Direct observation of multiple tautomers of oxythiamine and their recognition by the thiamine pyrophosphate riboswitch

Abstract

Structural diversification of canonical nucleic acid bases and nucleotide analogues by tautomerism has been proposed to be a powerful on/off switching mechanism allowing regulation of many biological processes mediated by RNA enzymes and aptamers. Despite the suspected biological importance of tautomerism, attempts to observe minor tautomeric forms in nucleic acid or hybrid nucleic acid-ligand complexes have met with challenges due to the lack of sensitive methods. Here, a combination of spectroscopic, biochemical, and computational tools probed tautomerism in the context of an RNA aptamer-ligand complex; studies involved a model ligand, oxythiamine pyrophosphate (OxyTPP), bound to the thiamine pyrophosphate (TPP) riboswitch (an RNA aptamer) as well as its unbound nonphosphorylated form, oxythiamine (OxyT). OxyTPP, similarly to canonical heteroaromatic nucleic acid bases, has a pyrimidine ring that forms hydrogen bonding interactions with the riboswitch. Tautomerism was established using two-dimensional infrared (2D IR) spectroscopy, variable temperature FTIR and NMR spectroscopies, binding isotope effects (BIEs), and computational methods. All three possible tautomers of OxyT, including the minor enol tautomer, were directly identified, and their distributions were quantitated. In the bound form, BIE data suggested that OxyTPP existed as a 4'-keto tautomer that was likely protonated at the N1'-position. These results also provide a mechanistic framework for understanding the activation of riboswitch in response to deamination of the active form of vitamin B1 (or TPP). The combination of methods reported here revealing the fine details of tautomerism can be applied to other systems where the importance of tautomerism is suspected.

Figure 1

A. Structures of thiamine (T) (top), oxythiamine (OxyT) (middle) and oxythiamine pyrophosphate (OxyTPP) (bottom) B. Secondary structure and sequence of the TPP riboswitch from Arabidosis thialana (adapted from reference (48)) used in the study.

Figure 2

Tautomers of OxyT and their relative Gibbs energies calculated in Gaussian09 using B3LYP functional and 6-31G (d, p) basis set in the presence of explicit water molecules and various implicit environments (vacuum, argon, acetonitrile and water).

Figure 3

A. FTIR spectra of OxyT in TPP buffer taken at various temperatures (5 °C – 90 °C); B. 2D IR spectrum of OxyT under the same solvent condition at 10 °C; C. DFT calculated IR spectra for the three OxyT tautomers using QChem. The frequencies have been scaled by 0.9614; D. Temperature dependence of populations of tautomers of OxyT. The populations were obtained from fitting the variable temperature FTIR spectra (Figure S6). Representative error bars are shown at 90 °C and are roughly independent of temperature.

Figure 4

A. Variable temperature ¹H NMR spectra of oxythiamine in DMF-d₇ (20 °C to -60 °C); B. Variable temperature ¹H NMR spectra of thiamine in DMF-d₇ (20 °C to -60 °C). The thiamine spectra were assigned according to SDBS database (Supplementary material; Note 3).

Figure 5

Structures of OxyTPP showing the numbering and the labeling schemes for different isotopes used for measuring BIEs and the values of 4'-¹⁸O BIEs measured using double labeled OxyTPP and the TPP riboswitch (as shown in Figure 1B).

Figure 6

Binding of the TPP riboswitch to ³³P labeled OxyTPP. The K_d value was measured after 2 hr incubation of binding reactions containing TPP buffer (methods section), 100 nM ³³P labeled OxyTPP and various concentrations of the TPP riboswitch (0.1, 0.5, 1, 5, 10, 30, 50 μM).

Figure 7

Proposed models showing interactions for both enol and keto tautomers of OxyTPP (4'-Enol-OxyTPP and 4'-keto-N1'H-OxyTPP) with the G28 of the TPP riboswitch, that are consistent with the crystal structure of the riboswitch with OxyTPP.(14)