Structural Investigation of a High‐Affinity Mn^IIBinding Site in the Hammerhead Ribozyme by EPR Spectroscopy and DFT Calculations. Effects of Neomycin B on Metal‐Ion Binding

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 Ke of 4.4 microM 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 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 Aiso(14N)=2.3 MHz. All three EPR parameters are sensitive to the arrangement of the Mn(II) ligand sphere and can therefore be used to determine the structure of the binding site. A possible location for 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 Scott et 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 Iglo-II basis set for all other atoms yielded values of kappa(14N)=+0.80 MHz, eta=0.324, and Aiso(14N)=+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 (Kd=1.2 microM) to the hammerhead ribozyme. Neomycin B has previously been shown to inhibit the catalytic activity of this ribozyme (Uhlenbeck et al., Biochemistry 1995, 34, 11 186).

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

“…Recent results from ESEEM experiments performed by our laboratory using site-specifically labeled RNA have precisely identified this binding site as the A9/G10.1 site [99]. The sole nitrogenous RNA-based ligand identified in earlier studies [94,109] is for the first time conclusively attributed to the N7 from G10.1. Another oxygen atom, likely from the phosphate 5′ to the adjacent adenosine (A9) also binds, making the pseudo-octahedral Mn(II) ion coordinatively saturated and rationalizing the relatively small ZFS constants determined from spectral simulation ( Table 1).…”

Binding of manganese(II) to a tertiary stabilized hammerhead ribozyme as studied by electron paramagnetic resonance spectroscopy

“…The extremely strong binding of the single Mn II prompted us to investigate whether the antibiotic neomycin B is capable of displacing it from the ribozyme, as was found for the single high-affinity Mn II in the mHHRz (Schiemann et al 2003). Figure 2B shows the titration of the tsHHRz/Mn II complex with neomycin B in the presence of 0.1 M NaCl (single nM binding site occupied by a single Mn II ).…”

“…If, however, the manganese ions form a large asymmetric complex, for example, with a protein or RNA, the relaxation times increase and the lines broaden significantly or may even become undetectable (Misra 1996). This effect can be used, as in the case of the mHHRz (Horton et al 1998;Schiemann et al 2003), to quantify the Mn II sites in the tertiary stabilized hammerhead ribozyme (tsHHRz) by monitoring the manganese cw EPR signal intensity while titrating Mn II into the ribozyme solution (pH = 7). The graphical representations of three such titrations are shown in Figure 2A.…”

“…Constructing for both the binding isotherms and fitting them with Equation 2 yielded for the mHHRz a single binding site with a K D of 4 µM. This dissociation constant of 4 µM does not change upon decreasing the NaCl concentration from 1 to 0.1 M, whereas the amount of Mn II binding sites increases from 1 to 4 (Horton et al 1998;Schiemann et al 2003). The fit of the binding isotherm of the tsHHRz yields an upper limit for the dissociation constant of 10 nM (best fit 1 nM) and 16 ± 4 low-affinity sites with K D s of 129 µM ± 31 µM (Fig.…”

“…Especially modern pulsed EPR methods like electron spin echo envelope modulation (ESEEM) (Dikanov and Tsvetkov 1992) and hyperfine sublevel correlation experiments (HYSCORE) (Benetis et al 2002) allow the gathering of information about the type and structure of surrounding ligands of metal centers, whereas pulsed electron double resonance (Schiemann et al 2004) could be used to localize these metal ions in the global context of the RNA fold. However, to be able to apply these methods, the naturally occurring diamagnetic Mg II has to be substituted by the paramagnetic Mn II (S = 5/2, I = 5/2) as done in previous studies on the mHHRz (Horton et al 1998;Schiemann et al 2003). The catalytic activity of RNA is usually reduced in the presence of Mn II despite the fact that both metal ions have some similar physical and chemical properties (Feig 2000).…”