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. 2013 May;20(5):555-65.
doi: 10.1038/nsmb.2547. Epub 2013 Apr 7.

Assembly, Analysis and Architecture of Atypical Ubiquitin Chains

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Free PMC article

Assembly, Analysis and Architecture of Atypical Ubiquitin Chains

Manuela K Hospenthal et al. Nat Struct Mol Biol. .
Free PMC article

Abstract

Ubiquitin (Ub) chains regulate many cellular processes, but several chain types including Lys6 linkages have remained unstudied. Here we analyze the bacterial effector E3 ligase NleL (non-Lee-encoded effector ligase) from enterohemorrhagic Escherichia coli (EHEC) O157:H7, which assembles Lys6- and Lys48-linked Ub polymers. Using linkage-specific human deubiquitinases (DUBs) we show that NleL generates heterotypic Ub chains, and branched chains are efficiently hydrolyzed by DUBs. USP family DUBs cleave Lys6-linked polymers exclusively from the distal end, whereas a DUB with preference for Lys6 can cleave Lys6-linked polymers at any position in the chain. We used NleL to generate large quantities of Lys6-linked polyUb. Crystallographic and NMR spectroscopy analyses revealed that an asymmetric interface between Ile44 and Ile36 hydrophobic patches of neighboring Ub moieties is propagated in longer Lys6-linked Ub chains. Interactions via the Ile36 patch can displace Leu8 from the Ile44 patch, leading to marked structural perturbations of Ub.

Figures

Figure 1
Figure 1. NleL-mediated Ub chain assembly
(a) NleL was used in Ub chain assembly reactions with E1, UBE2L3/UbcH7, and Ub mutants as indicated (see Methods). M, marker; WT, wild type Ub; Ub-lysine only, Ub mutants with Arg mutations in six out of seven Lys residues; Lys-less, Ub harbouring Arg mutations at all Lys residues. (b) A comparison of assembled unanchored Ub chains from WT Ub, Ub K6R and Ub K48R. A Ub K6R K48R double mutant is unable to assemble similar unanchored Ub products. Asterisks (*) indicate pentaUb species. (c) Time-course analysis of the NleL assembly reaction as in a with Ub K6R or Ub K48R.
Figure 2
Figure 2. Ub chain sequencing using linkage-specific DUBs
(a) Schematic illustrating potential tetramer complexity in NleL assembled WT Ub products, and the concept of Ub chain restriction mapping. Ub (yellow square) can be linked either via Lys6 (blue line; horizontal) or Lys48 (red line; vertical), leading to 14 distinct species. An orange dot indicates the free C-terminus of Ub (proximal moiety). Linkage-specific DUBs can be used as ‘Ub chain restriction enzymes’ to reveal building blocks within heterotypic Ub chains. (b, c) Specificity profile of OTUB1 at 4.7 μM (b) and OTUD3 at 5.5 μM (c) against Lys6 and Lys48 diUb and tetraUb. Hydrolysis of polyUb is followed during the indicated time-course, and resolved on a silver-stained SDS-PAGE gel. (d) Purified penta / hexaUb assembled from WT Ub (lane 1) is cleaved with 4.7 μM OTUB1, 5.5 μM OTUD3, 6.1 nM vOTU or combinations thereof. *, OTUD3 protein; $, OTUB1 protein. (e) NleL mediated ubiquitination of UBE2L3 variants as in Fig. 1a, detected by Western blotting with anti-UBE2L3 (left) and anti-Ub antibodies (right). noK UBE2L3, UBE2L3 mutant with 18 Lys residues changed to Arg. (f) DUB assays performed for 1 h as in d, on polyubiquitinated GST-tagged UBE2L3 from e, analysed by Western blotting as in e. Whole reaction, samples before GST-pulldown; supernatant, reaction after GST-pulldown containing free NleL assembled Ub chains. Lys6 polyUb detection is less efficient compared to Lys48 polyUb, compare f to d. Prox, proximal; Dist, distal; M, marker; WT, wild type.
Figure 3
Figure 3. NleL assembles branched Ub chains
(a) Schematic depicting the assembly of a branched triUb harbouring one Lys6- and one Lys48-linkage. (b) Time-course analysis of NleL assembly reaction (as in Fig. 1c) with a combination of 19 μM Ub ΔG76 (Acceptor) and 38 μM Ub K6R K48R (Donor). (c) NleL mediated assembly of Ub chains from Ub K6 only, Ub K48 only and a combination of Ub ΔG76 and Ub K6R K48R in a 1:2 molar ratio as in b. Resulting species are labelled. An emerging tetraUb band that also appears in b is labelled. M, marker. (d) DUB assay using the Lys11-linkage specific DUB Cezanne. A 30 min reaction is performed with noK UBE2L3 (Fig. 2e) to exclude autoubiquitination of the E2, and subsequently treated with 2 μM Cezanne for 30 min, which selectively removes the tetraUb species.
Figure 4
Figure 4. Hydrolysis of branched and Lys6-linked polyUb by DUBs
(a) Branched triUb was hydrolysed with 4.7 μM OTUB1, 5.5 μM OTUD3 or 6.1 nM vOTU. A silver-stained SDS-PAGE gel is shown. (b-d) Hydrolysis of homotypic Lys6 triUb, homotypic Lys48 triUb and branched triUb containing one Lys6- and one Lys48-linkage (see Fig. 3) by 6.1 nM vOTU (b), 2.5 μM USP7 (c) and 60 nM USP21 (d), performed as in a. All tested DUBs hydrolyse branched triUb similarly to homotypic Ub chains. (e) Structure of S. cerevisiae Otu1 (yOtu1, blue, pdb-id 3by4, ) covalently bound to Ub (orange). Ub Lys6 is shown in stick-representation, and is solvent exposed. (f) Structure of USP21 (green) in complex with Ub (orange) (pdb-id 2y5b,). Ub Lys6 (indicated with an arrow) is at the interface with the USP core, interacting with Glu427, which is highly conserved amongst USP DUBs. Ub is shown in the same orientation as in e. (g) Time-course DUB assay of OTUD3 cleavage of Lys6 chains. DiUb is generated at each time point, indicating that OTUD3 cleaves any linkage in the chain (endo-activity). (h) Time-course DUB assay of USP21 cleavage of Lys6 chains. DiUb is generated only at later time point, indicating that USP21 hydrolyses Lys6 polymers from the distal end with exo-activity, consistent with the structure. M, marker; INP, input.
Figure 5
Figure 5. Large-scale assembly and crystal structures of Lys6 polyUb
(a) Time-course analysis of a large-scale assembly reaction of Lys6 polyUb by NleL using 25 mg Ub K48R as input material, as described in Methods. (b) Chromatogram of purification of Lys6 polyUb species by size-exclusion chromatography (SEC). (c) Fractions corresponding to individual peaks from SEC purification in b are resolved by SDS-PAGE. (d) Comparison of the assembly reactions performed by NleL using WT Ub (lane 1), Ub K48R (lane 2) and WT Ub subsequently treated using 5.1 μM OTUB1 for 3 h (lane 3). Large-scale reactions can be treated with OTUB1 overnight. M, marker; WT, wild type. (e) Packing of Lys6 polyUb crystals, two neighbouring asymmetric units are shown, in which Ub molecules interact via a ‘tight’ interface (witin asymmetric unit) or via a ‘loose’ interface between asymmetric units. Green and blue spheres indicate the Cα positions of Ile36 and Ile44, respectively. Distances indicated by a black dotted line refer to Cα atoms of Leu73 and Lys6 in neighbouring moieties.
Figure 6
Figure 6. Solution studies of Lys6 diUb
(a) Schematic of Lys6 diUb species generated for NMR analysis. Ub moieties coloured in yellow are isotopically labelled. Mutations made to generate these Lys6 diUb species are indicated. (b) Selected region of 1H, 15N-HSQC spectra showing the 15N-labelled Lys6 diUb (blue) (i) overlaid onto Ub K48R (red). For full spectra see Supplementary Fig. 6a. Reassigned spectra from proximally (ii) and distally (iii) labelled Lys6 diUb (Supplementary Fig. 6b, c) allow unambiguous assignment of distal (d) and proximal (p) resonances in the uniformly 15N-labelled spectrum. (c, d) Weighted chemical shift perturbation (CSP) map from b for the proximal (c) and distal (d) molecule (see also Supplementary Fig. 5a). Grey bars, exchange broadened residues; (P), prolines. (e, f) Left, ‘tight’ interface of Lys6 polyUb indicating (e) proximal and (f) distal interfaces. Middle, significant CSPs were mapped onto the surface of Ub. Blue, CSPs >0.035 ppm; cyan, exchange broadened residues; green, prolines. Right, crystallographic interface according to PISA analysis (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html ) mapped onto the surface of Ub in red. Prox, proximal; Dist, distal.
Figure 7
Figure 7. Solution studies of Lys6 triUb by NMR spectroscopy
(a) Schematic and molecular model of Lys6 triUb where the ‘tight’ Ile44-Ile36 interface is propagated (compare Fig. 5e). The Cα atoms of Ile36 (green) and Ile44 (blue) are indicated as spheres. (b) NMR analysis of proximally labelled Lys6 triUb. Top, a schematic of the experiments showing the compared species. Ub moieties coloured in yellow are isotopically labelled. Weighted CSP map for Lys6 triUb with respect to Lys6 diUb. Grey bars, exchange-broadened residues; (P), prolines. For full spectra see Supplementary Fig. 7a. (c) Analysis as in b for distally labelled Lys6 triUb compared to monoUb K48R. For full spectra see Supplementary Fig. 7b. (d) Analysis as in b for a middle labelled Lys6 triUb. The CSP map indicates resonances that are shifted compared to monoUb K48R. Resonances that are shifted and overlay with the distal resonance position in uniformly labelled diUb (Fig. 6b) are indicated in red, and split resonances in cyan. For full spectra see Supplementary Fig. 8. (e) Mapping of perturbed residues onto the surface of Ub shown in two orientations. (f) NMR supports a model for longer Lys6 polyUb in which the asymmetric Ile36-Ile44 patch interactions are propagated. (g) Selected resonances from the middle labelled triUb species. Each panel shows a residue in the 1H,15N-HSQC spectra of middle labelled Lys6 triUb (blue) overlaid onto uniformly labelled Lys6 diUb (green, Fig. 6b) and monoUb K48R (red, Fig. 6b). In the diUb spectrum proximal (p) and distal (d) positions are indicated, whereas in the triUb spectrum split resonances are arbitrarily assigned a and b. See Supplementary Fig. 4 for additional resonances. Prox, proximal; Dist, distal.
Figure 8
Figure 8. Ile36 binding causes structural rearrangements in Ub
(a) Schematic representation and close-up of the ZnF UBP interaction with Ub. (b) Interacting residues according to PISA analysis are coloured on the Ub surface (red), and residues 12-14 on the β2-strand of Ub (purple, circled) are not involved in the interaction. (c) Selected region of 1H, 15N-HSQC spectra for a 1:1 molar complex of Ub with ZnF UBP (blue) overlaid onto Ub (red). Ub was 13C,15N-labelled and ZnF UBP was unlabelled. Resonances experience chemical shifts (Glu34), exchange broadening (Thr7) or are unperturbed (Ile3). For full spectra see Supplementary Fig. 9. (d) Top, schematic of the experiment showing the compared species. Weighted CSP map derived from spectra in c. Grey bars, residues completely exchange broadened in the Ub–ZnF UBP complex; Asterisk (*), residues exchange broadened already in Ub; (P), prolines. See also Supplementary Fig. 4b. (e) Peak height attenuation map derived from spectra in c indicating the degree of exchange broadening upon complex formation. Asterisk (*), residues exchange broadened already in Ub; (P), prolines. Thr9 (red arrow) is exchange broadened in monoUb but reappears when Ub is in complex with ZnF UBP. See also Supplementary Fig. 4b. (f) Significant CSPs were mapped onto the surface of Ub. Blue, CSPs >0.09 ppm; cyan, exchange broadened residues (intensity ratio <0.5); green, prolines. Circled residues Thr12, Ile13, Thr14 are perturbed but not involved in the interface.
Figure 9
Figure 9. Ub ‘loop-in’ and ‘loop-out’ conformations
(a) Ub in the ‘loop-out’ conformation with Leu8 contributing to the Ile44 patch. Left, proximal Ub from Lys6 diUb. Right, monoUb (pdb-id 1ubq). (b) Ub in the ‘loop-in’ conformation with Leu8 contributing to the Ile36 patch. Left, distal Ub from Lys6 diUb. Right, Ub bound to USP5 ZnF UBP (2g45). (c) Comparison of Ub from a (blue) with Ub from b (green), showing the displacement of the β2-strand it the ‘loop-in’ conformation. (d) Both loop conformations are found in the RDC ensemble of Ub. Four representative examples for each conformation are shown. Ile44 is in a more flexible region when Ub is in the ‘loop-in’ conformation. (e) Overlay of the eight Ub conformations from d, showing residues Thr12, Ile13 and Thr14 on the β2-strand of Ub. The ‘loop-in’ conformation shifts the β2-strand outwards. (f) Ub is shown in surface representation (white) and in identical orientations with the Ile44 patch coloured in blue, the Ile36 patch in green and Leu8 in orange. The ubiquitin interacting protein is shown in cartoon representation. The position of the β1/β2-loop in the ‘loop-in’ or ‘loop-out’ conformation is indicated. Structures from top left to bottom right: Lys6 diUb (2xk5), USP5 ZnF UBP (2g45), Vps9p CUE domain (1p3q), BIRC7 RING domain in complex with Ub-charged UBE2D2 (4auq), RNF4 RING domain bound to Ub-charged UBE2D1 (4ap4), Lys11 diUb (3nob), Ub bound to the NEDD4 N-lobe (2xbb), NEDD4L in complex with an UBE2D~Ub oxyester (3jw0).

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