Figure 4.

Models of two UIM-2 helices bound to (a) K48-linked Ub₂ and (b) K63-linked Ub₂. Side chains of Ub₂ residues with Δδ > 0.2 ppm are represented by red spheres, while CSPs on UIM-2 (in panel (a)) are colored as follows: Δδ ≥ 0.4 ppm (red), 0.4 ppm > Δδ > 0.125 ppm (orange). These structures were obtained assuming that each UIM-2 binds to the corresponding Ub unit in the chain in the same way/orientation as to monoUb (PDB code 1YX6 ⁸), and using the structures of K48-linked Ub₂ in its complex with UBA2 (PDB: 1ZO6 ²²) and of free K63-linked Ub₂¹⁸ as structural models for the open conformation of the corresponding chain. The resulting structures were obtained by direct superimposition of ubiquitin atoms in the UIM:Ub complex with those for each Ub unit in Ub₂. (c) The same complex as in panel (a), with Ub₂ shown in surface representation, painted red for those sites on both Ub units that show significant CSPs and/or signal attenuations (see Fig.1). (d) NMR characterization of the stoichiometry of Ub₂:S5a_263–307 binding using ¹⁵N longitudinal relaxation rate measurements. The “molecular weight calibration” curve representing the molecular mass dependence of ¹⁵N T₁ in proteins was calibrated as described in ²². The horizontal lines correspond to ¹⁵N T₁ values measured in Ub₂-P at various titration steps, as indicated (see also Table 1); the error bars represent standard deviations in T₁ over core residues in Ub. The dashed vertical lines are “molecular weight markers” indicating the expected mass of the corresponding constructs.