Figure 7. The interaction between K63-Ub2 and A20 ZnF4 at 313 K.
(A, C) NMR titrations of ZnF4 into wild type and mutant K63-Ub2 with 15N-labeling at the distal unit. Residues 50–62 (labeled) experience slow timescale exchange and gradually disappear upon ZnF4 titration. (B, D) Fittings of chemical shift perturbations to binding isotherms. The chemical shift perturbations are calculated as (ΔδH2 + ΔδN2)^0.5 in Hz units. Inset, ZnF4-binding surface on K63-Ub2 distal unit (residues 50–62) is mapped (colored orange).
Figure 7—figure supplement 1. Isothermal calorimetry (ITC) measurements for the bindings between A20 ZnF4 and (A) wild type and (B) E64RP mutant of K63-Ub2 proteins.
The heat exhausted could not be fitted to a binding isotherm.
Figure 7—figure supplement 2. Overlay of NMR spectra for K63-Ub2 and K63-Ub2 mixed with equimolar A20 ZnF4 at (A) 303 K and (B) 313 K.
The protein concentrations are 50 µM. The timescale of the exchange between ZnF4-free and bound species is slow for residues 50–62 of K63-Ub2 distal unit, whose peaks disappear upon ZnF4 titration. At 313 K, the exchange is slightly faster, which allows the fitting of the KD value.