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Acta Cryst. (2014). A70, C306
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The degradation of many short-lived proteins in eukaryotic cells is carried out by the Ubiquitin Proteasome System. The N-end rule pathway links the half-life of proteins to the identity of its N-terminal residue, also called N-degron. Destabilizing N-degrons, are recognized by E3 ubiquitin ligases termed N-recognins. N-degrons are grouped into type 1, composed of basic residues, and type 2, composed of bulky hydrophobic residues. In mammals, four N-recognins mediate the N-end rule pathway: UBR1, UBR2, UBR4 and UBR5. These proteins share a ~70-residue zinc finger-like motif termed the Ubiquitin Recognin (UBR) box, responsible for their specificity. The mammalian genome encodes at least three more UBR-box proteins: UBR3, UBR6/FBXO11 and UBR7. However, these UBRs cannot recognize any type of N-degrons. Our lab reported the crystal structures of the UBR boxes from the human UBR1 and UBR2, rationalizing the empirical rules for the classification of type 1 N-degrons. Despite the valuable information obtained from those structures there is not a clear explanation for the no recognition of N-degrons by other UBR-box proteins. Here we report the crystal structure of the UBR-box domain from UBR6 also known as FBXO11. UBR6 is a F-box protein of the SKP1-Cullin1-F-box (SCF) ubiquitin ligase complex and does not recognize any type of N-degrons. We crystallized a 77-residue fragment of the UBR-box of UBR6 and determined its structure at 1.7 Å resolution. Unexpectedly, this domain adopts an open conformation compared to UBR1-box, without any N-degron binding pockets. Its zinc-binding residues are conserved as in the N-recognins, but they are arranged in different zinc-binding motifs. Molecules form dimmers stabilized by zinc ions. The crystal had 4 molecules per asymmetric unit and space group P212121. For phasing we used Zn-SAD. With this structure we hope to obtain clues that explain the absence of N-degron recognition in some members of the UBR family.

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Acta Cryst. (2014). A70, C838
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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 cysteine- rich 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.

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Acta Cryst. (2014). A70, C845
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Mutations in the Parkin gene are responsible for an autosomal recessive form of Parkinson's disease. The parkin protein is a RING1-In-Between-RING2 (RBR) E3 ubiquitin ligase, which functions through a two-step mechanism involving a parkin~ubiquitin thioester intermediate [1]. However, compared to other ubiquitin ligases, parkin exhibits low basal activity and requires activation both in vitro and in cells. As parkin is neuroprotective in various models of Parkinson's disease, understanding how it catalyses ubiquitin transfer will be critical. We previously reported the crystal structure of full-length parkin [2]. The structure shows parkin in an auto-inhibited state and provides insight into how it is activated. The RING0 domain occludes the ubiquitin acceptor site Cys431 in RING2 whereas a novel Repressor Element of Parkin (REP) binds RING1 and blocks its E2-binding site. Remarkably, mutations that disrupt these inhibitory interactions activate parkin both in vitro and in cells. The structure also reveals that His433 and Glu444 form a catalytic dyad adjacent to Cys431. Here, we show that His433 catalyses the acyl transfer of ubiquitin carboxy terminus from Cys431 to a target lysine side-chain amino group. Mutation of His433 does not affect UbcH7~ubiquitin discharging or thioester intermediate formation, but prevents formation of polyubiquitin chains on parkin. However, mutation of His433 does not affect significantly parkin's mitochondrial recruitment and substrate ubiquitination, suggesting that other factors might be at play in vivo. We also investigate the catalytic role of other residues located around the Cys431, such as Trp462. The work provides insight into the mechanism of ubiquitination by RBR E3 ligases with important implications for Parkinson's disease.
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