Figure 6. NMR Titrations and Mapping of Interaction Interfaces in E4B U-box, UbcH5c and Ubc4.

(A, B) Superposition of the ¹H-¹⁵N HSQC titration spectra of ¹⁵N-labeled E4B U-box from free (black) to the bound (red) states with nonlabeled Ubc4 (A) and UbcH5c (B). The arrows indicate the directions of the titrations.

(C) In the crystal structure of E4B U-box and UbcH5c complex, E4B residues affected by UbcH5c titration are colored red (signals with chemical shift changes ≥ 0.02 ppm) or blue (weakened or disappeared signals, namely those of Asp1234, Leu1236, Asp1238, Leu1247, Ile1258, Asn1271 and Arg1272). The active site Cys85 is shown in yellow.

(D, E) Superposition of the ¹H-¹⁵N HSQC titration spectra of ¹⁵N-labeled Ubc4 (D) or UbcH5c (E) from the free (black) to the bound (red) states with nonlabeled E4B U-box. The arrows indicate the directions of the titrations.

(F) In the crystal structure of E4B U-box and UbcH5c complex, UbcH5c residues affected by E4B U-box titration are colored red (signals with chemical shift changes from 0.03 to 0.06 ppm) or blue (chemical shift changes from 0.07 to 0.16 ppm). The active site Cys85 is shown in yellow.

(G) UbcH5c residues distant from the binding interface with E4B U-box and for which changes in chemical shifts were detected are shown in gray. The active site Cys85 is shown in yellow.