Figure 9. Activity and interactions of the UBC13/RNF38^RING complex.

(A) The measured dissociation constants of mutants of UBC13/TRAF6^RING complex are given as Mean+/-SD. The difference of free energy of binding was calculated as ΔΔG_binding = RTln(K_d/K_d^wt), where T is 298K, and wt is the wild type complex. (B) In vitro ubiquitination assay was performed using UBC13/Mms2 (or dUBC13/Mms2) and RNF38. (C) Overlay of the ¹⁵N-edited HSQC spectra of free UBC13 (red) with different stoichiometric ratios of RNF38^RING as given in the top left-hand side of the spectra. (D) Regions of the HSQC spectra are expanded to show the UBC13 and dUBC13 peaks during titration with RNF38^RING. (E) The CSPs for each residue in UBC13 upon binding to RNF38^RING. The orange and red dashed lines correspond to Mean + SD and Mean + 2*SD, respectively. The secondary structure alignment of UBC13 against its sequence is provided above the plot. (F) The CSPs for each residue in dUBC13 upon binding to RNF38^RING. The dashed lines are replicated from (E). Significant CSPs were mapped on the UBC13 and dUBC13 structure using both the (G) ribbon and (H) surface representation. The UBC13 and dUBC13 are colored in light blue. The residues with CSPs above Mean + SD and Mean + 2*SD are colored in orange and red, respectively. The RNF38^RING domain is surface rendered and colored in magenta. (I) The measured dissociation constants of UBC13 and its mutants with RNF38^RING domain are given as Mean+/-SD. The difference in binding is calculated as in A).

Figure 9—figure supplement 1. R14-mediated intermolecular salt-bridges observed in crystallographic complexes of UBC13 with RING domains of other E3s.

Dashed red circles are indicative of intermolecular salt-bridges involving R14 in UBC13. RNF4 and RNF8 lack R14-mediated intermolecular salt-bridges.

Figure 9—figure supplement 2. Analysis of UBC13/RNF38^RING structure and charge distribution at and near the interface of RNF38/TRAF6^RING.

(A) Surface electrostatic potential reveals a lack of salt-bridge acceptors at the RNF38^RING interface (circled region). Color scale varies from −8 to +8 k_BT/e. (B) Negatively charged residues near the interface of RNF38^RING. (C) Negatively charged residues at/near the interface of TRAF6^RING. (D) The CSPs for residues in RNF38^RING upon binding to UBC13. The black dashed lines correspond to Mean + SD and Mean + 2*SD, respectively. The secondary structure alignment of RNF38^RING against its sequence is provided above the plot. A couple of amide resonances at the helix α2 broadened during titration, which is marked by asterisk. (E) The NMR data-driven model of UBC13/RNF38^RING complex calculated by HADDOCK (Dominguez et al., 2003). (F) The interface is zoomed in, and the R14/M417 contact at the interface is shown by the red line. The Zn atoms in RNF38 (magenta) and coordinating Cysteines/Histidines are also displayed.