Véronique Sauvé

Structure of Parkin Reveals Mechanisms for Ubiquitin Ligase Activation

Parkin Enhanced? Inactivation of parkin, an E3 ubiquitin ligase, is responsible for a familial form of Parkinsons disease and may be involved in sporadic forms as well. Trempe et al. (p. 1451, published online 9 May) present the crystal structure of full-length parkin in an autoinhibited configuration. Guided by the structure, mutations were designed that activated parkin both in vitro and in cells. Because parkin is neuroprotective, the structure provides a framework for enhancing parkin function as a therapeutic strategy in Parkinsons disease. The complete structure of a protein linked to Parkinson’s disease suggests how to activate it. Mutations in the PARK2 (parkin) gene are responsible for an autosomal recessive form of Parkinson’s disease. The parkin protein is a RING-in-between-RING E3 ubiquitin ligase that exhibits low basal activity. We describe the crystal structure of full-length rat parkin. The structure shows parkin in an autoinhibited state and provides insight into how it is activated. RING0 occludes the ubiquitin acceptor site Cys431 in RING2, whereas a repressor element of parkin binds RING1 and blocks its E2-binding site. Mutations that disrupted these inhibitory interactions activated parkin both in vitro and in cells. Parkin is neuroprotective, and these findings may provide a structural and mechanistic framework for enhancing parkin activity.

A Ubl/ubiquitin switch in the activation of Parkin

Mutations in Parkin and PINK1 cause an inherited early‐onset form of Parkinsons disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto‐inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho‐ubiquitin (pUb) and the ubiquitin‐like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86–130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin‐conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding.

The SoxYZ Complex Carries Sulfur Cycle Intermediates on a Peptide Swinging Arm.

The bacterial Sox (sulfur oxidizing) system allows the utilization of inorganic sulfur compounds in energy metabolism. Central to this process is the SoxYZ complex that carries the pathway intermediates on a cysteine residue near the C terminus of SoxY. Crystal structures have been determined for Paracoccus pantotrophus SoxYZ with the carrier cysteine in the underivatized state, conjugated to the polysulfide mimic β-mercaptoethanol, and as the sulfonate adduct pathway intermediate. The carrier cysteine is located on a peptide swinging arm and is bracketed on either side by diglycine dipeptides acting as molecular universal joints. This structure provides a novel solution to the requirement that the cysteine-bound intermediates be able to access and orient themselves within the active sites of multiple partner enzymes. Adjacent to the swinging arm there is a conserved, deep, apolar pocket into which the β-mercaptoethanol adduct extends. This pocket would be well suited to a role in protecting labile pathway intermediates from adventitious reactions.

Structure of the 16S rRNA pseudouridine synthase RsuA bound to uracil and UMP.

In Escherichia coli, the pseudouridine synthase RsuA catalyzes formation of pseudouridine (ψ) at position 516 in 16S rRNA during assembly of the 30S ribosomal subunit. We have determined the crystal structure of RsuA bound to uracil at 2.0 Å resolution and to uridine 5′-monophosphate (UMP) at 2.65 Å resolution. RsuA consists of an N-terminal domain connected by an extended linker to the central and C-terminal domains. Uracil and UMP bind in a cleft between the central and C-terminal domains near the catalytic residue Asp 102. The N-terminal domain shows structural similarity to the ribosomal protein S4. Despite only 15% amino acid identity, the other two domains are structurally similar to those of the tRNA-specific ψ-synthase TruA, including the position of the catalytic Asp. Our results suggest that all four families of pseudouridine synthases share the same fold of their catalytic domain(s) and uracil-binding site.

Mechanism for the Hydrolysis of a Sulfur-Sulfur Bond Based on the Crystal Structure of the Thiosulfohydrolase SoxB

SoxB is an essential component of the bacterial Sox sulfur oxidation pathway. SoxB contains a di-manganese(II) site and is proposed to catalyze the release of sulfate from a protein-bound cysteine S-thiosulfonate. A direct assay for SoxB activity is described. The structure of recombinant Thermus thermophilus SoxB was determined by x-ray crystallography to a resolution of 1.5 Å. Structures were also determined for SoxB in complex with the substrate analogue thiosulfate and in complex with the product sulfate. A mechanistic model for SoxB is proposed based on these structures.

Phosphorylated ubiquitin: a new shade of PINK1 in Parkin activation.

The Parkinsons disease (PD)-associated proteins, Parkin and PINK1, together comprise a mitochondrial quality control pathway that promotes neuronal survival through autophagy of damaged mitochondria. Three recent studies have found that Parkin recruitment to mitochondria and ubiquitin ligase activity is controlled by the phosphorylation of ubiquitin by PINK1.

Mechanism of parkin activation by phosphorylation

Mutations in the ubiquitin ligase parkin are responsible for a familial form of Parkinson’s disease. Parkin and the PINK1 kinase regulate a quality-control system for mitochondria. PINK1 phosphorylates ubiquitin on the outer membrane of damaged mitochondria, thus leading to recruitment and activation of parkin via phosphorylation of its ubiquitin-like (Ubl) domain. Here, we describe the mechanism of parkin activation by phosphorylation. The crystal structure of phosphorylated Bactrocera dorsalis (oriental fruit fly) parkin in complex with phosphorylated ubiquitin and an E2 ubiquitin-conjugating enzyme reveals that the key activating step is movement of the Ubl domain and release of the catalytic RING2 domain. Hydrogen/deuterium exchange and NMR experiments with the various intermediates in the activation pathway confirm and extend the interpretation of the crystal structure to mammalian parkin. Our results rationalize previously unexplained Parkinson’s disease mutations and the presence of internal linkers that allow large domain movements in parkin.Crystal structures of activated, phosphorylated fly parkin in complex with phosphorylated ubiquitin and human UbcH7 reveal large domain movements enabled by the parkin’s internal linkers. Results also explain some Parkinson’s disease mutations.

Publisher Correction: Mechanism of parkin activation by phosphorylation

In the version of this article initially published, RING2 in the schematic to the left in Fig. 1b was mislabeled as RING0. The error has been corrected in the HTML and PDF versions of the article.

Deciphering the activation of the E3 ubiquitin ligase parkin

Parkin is an E3 ubiquitin ligase responsible for some autosomal recessive forms of Parkinson’s disease. Even though parkin is a RINGtype E3 ligase, it uses a hybrid RING/HECT mechanism for its activity. The crystal structures of full-length and the RING0-RING1-InBetween-RING-RING2 module of parkin reveal a conformation of parkin in which its E2 binding site is too far from its catalytic cysteine for the transfer of ubiquitin [1]. Many intramolecular interactions occur between the different RING domains, as well as with a repressor element, which, with RING0, are unique to parkin. Mutations of residues involved in those interactions lead to an increase of parkin activity. This suggests that parkin adopts an auto-inhibited state in basal conditions. Therefore, under stress-response conditions, parkin needs to undergo molecular rearrangements, modulated by post-translational modification and/or interactions with other proteins, to become active. The phosphorylation of serine 65 in the Ubl domain of parkin by Pink1, a kinase also found mutated in some Parkinson’s patient, was shown to increase the activity of parkin. Recent publications have demonstrated that ubiquitin is also phosphorylated by Pink1 and, furthermore, that phosphorylated ubiquitin could activate parkin [2,3]. We have used different techniques of structural biology and protein-protein interactions to further characterize the interaction of phosphorylated ubiquitin with parkin. This work provides insight into the mechanism of activation of parkin and that causes Parkinsons disease.

Structure of parkin reveals the mechanism of autoinhibition

Mutations in the gene park2 that codes for a RING-In-Between-RING (RBR) E3 ubiquitin ligase are responsible for an autosomal recessive form of Parkinson’s disease (PD). Compared to other ubiquitin ligases, the parkin protein exhibits low basal activity and requires activation both in vitro and in cells. Parkin is a 465-residue E3 ubiquitin ligase promoting mitophagy of damaged mitochondria. Parkin has two RING motifs RING1 and RING2 linked by a cysteinerich in-between-RING (IBR) motif, a recently identified zinc-coordinating motif termed RING0, and an N-terminal ubiquitin-like domain (Ubl). It is believed that parkin may function as a RING/HECT hybrid, where ubiquitin is first transferred by the E2 enzyme onto parkin active cysteine and then to the substrate. Here, we report the crystal structure of full-length parkin at low resolution. This structure shows parkin in an auto-inhibited state and provides insight into how it is activated. In the structure RING0 occludes the ubiquitin acceptor site Cys431 in RING2 whereas a novel repressor element of parkin (REP) binds RING1 and blocks its E2-binding site. The ubiquitin-like domain (Ubl) binds adjacent to the REP through the hydrophobic surface centered around Ile44 and regulate parkin activity. Mutagenesis and NMR titrations verified interactions observed in the crystal. We also proposed the putative E2 binding site on RING1 and confirmed it by mutagenesis and NMR titrations. Importantly, mutations that disrupt these inhibitory interactions activate parkin both in vitro and in cells. The structure of the E3-ubiquitin ligase provides insights into how pathological mutations affect the protein integrity. Current work is directed towards obtaining high-resolution structure of full-length parkin in complex with E2 and substrates. The results will lead to new therapeutic strategies for treating and ultimately preventing PD.