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Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography	Mechanism of parkin activation by PINK1 Christina Gladkova , Sarah Maslen , J. Mark Skehel , David Komander Nature DOI: 10.1038/s41586-018-0224-x Diamond Proposal Number(s): [15916] Show Abstract ▼ Abstract: Mutations in the E3 ubiquitin ligase parkin (PARK2) and the protein kinase PINK1 (PARK6) are linked to autosomal-recessive juvenile Parkinsonism (AR-JP)1,2, and at the cellular level cause defects in mitophagy, the cellular process that organises destruction of damaged mitochondria3,4. Parkin is autoinhibited, and requires activation by PINK1, which phosphorylates Ser65 in ubiquitin and in the parkin ubiquitin-like (Ubl) domain. Parkin binds phospho-ubiquitin, which enables efficient Parkin phosphorylation; however, the enzyme remains autoinhibited with an inaccessible active site5,6. It is unclear how phosphorylation of parkin activates the molecule. Here we follow the activation of full-length human parkin by hydrogen deuterium exchange mass spectrometry, and reveal large-scale domain rearrangement in the activation process, in which the phospho-Ubl rebinds to the parkin core, and releases the catalytic RING2 domain. A 1.8 Å crystal structure of phosphorylated human parkin reveals the binding site of the phosphorylated Ubl on the unique parkin domain (UPD), involving a phosphate-binding pocket lined by AR-JP mutations. Strikingly, a conserved linker region between Ubl and UPD acts as an activating element (ACT) that contributes to RING2 release by mimicking RING2 interactions on the UPD, explaining further AR-JP mutations. Our data unveil how autoinhibition in parkin is resolved, and suggest how parkin ubiquitinates its substrates via an untethered RING2 domain. This opens exciting new avenues to design parkin activators for clinical use.	Jun 2018
I04-Macromolecular Crystallography	An invisible ubiquitin conformation is required for efficient phosphorylation by PINK1 Christina Gladkova , Alexander F. Schubert , James Wagstaff , Jonathan N. Pruneda , Stefan M. V. Freund , David Komander The Embo Journal DOI: 10.15252/embj.201797876 Diamond Proposal Number(s): [15916] Show Abstract ▼ Abstract: The Ser/Thr protein kinase PINK1 phosphorylates the well‐folded, globular protein ubiquitin (Ub) at a relatively protected site, Ser65. We previously showed that Ser65 phosphorylation results in a conformational change in which Ub adopts a dynamic equilibrium between the known, common Ub conformation and a distinct, second conformation wherein the last β‐strand is retracted to extend the Ser65 loop and shorten the C‐terminal tail. We show using chemical exchange saturation transfer (CEST) nuclear magnetic resonance experiments that a similar, C‐terminally retracted (Ub‐CR) conformation also exists at low population in wild‐type Ub. Point mutations in the moving β5 and neighbouring β‐strands shift the Ub/Ub‐CR equilibrium. This enabled functional studies of the two states, and we show that while the Ub‐CR conformation is defective for conjugation, it demonstrates improved binding to PINK1 through its extended Ser65 loop, and is a superior PINK1 substrate. Together our data suggest that PINK1 utilises a lowly populated yet more suitable Ub‐CR conformation of Ub for efficient phosphorylation. Our findings could be relevant for many kinases that phosphorylate residues in folded protein domains.	Nov 2017
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Structure of PINK1 in complex with its substrate ubiquitin Alexander Schubert , Christina Gladkova , Els Pardon , Jane L. Wagstaff , Stefan M. V. Freund , Jan Steyaert , Sarah L. Maslen , David Komander Nature DOI: 10.1038/nature24645 Show Abstract ▼ Abstract: Autosomal recessive juvenile Parkinsonism (AR-JP) is caused by mutations in a number of PARK genes, in particular in the E3 ubiquitin ligase Parkin (PARK2), and in its upstream protein kinase PINK1 (PARK6). PINK1 phosphorylates ubiquitin and the Parkin ubiquitin-like domain on structurally protected Ser65 to trigger mitophagy. We here report a crystal structure of a nanobody-stabilised complex between Pediculus humanus corporis (Ph)PINK1 with ubiquitin in the ‘C-terminally retracted’ (Ub-CR) conformation. The structure reveals many peculiarities of PINK1, including the architecture of the C-terminal region, and reveals how the PINK1 N-lobe binds ubiquitin via a unique insertion. The flexible Ser65-loop in the Ub-CR conformation reaches the activation segment, facilitating placement of Ser65 in a phosphate accepting position. The structure further explains how autophosphorylation in the N-lobe stabilises structurally and functionally important insertions, and reveals the molecular basis for AR-JP causing mutations, some of which disrupt ubiquitin binding.	Oct 2017
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Mechanism and regulation of the Lys6-selective deubiquitinase USP30 Malte Gersch , Christina Gladkova , Alexander F. Schubert , Martin A. Michel , Sarah Maslen , David Komander Nature Structural & Molecular Biology DOI: 10.1038/nsmb.3475 Show Abstract ▼ Abstract: Damaged mitochondria undergo mitophagy, a specialized form of autophagy that is initiated by the protein kinase PINK1 and the ubiquitin E3 ligase Parkin. Ubiquitin-specific protease USP30 antagonizes Parkin-mediated ubiquitination events on mitochondria and is a key negative regulator of mitophagy. Parkin and USP30 both show a preference for assembly or disassembly, respectively, of Lys6-linked polyubiquitin, a chain type that has not been well studied. Here we report crystal structures of human USP30 bound to monoubiquitin and Lys6-linked diubiquitin, which explain how USP30 achieves Lys6-linkage preference through unique ubiquitin binding interfaces. We assess the interplay between USP30, PINK1 and Parkin and show that distally phosphorylated ubiquitin chains impair USP30 activity. Lys6-linkage-specific affimers identify numerous mitochondrial substrates for this modification, and we show that USP30 regulates Lys6-polyubiquitinated TOM20. Our work provides insights into the architecture, activity and regulation of USP30, which will aid drug design against this and related enzymes.	Sep 2017

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