Fig. 6. Amide ¹⁵N relaxation dispersion experiments reveal msec-μsec conformational exchange in the ETS domain that is dampened by the CID.

(A) ΔR₂eff values for ETV6^R426′ from relaxation dispersion experiments with ν_CPMG of 50 Hz versus 1000 Hz, recorded using an 850 MHz NMR spectrometer. The cartoon representation of the secondary structure of ETV6^R426 is also given with coloring as in Figure 2; however, it is unknown if helix H4 remains stably folded in this variant. (B) When mapped on the predicted structure of ETV6^R426′, residues with an arbitrary cut-off of ΔR₂eff > 5 s⁻¹ (orange) or ΔR₂eff > 10 s⁻¹ (green) are broadly distributed through the ETS domain with some bias towards to the DNA-binding interface (centered around helix H3; see Fig. 7). In contrast, such exchange broadening is not seen for ETV6^D446′ (Supplemental Fig. S6) or ETV6^R458 (not shown) with an intact CID. (C) Complete relaxation dispersion profiles recorded with 500 MHz (blue boxes) and 850 MHz (green circles) spectrometers for uninhibited ETV6^R426′ (left) and inhibited ETV6^D446′ (right). Both Ala393 (helix H3) and Thr418 (between strand S4 and helix H4) show exchange broadening in ETV6^R426′ but not ETV6^D446′. Also presented is a control amide in Trp356 (strand S1) showing no dispersion in either protein. Error bars are smaller than the symbols, and the lines are best fits to a two-state exchange model.