Spectroscopic characterization of Mn2+ and Cd2+ coordination to phosphorothioates in the conserved A9 metal site of the hammerhead ribozyme

J Inorg Biochem. 2022 May:230:111754. doi: 10.1016/j.jinorgbio.2022.111754. Epub 2022 Feb 12.

Abstract

Phosphorothioate modifications have widespread use in the field of nucleic acids. As substitution of sulfur for oxygen can alter metal coordination preferences, the phosphorothioate metal-rescue experiment is a powerful method for identifying metal coordination sites that influence specific properties in a large RNAs. The A9/G10.1 metal binding site of the hammerhead ribozyme (HHRz) has previously been shown to be functionally important through phosphorothioate rescue experiments. While an A9-SRp substitution is inhibitory in Mg2+, thiophilic Cd2+ rescues HHRz activity. Mn2+ is also often used in phosphorothioate metal-rescue studies but does not support activity for the A9-SRp HHRz. Here, we use EPR, electron spin-echo envelope modulation (ESEEM), and X-ray absorption spectroscopic methods to directly probe the structural consequences of Mn2+ and Cd2+ coordination to Rp and Sp phosphorothioate modifications at the A9/G10.1 site in the truncated hammerhead ribozyme (tHHRz). The results demonstrate that while Cd2+ does indeed bind to S in the thio-substituted ligand, Mn2+ coordinates to the non‑sulfur oxo group of this phosphorothioate, regardless of isomer. Computational models demonstrate the energetic preference of MnO over MnS coordination in metal-dimethylthiophosphate models. In the case of the tHHRz, the resulting Mn2+ coordination preference of oxygen in either Rp or Sp A9 phosphorothioates differentially tunes catalytic activity, with MnO coordination in the A9-SRp phosphorothioate enzyme being inhibitory.

Keywords: Computation; DFT; EPR; ESEEM; EXAFS; Hammerhead; Metals; Phosphorothioate; RNA; Ribozyme; Spectroscopy.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Binding Sites
  • Cadmium* / chemistry
  • Metals
  • Nucleic Acid Conformation
  • Oxygen / chemistry
  • RNA, Catalytic* / chemistry
  • RNA, Catalytic* / genetics
  • RNA, Catalytic* / metabolism
  • Sulfur / chemistry

Substances

  • Metals
  • RNA, Catalytic
  • hammerhead ribozyme
  • Cadmium
  • Sulfur
  • Oxygen