha Weinberg,et al.  Widespread Genetic Switches and Toxicity Resistance Proteins for Fluoride , 2012, Science.

[38]  J. Šponer,et al.  Refinement of the Cornell et al. Nucleic Acids Force Field Based on Reference Quantum Chemical Calculations of Glycosidic Torsion Profiles , 2011, Journal of chemical theory and computation.

[39]  Wei Yang,et al.  Catalytic mechanism of RNA backbone cleavage by ribonuclease H from quantum mechanics/molecular mechanics simulations. , 2011, Journal of the American Chemical Society.

[40]  Daniel Herschlag,et al.  Biological phosphoryl-transfer reactions: understanding mechanism and catalysis. , 2011, Annual review of biochemistry.

[41]  Michael Gaus,et al.  DFTB3: Extension of the self-consistent-charge density-functional tight-binding method (SCC-DFTB). , 2011, Journal of chemical theory and computation.

[42]  Michal Otyepka,et al.  Performance of Molecular Mechanics Force Fields for RNA Simulations: Stability of UUCG and GNRA Hairpins. , 2010, Journal of chemical theory and computation.

[43]  D. York,et al.  Accurate proton affinity and gas-phase basicity values for molecules important in biocatalysis. , 2010, The journal of physical chemistry. B.

[44]  Jun Gao,et al.  QM/MM study on the reaction mechanism of O6-alkylguanine-DNA alkyltransferase. , 2010, The journal of physical chemistry. B.

[45]  Jeremy C. Smith,et al.  Magnesium-dependent active-site conformational selection in the Diels-Alderase ribozyme. , 2010, Journal of the American Chemical Society.

[46]  Piotr Cieplak,et al.  Free energy profile of RNA hairpins: a molecular dynamics simulation study. , 2010, Biophysical journal.

[47]  A. Ferré-D’Amaré,et al.  Ribozymes and riboswitches: modulation of RNA function by small molecules , 2009, Biochemistry.

[48]  Polina Georgieva,et al.  Density functional theory study of the reaction mechanism of the DNA repairing enzyme alkylguanine alkyltransferase , 2008 .

[49]  T. Cheatham,et al.  Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations , 2008, The journal of physical chemistry. B.

[50]  H. Adams,et al.  The Synthesis and Properties of Tricyclic Analogues of S6-Methylthioguanine and O6-Methylguanine , 2008 .

[51]  J. Tainer,et al.  DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O6-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy. , 2007, DNA repair.

[52]  J. Šponer,et al.  Refinement of the AMBER Force Field for Nucleic Acids: Improving the Description of α/γ Conformers , 2007 .

[53]  T. C. Bruice,et al.  Diels-Alder ribozyme catalysis: a computational approach. , 2007, Journal of the American Chemical Society.

[54]  M. Famulok,et al.  A ribozyme for the aldol reaction. , 2005, Chemistry & biology.

[55]  Greg L. Hura,et al.  Development of an improved four-site water model for biomolecular simulations: TIP4P-Ew. , 2004, The Journal of chemical physics.

[56]  M. Karplus,et al.  How Enzymes Work: Analysis by Modern Rate Theory and Computer Simulations , 2004, Science.

[57]  P. Bevilacqua.  Mechanistic considerations for general acid-base catalysis by RNA: revisiting the mechanism of the hairpin ribozyme. , 2003, Biochemistry.

[58]  D. Bartel,et al.  RNA-Catalyzed RNA Polymerization: Accurate and General RNA-Templated Primer Extension , 2001, Science.

[59]  M. Famulok,et al.  Novel RNA catalysts for the Michael reaction. , 2001, Chemistry & biology.

[60]  A. Jäschke,et al.  A small catalytic RNA motif with Diels-Alderase activity. , 1999, Chemistry & biology.

[61]  Sándor Suhai,et al.  Self-consistent-charge density-functional tight-binding method for simulations of complex materials properties , 1998 .

[62]  T. Tarasow,et al.  RNA-catalysed carbon–carbon bond formation , 1997, Nature.

[63]  T. Darden,et al.  A smooth particle mesh Ewald method , 1995 .

[64]  Jack W. Szostak,et al.  In vitro evolution of a self-alkylatlng ribozyme , 1995, Nature.

[65]  T. Darden,et al.  Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems , 1993 .

[66]  T. Cech,et al.  Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence of tetrahymena , 1982, Cell.

[67]  Maria T. Panteva,et al.  Multiscale methods for computational RNA enzymology. , 2015, Methods in enzymology.

[68]  Efthimios Kaxiras,et al.  A QM/MM Implementation of the Self-Consistent Charge Density Functional Tight Binding (SCC-DFTB) Method , 2001 .

[69]  B. Ganem.  RNA world , 1987, Nature.

[70]  D. Shugar,et al.  Spectrophotometric studies of nucleic acid derivatives and related compounds as a function of pH. I. Pyrimidines. , 1952, Biochimica et biophysica acta.