Publications by authors named "Gary S Shaw"

81 Publications

Calcium binds and rigidifies the dysferlin C2A domain in a tightly coupled manner.

Biochem J 2021 Jan;478(1):197-215

Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1.

The membrane protein dysferlin (DYSF) is important for calcium-activated plasma membrane repair, especially in muscle fibre cells. Nearly 600 mutations in the DYSF gene have been identified that are causative for rare genetic forms of muscular dystrophy. The dysferlin protein consists of seven C2 domains (C2A-C2G, 13%-33% identity) used to recruit calcium ions and traffic accessory proteins and vesicles to injured membrane sites needed to reseal a wound. Amongst these, the C2A is the most prominent facilitating the calcium-sensitive interaction with membrane surfaces. In this work, we determined the calcium-free and calcium-bound structures of the dysferlin C2A domain using NMR spectroscopy and X-ray crystallography. We show that binding two calcium ions to this domain reduces the flexibility of the Ca2+-binding loops in the structure. Furthermore, calcium titration and mutagenesis experiments reveal the tight coupling of these calcium-binding sites whereby the elimination of one site abolishes calcium binding to its partner site. We propose that the electrostatic potential distributed by the flexible, negatively charged calcium-binding loops in the dysferlin C2A domain control first contact with calcium that promotes subsequent binding. Based on these results, we hypothesize that dysferlin uses a 'calcium-catching' mechanism to respond to calcium influx during membrane repair.
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http://dx.doi.org/10.1042/BCJ20200773DOI Listing
January 2021

Recruitment of Ubiquitin within an E2 Chain Elongation Complex.

Biophys J 2020 04 15;118(7):1679-1689. Epub 2020 Feb 15.

Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada. Electronic address:

The ubiquitin (Ub) proteolysis pathway uses an E1, E2, and E3 enzyme cascade to label substrate proteins with ubiquitin and target them for degradation. The mechanisms of ubiquitin chain formation remain unclear and include a sequential addition model, in which polyubiquitin chains are built unit by unit on the substrate, or a preassembly model, in which polyubiquitin chains are preformed on the E2 or E3 enzyme and then transferred in one step to the substrate. The E2 conjugating enzyme UBE2K has a 150-residue catalytic core domain and a C-terminal ubiquitin-associated (UBA) domain. Polyubiquitin chains anchored to the catalytic cysteine and free in solution are formed by UBE2K supporting a preassembly model. To study how UBE2K might assemble polyubiquitin chains, we synthesized UBE2K-Ub and UBE2K-Ub covalent complexes and analyzed E2 interactions with the covalently attached Ub and Ub moieties using NMR spectroscopy. The UBE2K-Ub complex exists in multiple conformations, including the catalytically competent closed state independent of the UBA domain. In contrast, the UBE2K-Ub complex takes on a more extended conformation directed by interactions between the classic I44 hydrophobic face of the distal Ub and the conserved MGF hydrophobic patch of the UBA domain. Our results indicate there are distinct differences between the UBE2K-Ub and UBE2K-Ub complexes and show how the UBA domain can alter the position of a polyubiquitin chain attached to the UBE2K active site. These observations provide structural insights into the unique Ub chain-building capacity for UBE2K.
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http://dx.doi.org/10.1016/j.bpj.2020.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136282PMC
April 2020

The Zn and Ca -binding S100B and S100A1 proteins: beyond the myths.

Biol Rev Camb Philos Soc 2020 06 6;95(3):738-758. Epub 2020 Feb 6.

Department of Biochemistry, University of Western Ontario, London, Ontario, N6A5C1, Canada.

The S100 genes encode a conserved group of 21 vertebrate-specific EF-hand calcium-binding proteins. Since their discovery in 1965, S100 proteins have remained enigmatic in terms of their cellular functions. In this review, we summarize the calcium- and zinc-binding properties of the dimeric S100B and S100A1 proteins and highlight data that shed new light on the extracellular and intracellular regulation and functions of S100B. We point out that S100B and S100A1 homodimers are not functionally interchangeable and that in a S100A1/S100B heterodimer, S100A1 acts as a negative regulator for the ability of S100B to bind Zn . The Ca and Zn -dependent interactions of S100B with a wide array of proteins form the basis of its activities and have led to the derivation of some initial rules for S100B recognition of protein targets. However, recent findings have strongly suggested that these rules need to be revisited. Here, we describe a new consensus S100B binding motif present in intracellular and extracellular vertebrate-specific proteins and propose a new model for stable interactions of S100B dimers with full-length target proteins. A chaperone-associated function for intracellular S100B in adaptive cellular stress responses is also discussed. This review may help guide future studies on the functions of S100 proteins in general.
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http://dx.doi.org/10.1111/brv.12585DOI Listing
June 2020

Programmed ubiquitin acetylation using genetic code expansion reveals altered ubiquitination patterns.

FEBS Lett 2020 Apr 18;594(7):1226-1234. Epub 2019 Dec 18.

Department of Biochemistry, The University of Western Ontario, London, Canada.

Ubiquitination is a post-translational modification (PTM) capable of being regulated by other PTMs, including acetylation. However, the biological consequences of acetylated ubiquitin (acUb) variants are poorly understood, due to their transient nature in vivo and poor characterization in vitro. Since Ub is known to be acetylated in human cells, we produced all possible acUb variants using genetic code expansion. We also developed a protocol that optimizes acetyl-lysine addition to minimize mistranslated proteins and maximize site-specific acUb protein production. Purified acUb proteins were used in pilot ubiquitination assays and found to be competent with IpaH3CT and RNF8 E3 ligases. Overall, this work provides an optimized method to express and purify all acetyl-lysine variants for ubiquitin and shows these proteins can be used to identify potential unique ubiquitination patterns.
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http://dx.doi.org/10.1002/1873-3468.13702DOI Listing
April 2020

Optimized transformation, overexpression and purification of S100A10.

Biotechniques 2019 11 2;67(5):246-248. Epub 2019 Sep 2.

Department of Ophthalmology, Faculty of Medicine, Université Laval, Quebec, QC, Canada.

As a member of the S100 protein family, S100A10 has already been purified. However, its purity, or even yield, have often not been reported in the literature. To facilitate future biophysical experiments with S100A10, we aimed to obtain it at a purity of at least 95% in a reasonably large amount. Here, we report optimized conditions for the transformation, overexpression and purification of the protein. We obtained a purity of 97% and performed stability studies by circular dichroism. Our data confirmed that the S100A10 obtained is suitable for experiments to be performed at room temperature up to several days.
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http://dx.doi.org/10.2144/btn-2019-0081DOI Listing
November 2019

The mammalian CTLH complex is an E3 ubiquitin ligase that targets its subunit muskelin for degradation.

Sci Rep 2019 07 8;9(1):9864. Epub 2019 Jul 8.

Robarts Research Institute, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada.

The multi-subunit C-terminal to LisH (CTLH) complex is the mammalian homologue of the yeast Gid E3 ubiquitin ligase complex. In this study, we investigated the human CTLH complex and characterized its E3 ligase activity. We confirm that the complex immunoprecipitated from human cells comprises RanBPM, ARMC8 α/β, muskelin, WDR26, GID4 and the RING domain proteins RMND5A and MAEA. We find that loss of expression of individual subunits compromises the stability of other complex members and that MAEA and RMND5A protein levels are interdependent. Using in vitro ubiquitination assays, we demonstrate that the CTLH complex has E3 ligase activity which is dependent on RMND5A and MAEA. We report that the complex can pair with UBE2D1, UBE2D2 and UBE2D3 E2 enzymes and that recombinant RMND5A mediates K48 and K63 poly-ubiquitin chains. Finally, we show a proteasome-dependent increase in the protein levels of CTLH complex member muskelin in RMND5A KO cells. Furthermore, muskelin ubiquitination is dependent on RMND5A, suggesting that it may be a target of the complex. Overall, we further the characterization of the CTLH complex as an E3 ubiquitin ligase complex in human cells and reveal a potential autoregulation mechanism.
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http://dx.doi.org/10.1038/s41598-019-46279-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614414PMC
July 2019

A subset of calcium-binding S100 proteins show preferential heterodimerization.

FEBS J 2019 05 21;286(10):1859-1876. Epub 2019 Feb 21.

Department of Biochemistry, The University of Western Ontario, London, Canada.

The assembly of proteins into dimers and oligomers is a necessary step for the proper function of transcription factors, muscle proteins, and proteases. In uncontrolled states, oligomerization can also contribute to illnesses such as Alzheimer's disease. The S100 protein family is a group of dimeric proteins that have important roles in enzyme regulation, cell membrane repair, and cell growth. Most S100 proteins have been examined in their homodimeric state, yet some of these important proteins are found in similar tissues implying that heterodimeric molecules can also be formed from the combination of two different S100 members. In this work, we have established co-expression methods in order to identify and quantify the distribution of homo- and heterodimers for four specific pairs of S100 proteins in their calcium-free states. The split GFP trap methodology was used in combination with other GFP variants to simultaneously quantify homo- and heterodimeric S100 proteins in vitro and in living cells. For the specific S100 proteins examined, NMR, mass spectrometry, and GFP trap experiments consistently show that S100A1:S100B, S100A1:S100P, and S100A11:S100B heterodimers are the predominant species formed compared to their corresponding homodimers. We expect the tools developed here will help establish the roles of S100 heterodimeric proteins and identify how heterodimerization might alter the specificity for S100 protein action in cells.
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http://dx.doi.org/10.1111/febs.14775DOI Listing
May 2019

Monitoring Interactions Between S100B and the Dopamine D2 Receptor Using NMR Spectroscopy.

Methods Mol Biol 2019 ;1929:311-324

Department of Biochemistry, The University of Western Ontario, London, ON, Canada.

S100B is a dimeric EF-hand protein that undergoes a calcium-induced conformational change and interacts with a wide range of proteins to modulate their functions. The dopamine D2 receptor is one potential S100B binding partner that may play a key role in neurological processing. In this chapter, we describe the use of NMR spectroscopy to examine the interaction between calcium-bound S100B and the third intracellular loop (IC3) from the dopamine D2 receptor. We provide details that allow the strength of the interaction (K ) between the two proteins to be determined and the IC3 site of interaction on the structure of S100B to be identified. Both these characteristics can be identified from a single series of nondestructive experiments.
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http://dx.doi.org/10.1007/978-1-4939-9030-6_20DOI Listing
June 2019

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation.

EMBO J 2018 12 16;37(23). Epub 2018 Nov 16.

Department of Biochemistry, The University of Western Ontario, London, ON, Canada

The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early-onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N-terminal ubiquitin-like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho-ubiquitin-loaded C-terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re-modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity.
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http://dx.doi.org/10.15252/embj.2018100014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276879PMC
December 2018

Correction to "Calcium-Mediated Control of S100 Proteins: Allosteric Communication via an Agitator/Signal Blocking Mechanism".

J Am Chem Soc 2018 07 5;140(28):8998. Epub 2018 Jul 5.

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http://dx.doi.org/10.1021/jacs.8b06612DOI Listing
July 2018

Regulation of Shigella Effector Kinase OspG through Modulation of Its Dynamic Properties.

J Mol Biol 2018 07 16;430(14):2096-2112. Epub 2018 May 16.

Department of Biochemistry, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada; Department of Biochemistry, McGill University, Montreal, Quebec, Canada. Electronic address:

Gram-negative pathogens secrete effector proteins into human cells to modulate normal cellular processes and establish a bacterial replication niche. Shigella and pathogenic Escherichia coli possess homologous effector kinases, OspG and NleH1/2, respectively. Upon translocation, OspG but not NleH binds to ubiquitin and a subset of E2~Ub conjugates, which was shown to activate its kinase activity. Here we show that OspG, having a minimal kinase fold, acquired a novel mechanism of regulation of its activity. Binding of the E2~Ub conjugate to OspG not only stimulates its kinase activity but also increases its optimal temperature for activity to match the human body temperature and stabilizes its labile C-terminal domain. The melting temperature (T) of OspG alone is only 31 °C, as compared to 41 °C to NleH1/2 homologs. In the presence of E2~Ub, the T of OspG increases to ~42 °C, while Ub by itself increases the T to 39 °C. Moreover, OspG alone displays maximal activity at 26 °C, while in the presence of E2~Ub, maximal activity occurs at ~42 °C. Using NMR and molecular dynamics calculations, we have identified the C-terminal lobe and, in particular, the C-terminal helix, as the key elements responsible for lower thermal stability of OspG as compared to homologous effector kinases.
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http://dx.doi.org/10.1016/j.jmb.2018.05.015DOI Listing
July 2018

Impact of altered phosphorylation on loss of function of juvenile Parkinsonism-associated genetic variants of the E3 ligase parkin.

J Biol Chem 2018 04 12;293(17):6337-6348. Epub 2018 Mar 12.

From the Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada

Autosomal recessive juvenile Parkinsonism (ARJP) is an inherited neurodegenerative disease in which 50% of affected individuals harbor mutations in the gene encoding the E3 ligase parkin. Parkin regulates the mitochondrial recycling pathway, which is induced by oxidative stress. In its native state, parkin is auto-inhibited by its N-terminal ubiquitin-like (Ubl) domain, which blocks the binding site for an incoming E2∼ubiquitin conjugate, needed for parkin's ubiquitination activity. Parkin is activated via phosphorylation of Ser-65 in its Ubl domain by PTEN-induced putative kinase 1 (PINK1) and a ubiquitin molecule phosphorylated at a position equivalent to Ser-65 in parkin. Here we have examined the underlying molecular mechanism of phosphorylation of parkin's Ubl domain carrying ARJP-associated substitutions and how altered phosphorylation modulates parkin activation and ubiquitination. We found that three substitutions in the Ubl domain (G12R, R33Q, and R42P) significantly decrease PINK1's ability to phosphorylate the Ubl domain. We noted that two basic loss-of-function substitutions (R33Q and R42P) are close to acidic patches in the proposed PINK1-parkin interface, indicating that ionic interactions at this site may be important for efficient parkin phosphorylation. Increased auto-ubiquitination with unique ubiquitin chain patterns was observed for two other Ubl domain substitutions (G12R and T55I), suggesting that these substitutions, along with phosphorylation, increase parkin degradation. Moreover, Ubl domain phosphorylation decreased its affinity for the potential effector protein ataxin-3, which edits ubiquitin chain building by parkin. Overall, our work provides a framework for the mechanisms of parkin's loss-of-function, indicating an interplay between ARJP-associated substitutions and phosphorylation of its Ubl domain.
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http://dx.doi.org/10.1074/jbc.RA117.000605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5925814PMC
April 2018

Cytochrome c as a Peroxidase: Activation of the Precatalytic Native State by HO-Induced Covalent Modifications.

J Am Chem Soc 2017 11 30;139(44):15701-15709. Epub 2017 Oct 30.

Department of Chemistry and Department of Biochemistry, The University of Western Ontario , London, Ontario N6A 5B7, Canada.

In addition to serving as respiratory electron shuttle, ferri-cytochrome c (cyt c) acts as a peroxidase; i.e., it catalyzes the oxidation of organic substrates by HO. This peroxidase function plays a key role during apoptosis. Typical peroxidases have a five-coordinate heme with a vacant distal coordination site that permits the iron center to interact with HO. In contrast, native cyt c is six-coordinate, as the distal coordination site is occupied by Met80. It thus seems counterintuitive that native cyt c would exhibit peroxidase activity. The current work scrutinizes the origin of this structure-function mismatch. Cyt c-catalyzed peroxidase reactions show an initial lag phase that is consistent with the in situ conversion of a precatalyst to an active peroxidase. Using mass spectrometry, we demonstrate the occurrence of cyt c self-oxidation in the presence of HO. The newly generated oxidized proteoforms are shown to possess significantly enhanced peroxidase activity. HO-induced modifications commence with oxidation of Tyr67, followed by permanent displacement of Met80 from the heme iron. The actual peroxidase activation step corresponds to subsequent side chain carbonylation, likely at Lys72/73. The Tyr67-oxidized/carbonylated protein has a vacant distal ligation site, and it represents the true peroxidase-active structure of cyt c. Subsequent self-oxidation eventually causes deactivation. It appears that this is the first report that identifies HO-induced covalent modifications as an essential component for the peroxidase activity of "native" cyt c.
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http://dx.doi.org/10.1021/jacs.7b07106DOI Listing
November 2017

Calcium-Mediated Control of S100 Proteins: Allosteric Communication via an Agitator/Signal Blocking Mechanism.

J Am Chem Soc 2017 08 14;139(33):11460-11470. Epub 2017 Aug 14.

Department of Chemistry, The University of Western Ontario , London, Ontario N6A 5B7, Canada.

Allosteric proteins possess dynamically coupled residues for the propagation of input signals to distant target binding sites. The input signals usually correspond to "effector is present" or "effector is not present". Many aspects of allosteric regulation remain incompletely understood. This work focused on S100A11, a dimeric EF-hand protein with two hydrophobic target binding sites. An annexin peptide (Ax) served as the target. Target binding is allosterically controlled by Ca over a distance of ∼26 Å. Ca promotes formation of a [Ca S100 Ax] complex, where the Ax peptides are accommodated between helices III/IV and III'/IV'. Without Ca these binding sites are closed, precluding interactions with Ax. The allosteric mechanism was probed by microsecond MD simulations in explicit water, complemented by hydrogen exchange mass spectrometry (HDX/MS). Consistent with experimental data, MD runs in the absence of Ca and Ax culminated in target binding site closure. In simulations on [Ca S100] the target binding sites remained open. These results capture the essence of allosteric control, revealing how Ca prevents binding site closure. Both HDX/MS and MD data showed that the metalation sites become more dynamic after Ca loss. However, these enhanced dynamics do not represent the primary trigger of the allosteric cascade. Instead, a labile salt bridge acts as an incessantly active "agitator" that destabilizes the packing of adjacent residues, causing a domino chain of events that culminates in target binding site closure. This agitator represents the starting point of the allosteric signal propagation pathway. Ca binding rigidifies elements along this pathway, thereby blocking signal transmission. This blocking mechanism does not conform to the commonly held view that allosteric communication pathways generally originate at the sites where effectors interact with the protein.
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http://dx.doi.org/10.1021/jacs.7b04380DOI Listing
August 2017

Ubiquitin phosphorylated at Ser57 hyper-activates parkin.

Biochim Biophys Acta Gen Subj 2017 Nov 6;1861(11 Pt B):3038-3046. Epub 2017 Jul 6.

Department of Biochemistry, The University of Western Ontario, London, ON N6A 5C1, Canada; Department of Chemistry, The University of Western Ontario, London, ON N6A 5C1, Canada. Electronic address:

Malfunction of the ubiquitin (Ub) E3 ligase, parkin, leads to defects in mitophagy and protein quality control linked to Parkinson's disease. Parkin activity is stimulated by phosphorylation of Ub at Ser65 (pUb). Since the upstream kinase is only known for Ser65 (PINK1), the biochemical function of other phosphorylation sites on Ub remain largely unknown. We used fluorescently labelled and site-specifically phosphorylated Ub substrates to quantitatively relate the position and stoichiometry of Ub phosphorylation to parkin activation. Fluorescence measurements show that pUb-stimulated parkin is 5-fold more active than auto-inhibited and un-stimulated parkin, which catalyzes a basal level of auto-ubiquitination. We consistently observed a low but detectable level of parkin activity with pUb. Strikingly, pUb hyper-activates parkin, and our data demonstrate that parkin is able to selectively synthesize poly-pUb chains, even when 90% of the Ub in the reaction is un-phosphorylated. We further found that parkin ubiquitinates its physiological substrate Miro-1 with chains solely composed of pUb and more efficiently with pUb chains. Parkin hyper-activation by pUb demonstrates the first PINK1-independent route to active parkin, revealing the roles of multiple ubiquitin phosphorylation sites in governing parkin stimulation and catalytic activity. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.
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http://dx.doi.org/10.1016/j.bbagen.2017.06.023DOI Listing
November 2017

RBR Ubiquitin Transfer: Not Simply an "Open" and "Closed" Case?

Structure 2017 06;25(6):817-819

Department of Biochemistry, The University of Western Ontario, London, ON N6A 5C1, Canada. Electronic address:

The three distinct types of E3 ubiquitin ligases, RING, HECT, and RBR, employ different modes of ubiquitin transfer including E2∼Ub conjugate type and conformation. In this issue of Structure, Dove et al. (2017) provide a structural rationale for the preference and conformation of the UbcH7∼Ub conjugate by the RBR E3 ligase HHARI.
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http://dx.doi.org/10.1016/j.str.2017.05.013DOI Listing
June 2017

Parkin-phosphoubiquitin complex reveals cryptic ubiquitin-binding site required for RBR ligase activity.

Nat Struct Mol Biol 2017 May 17;24(5):475-483. Epub 2017 Apr 17.

MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, UK.

RING-between-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR ligase is Parkin, mutations in which lead to early-onset hereditary Parkinsonism. Parkin and other RBR ligases share a catalytic RBR module but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during Parkin activation. However, current data on active RBR ligases reflect the absence of regulatory domains. Therefore, it remains unclear how individual RBR ligases are activated, and whether they share a common mechanism. We now report the crystal structure of a human Parkin-phosphoubiquitin complex, which shows that phosphoubiquitin binding induces movement in the 'in-between RING' (IBR) domain to reveal a cryptic ubiquitin-binding site. Mutation of this site negatively affects Parkin's activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, which suggests a role for interdomain association in the RBR ligase mechanism.
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http://dx.doi.org/10.1038/nsmb.3400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5420311PMC
May 2017

Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation.

Proc Natl Acad Sci U S A 2017 01 22;114(2):298-303. Epub 2016 Dec 22.

Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, ON N6A 3K7, Canada

Mutations in PARK2 and PARK6 genes are responsible for the majority of hereditary Parkinson's disease cases. These genes encode the E3 ubiquitin ligase parkin and the protein kinase PTEN-induced kinase 1 (PINK1), respectively. Together, parkin and PINK1 regulate the mitophagy pathway, which recycles damaged mitochondria following oxidative stress. Native parkin is inactive and exists in an autoinhibited state mediated by its ubiquitin-like (UBL) domain. PINK1 phosphorylation of serine 65 in parkin's UBL and serine 65 of ubiquitin fully activate ubiquitin ligase activity; however, a structural rationale for these observations is not clear. Here, we report the structure of the phosphorylated UBL domain from parkin. We find that destabilization of the UBL results from rearrangements to hydrophobic core packing that modify its structure. Altered surface electrostatics from the phosphoserine group disrupt its intramolecular association, resulting in poorer autoinhibition in phosphorylated parkin. Further, we show that phosphorylation of both the UBL domain and ubiquitin are required to activate parkin by releasing the UBL domain, forming an extended structure needed to facilitate E2-ubiquitin binding. Together, the results underscore the importance of parkin activation by the PINK1 phosphorylation signal and provide a structural picture of the unraveling of parkin's ubiquitin ligase potential.
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http://dx.doi.org/10.1073/pnas.1613040114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240717PMC
January 2017

Generation of phospho-ubiquitin variants by orthogonal translation reveals codon skipping.

FEBS Lett 2016 05 4;590(10):1530-42. Epub 2016 May 4.

Department of Biochemistry, The University of Western Ontario, London, Canada.

The activity of the Parkinson's disease-linked E3 ligase parkin is stimulated by phosphorylation at ubiquitin Ser65 (pUb(S65) ). The role of other ubiquitin phospho-sites and their kinases are unknown. We produced pUb variants (pS7, pS12, pS20, pS57, pS65) by genetically encoding phosphoserine with the UAG codon. In release factor-deficient Escherichia coli (ΔRF1), intended to enhance UAG read-through, we discovered ubiquitin variants lacking the UAG-encoded residue, demonstrating previously undocumented +3 frame shifting. We successfully purified each pUb variant from mistranslated products. While pUb(S20) failed to stimulate parkin, parkin was partially active with pUb(S12) . We observed significant ubiquitination when pUb(S65) was the sole substrate.
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http://dx.doi.org/10.1002/1873-3468.12182DOI Listing
May 2016

Suramin inhibits cullin-RING E3 ubiquitin ligases.

Proc Natl Acad Sci U S A 2016 Apr 21;113(14):E2011-8. Epub 2016 Mar 21.

Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, New York, NY 10029; Jiangsu Center for the Collaboration and Innovation of Cancer Biotherapy, Cancer Institute, Xuzhou Medical College, Xuzhou, 221002, China;

Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3's cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1's conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2-E3 interface through small-molecule modulators.
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http://dx.doi.org/10.1073/pnas.1601089113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833235PMC
April 2016

Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis.

EMBO J 2015 Oct 7;34(20):2506-21. Epub 2015 Aug 7.

MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK

The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N-terminal ubiquitin-like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin-binding interface, provide a model for the phosphoubiquitin-parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin-binding site used by the E2~Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2~Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin.
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http://dx.doi.org/10.15252/embj.201592337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4609183PMC
October 2015

The HIP2~ubiquitin conjugate forms a non-compact monomeric thioester during di-ubiquitin synthesis.

PLoS One 2015 23;10(3):e0120318. Epub 2015 Mar 23.

Department of Biochemistry, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada, N6A 5C1.

Polyubiquitination is a post-translational event used to control the degradation of damaged or unwanted proteins by modifying the target protein with a chain of ubiquitin molecules. One potential mechanism for the assembly of polyubiquitin chains involves the dimerization of an E2 conjugating enzyme allowing conjugated ubiquitin molecules to be put into close proximity to assist reactivity. HIP2 (UBE2K) and Ubc1 (yeast homolog of UBE2K) are unique E2 conjugating enzymes that each contain a C-terminal UBA domain attached to their catalytic domains, and they have basal E3-independent polyubiquitination activity. Although the isolated enzymes are monomeric, polyubiquitin formation activity assays show that both can act as ubiquitin donors or ubiquitin acceptors when in the activated thioester conjugate suggesting dimerization of the E2-ubiquitin conjugates. Stable disulfide complexes, analytical ultracentrifugation and small angle x-ray scattering were used to show that the HIP2-Ub and Ubc1-Ub thioester complexes remain predominantly monomeric in solution. Models of the HIP2-Ub complex derived from SAXS data show the complex is not compact but instead forms an open or backbent conformation similar to UbcH5b~Ub or Ubc13~Ub where the UBA domain and covalently attached ubiquitin reside on opposite ends of the catalytic domain. Activity assays showed that full length HIP2 exhibited a five-fold increase in the formation rate of di-ubiquitin compared to a HIP2 lacking the UBA domain. This difference was not observed for Ubc1 and may be attributed to the closer proximity of the UBA domain in HIP2 to the catalytic core than for Ubc1.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0120318PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370575PMC
February 2016

Pivotal role for the ubiquitin Y59-E51 loop in lysine 48 polyubiquitination.

Proc Natl Acad Sci U S A 2014 Jun 27;111(23):8434-9. Epub 2014 May 27.

Departments of Oncological Sciences,Xuzhou Medical College, Jiangsu Key Laboratory of Biological Cancer Therapy, Jiangsu 221002, China

Lysine 48 (K48)-polyubiquitination is the predominant mechanism for mediating selective protein degradation, but the underlying molecular basis of selecting ubiquitin (Ub) K48 for linkage-specific chain synthesis remains elusive. Here, we present biochemical, structural, and cell-based evidence demonstrating a pivotal role for the Ub Y59-E51 loop in supporting K48-polyubiquitination. This loop is established by a hydrogen bond between Ub Y59's hydroxyl group and the backbone amide of Ub E51, as substantiated by NMR spectroscopic analysis. Loop residues Y59 and R54 are specifically required for the receptor activity enabling K48 to attack the donor Ub-E2 thiol ester in reconstituted ubiquitination catalyzed by Skp1-Cullin1-F-box (SCF)(βTrCP) E3 ligase and Cdc34 E2-conjugating enzyme. When introduced into mammalian cells, loop-disruptive mutant Ub(R54A/Y59A) diminished the production of K48-polyubiquitin chains. Importantly, conditional replacement of human endogenous Ub by Ub(R54A/Y59A) or Ub(K48R) yielded profound apoptosis at a similar extent, underscoring the global impact of the Ub Y59-E51 loop in cellular K48-polyubiquitination. Finally, disulfide cross-linking revealed interactions between the donor Ub-bound Cdc34 acidic loop and the Ub K48 site, as well as residues within the Y59-E51 loop, suggesting a mechanism in which the Ub Y59-E51 loop helps recruit the E2 acidic loop that aligns the receptor Ub K48 to the donor Ub for catalysis.
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http://dx.doi.org/10.1073/pnas.1407849111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060658PMC
June 2014

Switching on ubiquitylation by phosphorylating a ubiquitous activator.

Authors:
Gary S Shaw

Biochem J 2014 Jun;460(3):e1-3

*Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada, N6A 5C1.

The dysfunction of the E3 ubiquitin ligase Parkin is a key contributor to the development of early-onset Parkinson's disease. Parkin is responsible for the labelling of outer mitochondrial membrane proteins with the small modifier protein ubiquitin in response to oxidative stress. This ubiquitylation signals the clearance of the damaged mitochondria to preserve overall cell health. Recent structural and biochemical experiments have shown that native Parkin exists in an autoinhibited state that must be activated in order to unmask its full ubiquitylation potential. In a recent article in the Biochemical Journal (vol. 460, pp. 127-139), Kazlauskaite and co-workers identified that the Parkinson's disease-associated kinase PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1] can phosphorylate ubiquitin in response to mitochondrial depolarization. Furthermore, the authors demonstrated that phosphorylated ubiquitin can activate Parkin's E3 ligase activity and promote both increased autoubiquitylation and substrate ubiquitylation of the mitochondrial protein Miro1. The study provides exciting initial insights that show how PINK1 might activate ubiquitin through phosphorylation, and how this important regulatory step might switch on Parkin-mediated ubiquitylation.
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http://dx.doi.org/10.1042/BJ20140459DOI Listing
June 2014

Structural basis for the inhibition of host protein ubiquitination by Shigella effector kinase OspG.

Structure 2014 Jun 22;22(6):878-88. Epub 2014 May 22.

Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada; Department of Biochemistry, McGill University, Montreal, QC H3G 1Y6, Canada. Electronic address:

Shigella invasion of its human host is assisted by T3SS-delivered effector proteins. The OspG effector kinase binds ubiquitin and ubiquitin-loaded E2-conjugating enzymes, including UbcH5b and UbcH7, and attenuates the host innate immune NF-kB signaling. We present the structure of OspG bound to the UbcH7∼Ub conjugate. OspG has a minimal kinase fold lacking the activation loop of regulatory kinases. UbcH7∼Ub binds OspG at sites remote from the kinase active site, yet increases its kinase activity. The ubiquitin is positioned in the "open" conformation with respect to UbcH7 using its I44 patch to interact with the C terminus of OspG. UbcH7 binds to OspG using two conserved loops essential for E3 ligase recruitment. The interaction of the UbcH7∼Ub with OspG is remarkably similar to the interaction of an E2∼Ub with a HECT E3 ligase. OspG interferes with the interaction of UbcH7 with the E3 parkin and inhibits the activity of the E3.
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http://dx.doi.org/10.1016/j.str.2014.04.010DOI Listing
June 2014

RBR E3 ubiquitin ligases: new structures, new insights, new questions.

Biochem J 2014 Mar;458(3):421-37

*Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada, N6A 5C1.

The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology.
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http://dx.doi.org/10.1042/BJ20140006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940038PMC
March 2014

A snapshot of ubiquitin chain elongation: lysine 48-tetra-ubiquitin slows down ubiquitination.

J Biol Chem 2014 Mar 24;289(10):7068-81. Epub 2014 Jan 24.

From the Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, New York 10029-6574.

We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IκBα and β-catenin by the Skp1-cullin 1-βTrCP F-box protein (SCF(βTrCP)) E3 ubiquitin (Ub) ligase complex. Competition experiments revealed that SCF(βTrCP) formed a complex with IκBα and that the Nedd8 modified E3-substrate platform engaged in dynamic interactions with the Cdc34 E2 Ub conjugating enzyme for chain elongation. Using "elongation intermediates" containing β-catenin linked with Ub chains of defined length, it was observed that a Lys-48-Ub chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional Ub conjugation. Thus, the Ub chain length and linkage impact kinetic rates of chain elongation. Given that Lys-48-tetra-Ub is packed into compact conformations due to extensive intrachain interactions between Ub subunits, this topology may limit the accessibility of SCF(βTrCP)/Cdc34 to the distal Ub Lys-48 and result in slowed elongation.
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http://dx.doi.org/10.1074/jbc.M113.530576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945367PMC
March 2014

Ataxin-3 is a multivalent ligand for the parkin Ubl domain.

Biochemistry 2013 Oct 9;52(42):7369-76. Epub 2013 Oct 9.

Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario , London, Ontario, Canada N6A 5C1.

The ubiquitin signaling pathway consists of hundreds of enzymes that are tightly regulated for the maintenance of cell homeostasis. Parkin is an E3 ubiquitin ligase responsible for conjugating ubiquitin onto a substrate protein, which itself can be ubiquitinated. Ataxin-3 performs the opposing function as a deubiquitinating enzyme that can remove ubiquitin from parkin. In this work, we have identified the mechanism of interaction between the ubiquitin-like (Ubl) domain from parkin and three C-terminal ubiquitin-interacting motifs (UIMs) in ataxin-3. (1)H-(15)N heteronuclear single-quantum coherence titration experiments revealed that there are weak direct interactions between all three individual UIM regions of ataxin-3 and the Ubl domain. Each UIM utilizes the exposed β-grasp surface of the Ubl domain centered around the I44 patch that did not vary in the residues involved or the surface size as a function of the number of ataxin-3 UIMs involved. Further, the apparent dissociation constant for ataxin-3 decreased as a function of the number of UIM regions used in experiments. A global multisite fit of the nuclear magnetic resonance titration data, based on three identical binding ligands, resulted in a KD of 669 ± 62 μM for each site. Our observations support a multivalent ligand binding mechanism employed by the parkin Ubl domain to recruit multiple UIM regions in ataxin-3 and provide insight into how these two proteins function together in ubiquitination-deubiquitination pathways.
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http://dx.doi.org/10.1021/bi400780vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807529PMC
October 2013

Structure of the HHARI catalytic domain shows glimpses of a HECT E3 ligase.

PLoS One 2013 15;8(8):e74047. Epub 2013 Aug 15.

Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn²⁺-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074047PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772753PMC
June 2014

A molecular explanation for the recessive nature of parkin-linked Parkinson's disease.

Nat Commun 2013 ;4:1983

Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.

Mutations in the park2 gene, encoding the RING-inBetweenRING-RING E3 ubiquitin ligase parkin, cause 50% of autosomal recessive juvenile Parkinsonism cases. More than 70 known pathogenic mutations occur throughout parkin, many of which cluster in the inhibitory amino-terminal ubiquitin-like domain, and the carboxy-terminal RING2 domain that is indispensable for ubiquitin transfer. A structural rationale showing how autosomal recessive juvenile Parkinsonism mutations alter parkin function is still lacking. Here we show that the structure of parkin RING2 is distinct from canonical RING E3 ligases and lacks key elements required for E2-conjugating enzyme recruitment. Several pathogenic mutations in RING2 alter the environment of a single surface-exposed catalytic cysteine to inhibit ubiquitination. Native parkin adopts a globular inhibited conformation in solution facilitated by the association of the ubiquitin-like domain with the RING-inBetweenRING-RING C-terminus. Autosomal recessive juvenile Parkinsonism mutations disrupt this conformation. Finally, parkin autoubiquitinates only in cis, providing a molecular explanation for the recessive nature of autosomal recessive juvenile Parkinsonism.
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http://dx.doi.org/10.1038/ncomms2983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3709501PMC
December 2013