Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin-like domain

Abstract

Parkin, a product of the causative gene of autosomal-recessive juvenile parkinsonism (AR-JP), is a RING-type E3 ubiquitin ligase and has an amino-terminal ubiquitin-like (Ubl) domain. Although a single mutation that causes an Arg to Pro substitution at position 42 of the Ubl domain (the Arg 42 mutation) has been identified in AR-JP patients, the function of this domain is not clear. In this study, we determined the three-dimensional structure of the Ubl domain of parkin by NMR, in particular by extensive use of backbone (15)N-(1)H residual dipolar-coupling data. Inspection of chemical-shift-perturbation data showed that the parkin Ubl domain binds the Rpn10 subunit of 26S proteasomes via the region of parkin that includes position 42. Our findings suggest that the Arg 42 mutation induces a conformational change in the Rpn10-binding site of Ubl, resulting in impaired proteasomal binding of parkin, which could be the cause of AR-JP.

Figure 1

The solution structure of the ubiquitin-like (Ubl) domain of parkin. (A) Stereo view of ten converged structures of the parkin Ubl domain. (B) Ribbon representation of the average structure. β-strands and α-helices are coloured yellow and pink, respectively, in (A) and (B). Numbers in (A) and (B) indicate amino-acid positions in the Ubl domain sequence. (C) Sequence alignment and secondary-structure elements of the parkin Ubl domain and ubiquitin.

Figure 2

Identification of the binding site for Rpn10_196–306 in the parkin ubiquitin-like (Ubl) domain. (A) ¹H-¹⁵N heteronuclear single-quantum coherence (HSQC) spectrum of the parkin Ubl domain in the presence (red) and absence (black) of equimolar quantities of Rpn10_196–306. The peaks labelled with L-2, G-1 and S0 originate from the amino-terminal tag. (B) NMR chemical-shift-perturbation data for the parkin Ubl domain. The data are displayed for each residue according to the equation (0.2 δ_N² + δ_H²)^1/2, where δ_N and δ_H represent the change in nitrogen and proton chemical shifts on addition of Rpn10_196–306. Asterisks indicate residues the peaks of which became undetectable due to broadening. Secondary structure elements for the parkin Ubl are shown below the graph. (C) Mapping of the perturbed residues of the Ubl domains of parkin and PLIC2 (Walters et al., 2002) on binding to Rpn10. Residues showing a chemicalshift-perturbation are coloured in red, with the colour gradient indicating the strength of the perturbation. Residues the peaks of which became undetectable on binding to Rpn10 are shown in purple.