Mechanism of allosteric activation of human mRNA cap methyltransferase (RNMT) by RAM: Insights from accelerated molecular dynamics simulations

Juan Bueren-Calabuig (Lead / Corresponding author), Marcus Bage, Victoria Cowling, Andrei Pisliakov (Lead / Corresponding author)

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Abstract

The RNA guanine-N7 methyltransferase (RNMT) in complex with RNMT-Activating Miniprotein (RAM) catalyses the formation of a N7-methylated guanosine cap structure on the 5’ end of nascent RNA polymerase II transcripts. The mRNA cap protects the primary transcript from exonucleases and recruits cap-binding complexes that mediate RNA processing, export and translation. By using microsecond standard and accelerated molecular dynamics simulations, we provide for the first time a detailed molecular mechanism of allosteric regulation of RNMT by RAM. We show that RAM selects the RNMT active site conformations that are optimal for binding of substrates (AdoMet and the cap), thus enhancing their affinity. Furthermore, our results strongly suggest the likely scenario in which the cap binding promotes the subsequent AdoMet binding, consistent with the previously suggested cooperative binding model. By employing the network community analyses, we revealed the underlying long-range allosteric networks and paths that are crucial for allosteric regulation by RAM. Our findings complement and explain previous experimental data on RNMT activity. Moreover, this study provides the most complete description of the cap and AdoMet binding poses and interactions within the enzyme’s active site. This information is critical for the drug discovery efforts that consider RNMT as a promising anti-cancer target.
Original languageEnglish
Article numbergkz613
Pages (from-to)8675-8692
Number of pages18
JournalNucleic Acids Research
Volume47
Issue number16
DOIs
Publication statusPublished - 19 Sep 2019

Keywords

  • RNMT
  • allosteric activation
  • accelerated molecular dynamics
  • conformational selection
  • mRNA cap
  • Allosteric Regulation
  • Humans
  • Substrate Specificity
  • RNA Caps/chemistry
  • Recombinant Proteins/chemistry
  • Thermodynamics
  • RNA Polymerase II/genetics
  • Methyltransferases/chemistry
  • S-Adenosylmethionine/chemistry
  • Cloning, Molecular
  • Transcription, Genetic
  • Protein Interaction Domains and Motifs
  • Binding Sites
  • Genetic Vectors/chemistry
  • Amino Acid Sequence
  • Protein Conformation, alpha-Helical
  • Gene Expression
  • RNA-Binding Proteins/chemistry
  • S-Adenosylhomocysteine/chemistry
  • Escherichia coli/genetics
  • Molecular Dynamics Simulation
  • Sequence Homology, Amino Acid
  • Sequence Alignment
  • Protein Conformation, beta-Strand
  • Protein Binding
  • Kinetics

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