3) The Structural Basis of Parkin-Mediated Ubiquitination

 

            We have been interested in how the different domains within the parkin protein affect its function. Parkin contains a Ub-like domain (Ubl) at its an N-terminus. We have shown that this Ubl interacts with a subset of UIM and SH3 domain proteins involved in intracellular trafficking (Fallon et al. 2006 Nat. Cell Biol.; Trempe et al. 2009 Mol. Cell). However, many features of the parkin protein remain to be resolved. Based on its sequence, parkin contains several zinc-binding “RING” domains termed RING0, RING1, In-Between-RING (IBR) and RING2. This configuration defines a subset of RING-type E3 Ub ligases called the RBR family. RBR proteins appear to behave as hybrids between classic RING E3s, which function largely as scaffolds connecting the E2 Ub conjugating enzyme with the substrate, and HECT E3s, the other large family of E3 Ub ligases. Similar to HECTs, RBR E3s appear to contain a catalytic cysteine that forms a thioester intermediate with Ub prior to conjugation onto the substrate. Given these unusual features, understanding the structural basis of parkin function will have enormous implications for our capacity to regulate and manipulate its activity in PD. Moreover, parkin E3 activity appears unusually low in the basal state compared to other E3s. In a project funded by Parkinson Society Canada and CIHR, in collaboration with Dr Kalle Gehring at McGill, we used a combination of X-ray crystallography, Nuclear Magnetic Resonance (NMR) and Small-Angle X-Ray Scattering (SAXS) to resolve the structure of full-length parkin (Trempe et al. 2013 Science). The structure shows parkin in an auto-inhibited state (Figure 1). Whereas most PD mutations inactivate parkin (Figure 2), knowing the structure allowed us to design mutations to activate parkin both in vitro and in cells (Figure 3). As parkin is neuroprotective, the structure-based approach provides a framework for enhancing parkin function as a therapeutic strategy in PD.

See movie about the parkin structure: https://www.dropbox.com/s/i4p79zi4rpxela2/ParkinStructureEnglish.mov

 Figure 1

 Figure 2