Structure of the Parkin in-between-ring domain provides insights for E3-ligase dysfunction in autosomal recessive Parkinson's disease

Abstract

Mutations in Parkin are one of the predominant hereditary factors found in patients suffering from autosomal recessive juvenile Parkinsonism. Parkin is a member of the E3 ubiquitin ligase family that is defined by a tripartite RING1-in-between-ring (IBR)-RING2 motif. In Parkin, the IBR domain has been shown to augment binding of the E2 proteins UbcH7 and UbcH8, and the subsequent ubiquitination of the proteins synphilin-1, Sept5, and SIM2. To facilitate our understanding of Parkin function, the solution structure of the Parkin IBR domain was solved by using NMR spectroscopy. Folding of the IBR domain (residues M327-S378) was found to be zinc dependent, and the structure reveals the domain forms a unique pair scissor-like and GAG knuckle-like zinc-binding sites, different from other zinc-binding motifs such as the RING, LIM, PHD, or B-box motifs. The N terminus of the IBR domain, residues E307-E322, is unstructured. The disease causing mutation T351P causes global unfolding, whereas the mutation R334C causes some structural rearrangement of the domain. In contrast, the protein containing the mutation G328E appears to be properly folded. The structure of the Parkin IBR domain, in combination with mutational data, allows a model to be proposed where the IBR domain facilitates a close arrangement of the adjacent RING1 and RING2 domains to facilitate protein interactions and subsequent ubiquitination.ubiquitination ͉ zinc-binding ͉ NMR spectroscopy ͉ protein folding ͉ protein interactions P arkinson's disease (PD) is a common neurodegenerative disease characterized by damage to the dopaminergic neurons of the substantia nigra of the midbrain manifesting itself with symptoms such as tremors, rigidity, and bradykinesia (1, 2). Autosomal recessive juvenile PD (ARJP) is an early-onset (Ͻ40 years of age) form of the disease that is caused by hereditary factors including mutations in the genes DJ-1, PINK1, and in about half of the cases, parkin. The protein Parkin has been identified as an E3 ubiquitin-protein ligase of the ubiquitin-proteosome system that is required to maintain cellular protein quality control by removing misfolded or damaged proteins (3, 4). Ubiquitin mediated proteolysis occurs through a cascade of ubiquitin transfers from an E1 activating enzyme, to an E2 conjugating enzyme and finally in complex with an E3 ligase to the target substrate (5, 6). At least 40 different missense and deletion mutations in the parkin gene have been identified and correlated to dysfunction of the ubiquitination system, manifesting itself as ARJP due to the loss of the dopaminergic neurons (7).Parkin is a 465-residue protein comprising an N-terminal ubiquitin-like domain, a unique Parkin-specific domain in the central region, and two RING finger domains (RING1, RING2) separated by an in-between ring (IBR) or double ring linked domain near its C terminus. The cysteine and histidine rich RING-IBR-RING (RBR) domain architecture is highly conserved and found only in eukaryotes. To date, all characterized proteins containing the ...

RBR Ubiquitin Ligases: Diversification and Streamlining in Animal Lineages

“…The second domain, known as IBR, typically has a C 6 HC signature and is present only in RBR proteins. Recent structural data have shown that the IBR domain in fact consists of two consecutive zinc-binding domains, respectively, based on C 4 and C 2 HC ligands (Beasley et al 2007). Finally, the third, C-terminal domain, often called RING2, resembles a RING finger (e.g., it also contains a C 3 HC 4 signature).…”

Identification of a Novel Zn2+-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin

“…Of these, the only experimental evidence exists for the IBR domain that has been shown to coordinate two Zn 2ϩ ions (23). The RING2 domain of the RBR family member HHARI has been observed to coordinate only a single Zn 2ϩ ion, although it was originally thought to ligate two metal ions (36).…”

“…A New Domain in Parkin Identified by V8 and Trypsin Limited Proteolysis-Sequence similarity and alignment methods have identified the N-terminal UblD (1-75) and C-terminal RBR domains (238 -465) in parkin, whereas the central UPD (76 -238) conforms to no known domain sequence or structural motif (22,46). However, to date only the UblD and IBR domains (23,38,39) of parkin have actually been purified and characterized by protein chemistry methods. The purification of parkin now allows a more comprehensive examination of the remaining domain architecture within this E3 ligase protein, possibly establishing new domains and/or refining theoretical boundaries.…”

“…The currently accepted domain architecture of parkin, deduced from multiple sequence alignment, shows that the C terminus of the protein is characterized by two ϳ50-residue RING (really interesting new gene) domains separated by a 51-residue IBR (In-Between-RING) domain (22,23). The RING domains of parkin are proposed to interact with the ubiquitinconjugating enzymes UbcH7, UbcH8, Ubc7, and Ubc13 and control parkin-mediated ubiquitination of a variety of substrates such as Pael-R, synphilin-1, Sept5, and PICK1 among others (24 -31).…”

“…These mutations would be expected to alter Zn 2ϩ binding and have been shown to decrease parkin E3 ligase activity, indicating that Zn 2ϩ coordination is crucial for protein function. In addition, removal of Zn 2ϩ ions by EDTA addition to the IBR domain (23) or the HHARI RING2 domain (36) causes complete unfolding of these domains. In light of this, we investigated the effect of EDTA-induced Zn 2ϩ removal on the secondary structure of parkin by CD spectropolarimetry.…”