Figure 4
Characterization of the hDdi2 UBL interaction with UBQ.
(A) 15N/1H-HSQC titration spectra of Ddi2 UBL with addition of a 1-, 2-, 4-, 6-, 8-, or 10-fold molar excess of UBQ. Residues Cys7, Val8, Thr16, Phe17, Val21, Phe25, Phe30, Gln46, Asp70 and Ile73 were used for Kd calculation (0.42–1.1 mM). (B) The mapped interaction site shown on the UBL structure is most likely located in the β-sheet area, according to shifts in Leu3, Cys7, Val8, Thr16, Phe17 and Ile73 upon UBQ binding. Additional shifts in backbone amides observed in the spectra (Val21, Ala23, Phe25, Glu26, Phe30 and Asp70) at the other site of the domain could be the result of a structural change upon binding. Amino acids that could not be used for evaluation are marked black. (C) Titration curves of selected hDdi2 UBL amino acids used for Kd calculation according to the 1:1 stoichiometry model for specific binding. (D) CSP plot showing perturbation at the titration endpoint. Residues not considered in the evaluation are marked with red crosses. (E) 15N/1H-HSQC titration spectra of UBQ with final 6-fold excess of hDdi2 ΔUIM with close-ups of the shifted signals of individual amino acids mapped (F) onto UBQ (PDB entry 1D3Z) (G) Plots of chemical shift perturbations of individual amino acids of UBQ.
