Supplementary Figure 2: Effect of phosphorylation and ATP titration on p38α.

(a) Overlay of the annotated Leu/Val methyl ¹H-¹³C SOFAST-HMQC spectra of p38α-2P in the absence (black) and presence (red) of 5 mM ATP analog. Large chemical shift changes are indicated by blue arrows. (b) Left, center: Overlay of the Ile(δ1) and Leu/Val methyl ¹H-¹³C SOFAST-HMQC spectra of 20 μM [ILV-methyl] p38α-2P in the presence (red) and absence (black) of 5 mM ATP. Large chemical shift changes in the Ile(δ1) region are indicated by blue arrows. Spectra were measured with an 800 MHz ¹H resonance frequency spectrometer at 10 °C. Right: Methyl sites that exhibited chemical shift changes larger than the linewidth or were not assigned only in the ATP-bound state are indicated as red spheres on the crystal structure (PDB code: 1A9U⁵¹). The ATP-binding site is highlighted in the orange oval. (c) Overlay of the annotated Leu/Val methyl ¹H-¹³C SOFAST-HMQC spectra of p38α-2P (blue) and unphosphorylated p38α (black) in the apo state. (d) Comparison of the Ile(δ1) and Leu/Val methyl regions of ¹H-¹³C HMQC spectra of 80 μM [ILVM-methyl] unphosphorylated p38α, in the presence (green) and absence (red) of 4 mM ATP. Spectra were measured at 800 MHz (¹H frequency) at 25 °C. For the methyl resonances of Leu and Val residues, only those dispersed in the spectra are labeled, for clarification. (e) Met methyl regions of ¹H-¹³C HMQC spectra of unphosphorylated p38α titrated with various concentration of ATP. The resonance of Met109, which was traced during the titration to determine the affinity of unphosphorylated p38α for ATP, is expanded as an inset, in which all of the titration points are overlaid. The dissociation constant of unphosphorylated p38α for ATP was estimated to be larger than 15 mM, based on the CSPs of the Met109 methyl ¹H and ¹³C resonances. Since the chemical shift changes did not reach a plateau at the highest ATP concentration (15 mM), a unique dissociation constant was not obtained.