Figure 7. 2D-HMQC NMR confirms R-state selection by extracellular signal-regulated kinase (ERK) inhibitors.

2D-HMQC NMR spectra were collected on 2P-ERK2 at 25°C and 5°C. Effects on [methyl ¹³C,¹H] peaks of residues (A) I72, (B) L220, and (C) L242 are shown for the representative set of inhibitors in Figure 5. Inhibitors #5, #6, #8, and #16 (shown in blue) shifted the R↔L equilibrium to 100% R at all temperatures, confirming R-state selection as suggested by their HDX behaviors in Figure 5 and Figure 6. Inhibitors #4 and #15 (in gray) retained R:L populations comparable to those with GDC0994. Inhibitor #1 (in cyan) showed partial selection for the R state but retained conformational exchange, as evidenced by an L-state population present at 5 °C. Full NMR spectra are shown in Figure 7—figure supplement 1 and Figure 7—figure supplement 2.

Figure 7—figure supplement 1. 2D-HMQC NMR spectra of inhibitor-bound 2P-ERK2.

2D [¹³C,¹H] HMQC spectra for 2P-ERK2:Inh#5, 2P-ERK2:Inh#6, 2P-ERK2:Inh#8, 2P-ERK2:Inh#16. Each complex was formed from 150 μM extracellular signal-regulated kinase-2 (ERK2) and 180 μM inhibitor.

Figure 7—figure supplement 2. 2D-HMQC NMR spectra of inhibitor-bound 2P-ERK2.

2D [¹³C,¹H] HMQC spectra for 2P-ERK2:Inh#1, 2P-ERK2:Inh#4, and 2P-ERK2:Inh#15. Each complex was formed from 150 μM extracellular signal-regulated kinase-2 (ERK2) and 180 μM inhibitor.