Interaction Of The Parkin UBL Domain With SH3-containing Proteins Involved In Synaptic Vesicle Endocytosis: Structure And Role In Protein Ubiquitination

Abstract

Mutations in the ubiquitin ligase Parkin have been associated with the development of autosomal recessive juvenile Parkinsonism (ARJP). Mutations have been found in the RING domains, but also in the N-terminal ubiquitin-like (UBL) domain. In an effort to identify protein substrates implicated in ARJP, we identified Endophilin-A as a ligand of the rat Parkin-UBL domain. Endophilin-A is a BAR-domain containing protein that induces membrane curvature and is implicated in synaptic vesicle endocytosis and recycling. Parkin-UBL interacts directly with the Endophilin-A C-terminal SH3 domain with an affinity of 10-15 uM. The interaction is specific as ubiquitin, Nedd8 and the Plic1-UBL domain do not interact with the SH3 domain. The Parkin-UBL domain only interacts with a subset of SH3 domains. We determined the crystal structure of rat Endophilin-A1 SH3 domain at 1.4 Angstrom resolution. Using NMR spectroscopy, we mapped the protein-protein interaction surfaces on both the rat UBL and SH3 domains. Using our SH3 structure and the crystal structure of murine Parkin-UBL, we calculated a docking model of the UBL:SH3 complex using NMR chemical shift perturbations and residual dipolar couplings. The UBL surface consists of the hydrophobic patch located around Ile44 as well as the basic C-terminal tail of the UBL domain, which is major specificity determinant. Using single-site mutagenesis, we identified key specificity and affinity determinants in the SH3 domain that explains that specificity of Parkin-UBL towards different SH3 domains. The SH3 surface involved in binding the UBL domain is centered around an invariant proline previously shown to be involved in proline-rich domain (PRD) binding. Parkin-UBL effectively competes with synaptojanin PRD for binding Endophilin-A1 SH3 domain. Finally, we show that the UBL:SH3 interaction is required for Endophilin-A ubiquitination by Parkin.