Highlights•The HECT E3 ligases UBE3C and AREL1 assemble K29- and K33-linked polyubiquitin, respectively•K29- and K33-linked chains adopt open conformations in solution•The N-terminal NZF1 domain of TRABID specifically recognizes K29/K33-diubiquitin•A structure of a K33 filament bound to NZF1 domains explains TRABID specificitySummaryProtein ubiquitination regulates many cellular processes via attachment of structurally and functionally distinct ubiquitin (Ub) chains. Several atypical chain types have remained poorly characterized because the enzymes mediating their assembly and receptors with specific binding properties have been elusive. We found that the human HECT E3 ligases UBE3C and AREL1 assemble K48/K29- and K11/K33-linked Ub chains, respectively, and can be used in combination with DUBs to generate K29- and K33-linked chains for biochemical and structural analyses. Solution studies indicate that both chains adopt open and dynamic conformations. We further show that the N-terminal Npl4-like zinc finger (NZF1) domain of the K29/K33-specific deubiquitinase TRABID specifically binds K29/K33-linked diUb, and a crystal structure of this complex explains TRABID specificity and suggests a model for chain binding by TRABID. Our work uncovers linkage-specific components in the Ub system for atypical K29- and K33-linked Ub chains, providing tools to further understand these unstudied posttranslational modifications.Graphical abstract
We report the successful application of selective excitation sculptured TOCSY NMR (SXS-TOCSY) to identify individual solution components from a heterogeneous system using selectively acquired 1H NMR spin system patterns. SXS-TOCSY application is illustrated by detection of the simultaneous presence of poly-β-(1,6)-N-acetylglucosamine (PNAG) and poly(glycerol phosphate) teichoic acid (TA) carbohydrate polymer components in crude biofilm extracts from Staphylococcus epidermidis without the need for further sample purification and component separation. Biofilms are implicated in the barriers for resistance of microbes toward antibiotics and immune responses, therefore efficient rapid detection and quantification of key components are important to assist in the design of a clinical infection response.
Article13 November 2017Open Access Source DataTransparent process An invisible ubiquitin conformation is required for efficient phosphorylation by PINK1 Christina Gladkova Christina Gladkova Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Alexander F Schubert Alexander F Schubert Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Jane L Wagstaff Jane L Wagstaff Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Jonathan N Pruneda Jonathan N Pruneda orcid.org/0000-0002-0304-4418 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Stefan MV Freund Stefan MV Freund Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author David Komander Corresponding Author David Komander [email protected] orcid.org/0000-0002-8092-4320 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Christina Gladkova Christina Gladkova Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Alexander F Schubert Alexander F Schubert Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Jane L Wagstaff Jane L Wagstaff Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Jonathan N Pruneda Jonathan N Pruneda orcid.org/0000-0002-0304-4418 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Stefan MV Freund Stefan MV Freund Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author David Komander Corresponding Author David Komander [email protected] orcid.org/0000-0002-8092-4320 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Author Information Christina Gladkova1,‡, Alexander F Schubert1,‡, Jane L Wagstaff1, Jonathan N Pruneda1, Stefan MV Freund1 and David Komander *,1 1Medical Research Council Laboratory of Molecular Biology, Cambridge, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +44 1223 267160; E-mail: [email protected] The EMBO Journal (2017)36:3555-3572https://doi.org/10.15252/embj.201797876 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis Phosphorylation of stably folded ubiquitin has been found to affect its conformational equilibrium. New NMR techniques reveal that unmodified ubiquitin also exists in two distinct conformational states, whose mutational stabilization differentially affects conjugation and phosphorylation reactions. A small population of wild-type ubiquitin exhibits a C-terminally retracted (Ub-CR) conformation in Chemical Exchange Saturation Transfer (CEST)-NMR experiments. Structure-guided point mutations can shift the equilibrium in favor of either canonical or Ub-CR conformation. Stabilization of the Ub-CR conformation compromises ubiquitin conjugation reactions. The Ub-CR conformation is a superior substrate for phosphorylation by the PINK1 kinase. Previous ArticleNext Article Read MoreAbout the coverClose modalView large imageVolume 36,Issue 24,15 December 2017Cover: Ubiquitin adopts a distinct, lowly populated conformation in which the C-terminus is retracted into the core. The kinase PINK1 exploits this ubiquitin conformation to phosphorylate Ser65, a crucial signal for the initiation of mitophagy. From Christina Gladkova, Alexander F Schubert, David Komander and colleagues: An invisible ubiquitin conformation is required for efficient phosphorylation by PINK1. For details, see the Article on p 3555. Cover illustration by Christina Gladkova. Volume 36Issue 2415 December 2017In this issue RelatedDetailsLoading ...
Abstract Intermediate species in the assembly of amyloid filaments are believed to play a central role in neurodegenerative diseases and may constitute important targets for therapeutic intervention. However, structural information about intermediate species has been scarce and the molecular mechanisms by which amyloids assemble remain largely unknown. Here, we use time-resolved electron cryo-microscopy (cryo-EM) to study the in vitro assembly of recombinant truncated tau (amino acids 297-391) into paired helical filaments of Alzheimer’s disease or into filaments of chronic traumatic encephalopathy. We report the formation of a shared first intermediate amyloid (FIA), with an ordered core comprising amino acids 302-316. Nuclear magnetic resonance indicates that the same amino acids adopt rigid, β-strand-like conformations in monomeric tau. At later time points, the FIAs disappear and we observe many different intermediate amyloid filaments, with structures that depend on the reaction conditions. At the end of both reactions, most intermediate amyloids disappear and filaments with the same ordered cores as those from human brains remain. Our results provide structural insights into the processes of primary and secondary nucleation of amyloid assembly, with implications for the design of novel therapies.
ABSTRACT The Ser/Thr protein kinase PINK1 phosphorylates the well-folded, globular protein ubiquitin (Ub) at a relatively protected site, Ser65. We had previously shown 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 in which the last β-strand is retracted to extend the Ser65 loop and shorten the C-terminal tail. We here show using Chemical Exchange Saturation Transfer (CEST) NMR experiments, that a similar, C-terminally retracted (Ub-CR) conformation exists in wild-type Ub. Ub point mutations in the moving β5-strand and in neighbouring strands shift the Ub/Ub-CR equilibrium. This enabled functional studies of the two states, and we show that the Ub-CR conformation binds to the PINK1 kinase domain 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 proteins or domains.
This review aims to illustrate that STD NMR is not simply a method for drug screening and discovery, but has qualitative and quantitative applications that can answer fundamental and applied biological and biomedical questions involving molecular interactions between ligands and proteins. We begin with a basic introduction to the technique of STD NMR and report on recent advances and biological applications of STD including studies to follow the interactions of non-steroidal anti-inflammatories, minimum binding requirements for virus infection and understating inhibition of amyloid fibre formation. We expand on this introduction by reporting recent STD NMR studies of live-cell receptor systems, new methodologies using scanning STD, magic-angle spinning STD and approaches to use STD NMR in a quantitative fashion for dissociation constants and group epitope mapping (GEM) determination. We finish by outlining new approaches that have potential to influence future applications of the technique; NMR isotope-editing, heteronuclear multidimensional STD and (19)F STD methods that are becoming more amenable due to the latest NMR equipment technologies.
Integrin αvβ6 is an important emerging target for both imaging and therapy of cancer that requires specific ligands based on Arg-Gly-Asp (RGD) peptides. There remains little correlation between integrin-RGD ligand specificity despite studies suggesting an RGD-turn-helix ligand motif is required. Here, we describe the application of 15N NMR relaxation analyses and structure determination of αvβ6 peptide ligands in the presence and absence of trifluoroethanol (TFE) to identify their critical molecular nature that influences specificity, interaction and function. Two linear peptides; one known to demonstrate αvβ6 specificity (FMDV2) and the other based on a natural RGD ligand (LAP2), were compared to two additional peptides based on FMDV2 but cyclised in different positions using a disulphide bond (DBD1 and DBD2). The cyclic adaptation in DBD1 produces a significant alteration in backbone dynamic properties when compared to FMDV2; a potential driver for the loss in αvβ6 specificity by DBD1. The importance of ligand dynamics are highlighted through a comprehensive reduced spectral density and ModelFree analysis of peptide 15N NMR relaxation data and suggest αvβ6 specificity requires the formation of a structurally rigid helix preceded by a RGD motif exhibiting slow internal motion. Additional observations include the effect of TFE/water viscosity on global NMR dynamics and the advantages of using spectral density NMR relaxation data to estimate correlation times and motional time regimes for peptides in solution.
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