Figure 7.

Structure of the Ub:Ddi1UBL complex. (A) NMR-based mapping of the Ub:Ddi1UBL interface. Left: Ddi1UBL-binding site (colored green) on the surface of Ub (orange). Right: Ub-binding site (orange) on the surface of Ddi1UBL (green). CSPs > 0.075 ppm were used for mapping in both cases. (B) HADDOCK-derived structure of the complex of Ub (orange) and Ddi1UBL (green). (C) The Ub:Ddi1UBL interface is formed by both hydrophobic and polar/charged amino acids (shown as spheres colored orange and blue, respectively, for Ub, green and red for Ddi1UBL). Ribbon colors are as in (A–B). (D) Specific side-chain contacts at the interface of the Ub:Ddi1UBL complex. Hydrophobic amino acids of Ub: L8, I44, V70 (orange) and Ddi1: I13, L70, L72 (green) are shown as spheres. Shown in stick representation are K6, R42, H68, R72 of Ub (pink) and D2, E8, D68 of Ddi1UBL (green) that form polar/electrostatic contacts (shown as yellow dash lines). To distinguish between Ub and Ddi1UBL residues, the latter are indicated in italic. (E, F) Validation of the Ub:Ddi1UBL complex by measurements at physiological ionic strength and by site-directed mutagenesis. (E) Amide CSPs in Ddi1UBL at the endpoint of titration with WT Ub at 0 mM NaCl (top) and at 150 mM NaCl (center), and in the presence of 4-fold molar excess of Ub^{K6A,R42A,R72A}, also at 150 mM NaCl (bottom). (F) Overlay of the NMR signals of Tyr14 of Ddi1UBL free in solution and upon addition of WT Ub or indicated Ub mutants, at [Ub]:[UBL]=4 and 150 mM NaCl. (See also Fig S7)