Structural investigation of a high-affinity Mn-II binding site in the hammerhead ribozyme by EPR spectroscopy and DFT calculations. Effects of neomycin B on metal-ion binding

Olav Schiemann, J Fritscher, N Kisseleva, ST Sigurdsson, TF Prisner

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57 Citations (Scopus)

Abstract

Electron paramagnetic resonance spectroscopy and density functional theory methods were used to study the structure of a single, high-affinity Mn-II binding site in the hammerhead ribozyme. This binding site exhibits a dissociation constant K-d of 4.4 muM in buffer solutions containing 1 M NaCl, as shown by titrations monitored by continuous wave (cw) EPR. a combination of electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) experiments revealed that the <LF>paramagnetic manganese(II) ion in this binding site is coupled to a single nitrogen atom with a quadrupole coupling constant kappa of 0.7 MHz, an asymmetry parameter eta of 0.4, and an isotropic hyperfine coupling constant of A(iso)(N-14) = 2.3 MHz. All three EPR parameters are sensitive to the arrangement of MNII ligand sphere and can therefore be used to determine the structure of the binding site. A possible location of this binding site may be at the G10.1, A9 site found to be occupied by Mn-II in crystals (MacKay et al., Nature 1994, 372, 68 and Scottet al., Science 1996, 274, 2065). To determine whether the structure of the binding site is the same in frozen solution, we performed DFT calculations for the EPR parameters, based on the structure of the Mn-II site in the crystal. Computations with the BHPW91 density function in combination with a 9s7p4d basis set for the manganese (ii) center and the Igio-II basis set for all other atoms yielded values of kappa((1)4N) = +0.80 MHz, eta - 0.324, and A(iso) (N-14) = +2.7 MHz, in excellent agreement with the experimentally obtained EPR parameters, which suggests that the binding site found in the crystal and in frozen solution are the same. In addition, we demonstrated by EPR that Mn-II is released from this site upon binding of the aminoglycoside antibiotic neomycin B (K-d = 1.2 muM) to the hammerhead ribozyme. Neomycin B has previouisly been shown to inhibit the catalytic activity of this ribozyme.

Original languageEnglish
Pages (from-to)1057-1065
Number of pages9
JournalChemBioChem
Volume4
Issue number10
DOIs
Publication statusPublished - 6 Oct 2003

Keywords

  • antibiotics
  • density functional calculations
  • EPR spectroscopy
  • manganese
  • RNA
  • RESONANCE ENERGY-TRANSFER
  • HAIRPIN RIBOZYME
  • GLOBAL CONFORMATION
  • CATALYTIC RNA
  • ELECTRON-GAS
  • DENSITY
  • INHIBITION
  • EXCHANGE
  • NMR
  • IDENTIFICATION

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