Synergistic Recruitment of UbcH7~Ub and Phosphorylated Ubl Domain Triggers Parkin Activation (2018)
Figure 2

Figure 2. Model of the E2-Ub conjugate bound to pUb-activated parkin

Portion of the 1H-15N TROSY NMR spectrum showing signals from R0RBR parkin within the R0RBR:pUb complex (black) and in the presence of one equivalent of the unlabelled UbcH7-Ub conjugate (blue). Signals that shift in the presence of UbcH7-Ub are indicated by arrows. Some signals shift and broaden and cannot be identified in the bound state. Boxes indicate signals that have shifted and are visible at very low contour level.

Two distinct surfaces are revealed on parkin upon binding of the UbcH7-Ub conjugate. Chemical shift perturbations were measured from 1H-15N TROSY spectra of 2H,13C,15N-labelled R0RBR parkin bound to unlabelled pUb in the absence and presence of one equivalent unlabelled UbcH7-Ub. Chemical shift perturbations (absence of signal, average shift + 1 SD) are modelled onto the surface of R0RBR bound to pUb (PDB 5N2W). The UbcH7-Ub binding site comprises the RING1 and IBR regions (magenta). An adjacent site composed of many buried residues was also observed (cyan) that results from E2-Ub binding but does not include the E2 binding site. Sections of the tether and linker regions not visible in the crystal structure were added using Modeller (Eswar et al, 2006) so that chemical shift changes for residues not observed in crystal structures could be mapped.

Model of R0RBR:pUb in complex with UbcH7-Ub derived from NMR chemical shift data and HADDOCK docking (Dominguez et al, 2003). The lowest energy structure is shown and the top 20 structures all showed excellent agreement (RMSD 0.71 ± 0.1Å) although some minor differences were noted for the orientation of the Ub molecule. The structure shows that UbcH7-Ub binds in an open conformation. UbcH7 uses canonical E2-RING E3 interactions that include residues from two loops in the RING1 domain (L1, L2) and two loops in the E2 enzyme (L4, L7) to stabilize the interaction. Ub binds to a RING1/IBR pocket. In this model no attempt was made to move or re-orient any of the domains in parkin.

Effects of ubiquitin surface mutants based on the UbcH7-Ub:R0RBR:pUb model for the ubiquitin loading and off-loading potential of pParkinCH (pParkinC431S,H433A). Coomassie stained gels (top) depict the formation of a pParkinCH ~ Ub oxyester intermediate after 60 minutes in the absence and presence of pUb using the indicated ubiquitin species. Anti-HA blots (bottom) depict the ubiquitination of HA-tagged Miro1181-579 by pParkin after 10 minutes in the absence and presence of pUb using the indicated ubiquitin species. The E24A substitution in Ub is a negative control as this residue faces away from the interface with parkin.

Auto-ubiquitination assay for parkin using ARJP and non-ARJP substitutions in parkin observed near the interface with UbcH7-Ub. Assays were done in the absence and presence of pUb. Experiments with PINK1 were done by treating parkin:pUb with PINK1 for 30 minutes prior to adding other reagents needed for ubiquitination. Assays were monitored using a DyLight-labelled Ub protein and measuring fluorescence at 800 nm.