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.

Fig. 1.

Shown are 600 MHz ¹H-¹⁵N HSQC spectra of Parkin IBR^307–384. (a) Folded 0.2 mM IBR^307–384 in 25 mM phosphate buffer (pH 6.95), 100 mM NaCl, 1 mM DTT at 25°C. (b) Spectrum of IBR^307–384 from a upon EDTA addition showing the protein becomes unfolded. Peaks resulting from the leader sequence, an artifact of the cloning process, are indicated by an asterisk.

Fig. 2.

The structure of the Parkin IBR^307–384 domain. (a) Backbone superposition of 20 structures having the lowest target function from CYANA. The superposition used residues G329–C332, C337–C352, and C360–S377 resulting in a backbone rmsd of 0.41 ± 0.09 Å. Zinc-binding site I comprises the extended loop regions L1 (blue) and L2 (green), and zinc site II is shown in red. (b) Ribbon diagram of Parkin IBR showing the position of the coordinating zinc residues. The unstructured N- (307–319) and C-terminal (378–384) regions are not shown.

Fig. 3.

Conserved zinc coordination and core regions of IBR domains. (a) A ribbon structure showing the packing of conserved core hydrophobic residues in Parkin. The structure shows residues V330, A339, and L341 (L1) pack with residues V350 and F364 (L2) allowing formation of a scissor-like arrangement. Two peripheral residues, P343 and R366 provide auxiliary interactions whereas Y372 helps stabilize site II to site I. (b) Multiple sequence alignment of IBR domains from several human paralogs constructed from ClustalW (43) and Jalview (44). Underlined proteins interact with human UbcH7 and UbcH8, whereas RNF14 interacts with UbcH7 alone. Conserved metal binding ligands (yellow) and hydrophobic core residues (green) are highlighted. Residues where mutations have been found in ARJP (open stars) and mutated in this study (filled stars) are indicated.

Fig. 4.

Selected regions of the 600 MHz ¹H NMR spectra of Parkin IBR^307–384 and missense mutations found in patients suffering from ARJP. Residues are labeled for the downfield amide region (a), aromatic region (b), and upfield aliphatic methyl proton region (c). Spectra were scaled to provide approximately equal peak intensities. A residual peak from the internal standard DSS is indicated with an asterisk.

Fig. 5.

Proposed Parkin interactions and arrangement of IBR and RING domains. Surface representation of the IBR structure showing the positions of ARJP mutations (G328E, R334C, V380L) is shown in red. Two of these mutations (G328E, R334C), and the conserved residue L331 (blue) lie along the L1 loop and result in decreased binding and ubiquitination of substrates such as synphilin-1 and p38. Other substrates are listed based on biochemical experiments that indicate interaction with either the RING1–IBR or IBR–RING2 domain pairs. Residues where S-nitrosylation is proposed (C323) or phosphorylation has been identified (S378) are shown in yellow. These residues, along with the missense mutation site V380 lie at the crown of the IBR domain where the N and C termini are close in space bringing the RING1 and RING2 domains within close proximity to each other and the IBR domain.