Mechanistic characterization of the 5′-triphosphate-dependent activation of PKR: Lack of 5′-end nucleobase specificity, evidence for a distinct triphosphate binding site, and a critical role for the dsRBD

The ATP binding site has high affinity: ITC experiments. ITC titration curves for NTP binding to K296R. (A) GTP (green trace), UTP (blue trace), and CTP (orange trace) binding to K296R. Titration of each NTP into buffer is included (black traces). NTP traces are offset from buffer traces by ∼0.1 μcal/sec in raw data (upper panels) for clarity. Legends are provided in plots. (B) ATP binding to K296R (red trace). Titration of ATP into buffer is included (black trace). ATP titration curve is fitted to a two-site binding model (see Materials and Methods), and thermodynamic parameters are provided to the right of the plot. The major contribution is with site 1 (n = 1.32 ± 0.09) and a K_d of 19 ± 1.8 μM. Note that uncertainty in ΔH is due to the lack of a good lower baseline, which is common with micromolar K_d experiments. However, this does not affect the K_d value because that is mainly based on the slope of the transition region.