Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases

Abstract

Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson's disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed 'RING/HECT hybrid' enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin's cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein.

Figure 1. Overall Parkin domain organization and RING structures.

(a) Schematic diagram of Parkin indicating linear domain organization and structural domain boundaries. L denotes linker and T, the tether. (b) Overall ribbon diagram of R0RBR (left) and overall surface structure (right). (c) View of individual RING domains. See Supplementary Fig. S2 for further details.

Figure 2. R0RBR is assembled into two compact domain groups separated by linkers.

(a) The R0 (blue) and R1 (green) interface is relatively hydrophilic and separated by the R0–R1 linker (beige), suggesting this area may have some structural flexibility. (b) The tether (beige) residue W403 sits in a hydrophobic pocket on R1 and may serve as a ‘pin’ to anchor the two turn helix of the tether to R1. W403 also forms a hydrogen bond with the terminal carboxylate of V465 (pink). R256 is the site of a human PD mutation. (c) The R0 (blue) domain forms a hydrophobic interface with the catalytic domain R2 (pink), inserting residues W462 and F463 into the hydrophobic core of R0. The catalytic cysteine, C431, is adjacent to this interface.

Figure 3. Catalytic machinery of Parkin.

(a) The activity-probe HA-Ub-VS was incubated with various Parkin constructs (or USP2 control) to determine intrinsic Parkin enzymatic activity. Reactions were allowed to proceed for 3 h, or for the Parkin blot (loading control) samples were removed at time zero (t0) hours and terminated by the addition of SDS-loading dye. For RBR samples, the reaction proceeds so quickly that even at time zero, the reaction is observed. (b) The potential catalytic triad residues C431, H433 and E444 are misaligned. H433 is engaged in a water-mediated hydrogen bond with W462 and is ~5.1 Å from C431. A GG-C431 motif is present (asterisks), which could serve as a classical oxyanion hole during catalysis. (c) Parkin probe reactivity requires elements of a classical catalytic triad. Ub-VS probe reactivity was assessed in various Parkin mutants over a range of pH.

Figure 4. Mitochondrial stress activates Parkin and drives exposure of the active site C431.

(a) Parkin active site mutants C431S/C431A compromise Parkin’s ability to decrease cellular Tom20 levels. Mutations in Parkin’s Zn binding residue C449 also inhibited Parkin’s ability to function in this assay. Full-length wild-type Parkin-induced Tom20 loss after CCCP treatment. DMSO treated was set to 100%. Data shown are representative of three-independent experiments (error bars represents s.e.m.). The significance levels were determined using the heteroscedastic Student’s t-test with two-tailed distribution. Triple asterisk denotes P≤0.005 (C449A, P=0.00006; C431A, P=0.00004; C431S P=0.001). (b) Western blot showing formation of ~8 kDa Parkin immunoreactive species during mitochondrial stress (CCCP) only in cells expressing full-length Parkin C431S. (c) Western blot showing the ~8 kDa Parkin immunoreactive species is sensitive to sodium hydroxide treatment indicative of ubiquitin oxyester formation on full-length Parkin C431S. (d) Enhanced cellular activity of full-length Parkin F463Y compared to full-length wild-type Parkin. Data shown are representative of three-independent experiments (error bars represent s.e.m.). The significance levels were determined using the heteroscedastic Student’s t-test with two-tailed distribution. Triple asterisk denotes P≤0.005 (F463Y, P=0.002). (e) Autoubiquitination of R0RBR F463Y is increased compared with wild-type R0RBR. (f) Increased HA-Ub-VS probe labelling of R0RBR F463Y compared with wild-type R0RBR.

Figure 5. Human genetic PD mutations mapped on Parkin-R0RBR.

(a) Schematic diagram of Parkin-R0RBR indicating residues that can be mutated in PD. (b) R0RBR ribbon representation (left) and space filling model (right) with two 180° views. PD mutations are shown in red and the catalytic cysteine C431 is shown in black. One face of Parkin has a higher number of mutations than the other face. Several areas contain higher densities of mutations, and these regions are circled. These functional regions include the area near the R1:IBR interface, the putative E2-binding site, and the area around the catalytic C431.