K29-selective ubiquitin binding domain reveals structural basis of specificity and heterotypic nature of k29 polyubiquitin

Abstract

Polyubiquitin chains regulate diverse cellular processes through the ability of ubiquitin to form chains of eight different linkage types. Although detected in yeast and mammals, little is known about K29-linked polyubiquitin. Here we report the generation of K29 chains in vitro using a ubiquitin chain-editing complex consisting of the HECT E3 ligase UBE3C and the deubiquitinase vOTU. We determined the crystal structure of K29-linked diubiquitin, which adopts an extended conformation with the hydrophobic patches on both ubiquitin moieties exposed and available for binding. Indeed, the crystal structure of the NZF1 domain of TRABID in complex with K29 chains reveals a binding mode that involves the hydrophobic patch on only one of the ubiquitin moieties and exploits the flexibility of K29 chains to achieve linkage selective binding. Further, we establish methods to study K29-linked polyubiquitin and find that K29 linkages exist in cells within mixed or branched chains containing other linkages.

Graphical abstract

Figure 1

Assembly and Purification of K29-Linked PolyUb (A) A schematic illustrates how E3 ligase and DUB(s) are used to assemble free polyUb chains. (B) Large-scale assembly of polyUb chains by UBE3C in the presence of UBE1, UBE2D3, and Ub. The addition of vOTU releases free polyUb chains. (C) Ubiquitylation assays of UBE3C in the presence of UBE1, UBE2D3, and wild-type Ub or K-to-R Ub mutants. vOTU was added at the end of the ubiquitylation reaction. (D) Time course of ubiquitylation by UBE3C in the presence of UBE1, UBE2D3, vOTU, and wild-type Ub or K29only Ub mutant. To one half of the reaction stopped at 60 min, apyrase was added to quench ATP followed by DUB hydrolysis with TRABID. (E) Purification of K29-linked diUb, triUb, tetraUb, and pentaUb by cation-exchange chromatography. Proteins from the corresponding peak fractions were visualized on Coomassie-stained SDS-PAGE gel. (F) The K29-linked diUb and triUb purified in (E) are visualized in silver-stained SDS-PAGE gel. See also Figure S1.

Figure 2

Crystal Structure of K29-Linked diUb (A) The crystal structure of K29-linked diUb is shown in two orientations. (B) Residues at the interface between the two Ub moieties are shown in stick representation. Polar interactions are shown as dotted lines. (C) Positioning of surface hydrophobic patches in the structure of K29-linked diUb. A semitransparent surface, colored blue for I44 patch (I44, L8, H68, and V70) and green for I36 patch (I36, L71, and L73), shows that the hydrophobic patches are not involved in the interface. (D and E) Relative orientations of hydrophobic patches in K63-linked (D) and M1-linked (E) diUb. A semitransparent surface as in (C), showing the I36 and I44 patches (PDB accession numbers 2JF5 and 2W9N) (Komander et al., 2009). See also Figure S2.

Figure 3

The NZF1 Domain of TRABID Is Highly Selective for K29 and K33 Chains (A) Purified tetraUb of M1, K6, K11, K29, K33, K48, and K63 linkages were separated on 4% to 12% SDS-PAGE gel and visualized by Coomassie and silver staining, and anti-Ub immunoblotting. (B) Linkage selectivity of different UBDs was investigated using pull-down assays. Halo-tagged RAD23B UBA1-2, Ubiquilin-1 UBA, NPL4 NZF, and TRABID NZF1-3 were used in pull-down assays with tetraUb of the indicated linkage types. The chains were visualized by silver-staining. Fifty percent of tetraUb input used in the pull-down assay was included as control. Asterisk shows NPL4 released from Halo resins. (C) Pull-down assays to determine the linkage selectivity of the individual or tandem NZF domains of TRABID. Halo-tagged fusions of the indicated TRABID NZF domains were used in pull-down assays with tetraUb of seven different linkage types. One hundred percent of tetraUb input used in the pull-down assay was included as control. (D) Linkage selectivity of TRABID NZF1 assayed as in (B). (E) Linkage preference of TRABID NZF domains was assayed using a competition pull-down assay. K29- or K33-linked tetraUb was mixed with M1- and K63-tetraUb chains, and pull-downs performed using Halo-TRABID NZF1-3 or NZF1. Asterisk shows NZF1 released from Halo beads. (F–J) ITC measurements for the NZF1 domain of TRABID with K29-Ub2 (F), K33-Ub2 (G), M1-Ub2 (H), K63-Ub2 (I), and monoUb (J). The K_d value for each measurement is indicated. See also Figure S3.

Figure 4

Crystal Structure of TRABID NZF1 in Complex with K29-Linked diUb (A) Structure of TRABID NZF1 (cyan; zinc as gray sphere coordinated by cysteine residues [green]) bound to diUb (orange, proximal Ub; yellow, distal Ub). I44, V70, and L8 are shown as blue sticks. (B) Selected residues of TRABID NZF1 (gray) and K29-diUb (blue) in the interface are shown. (C) A semitransparent surface shows that only the Ile44 patch (blue) of the distal diUb interacts with TRABID NZF1. (D) Sequence alignment of TRABID NZF1 from different species. Green and red circles indicate residues of TRABID NZF1 involved in binding to the distal and proximal Ub, respectively. (E) Residues of TRABID NZF1 involved in Ub binding were mutated to alanine and the binding to K29- and K33-tetraUb was assayed as in Figure 3C. Halo-fusion proteins coupled to the resin were visualized on Coomassie-stained SDS-PAGE gel. See also Figure S4.

Figure 5

Determinants of NZF Binding to PolyUb (A) Superposition of the distal Ub moieties (yellow) of K29-diUb alone and K29-diUb in the complex with TRABID NZF1. In the complex, the proximal moiety of free K29-diUb (gray) rotates approximately 45° to interact with NZF1 (cyan). (B) Sequence alignment of TRABID NZF1, NZF2, and NZF3 with NZF domains of TAB2, TAB3, and NPL4. The hydrophobic residues making up the T-F/Φ binding patch are highlighted by red spheres. (C and D) TRABID NZF1 mutants Y15 (C) and M26 (D) were assayed for binding to K29, K33, and K63 tetraUb. (E) TAB2 NZF mutants were assayed as in (C). (F) T25 of TRABID NZF1 was mutated and polyUb binding was assayed as in (C). (G) Linkage specificity of TRABID NZF1 T25E mutant was assayed as in Figure 3B. See also Figure S5.

Figure 6

Isolation and Analysis of K29 Chains from Mammalian Cells (A) Analysis of polyUb captured from HEK293 cells transiently expressing Flag-tagged full-length TRABID (FL-WT), full-length catalytically dead (FL-CA), or tandem NZF1-3 domains (NZF1-3). One half of the Flag-immunopurified sample was incubated with vOTU that hydrolyzes all linkage types excepting M1, K27, and K29 linkages and visualized by anti-Ub immunoblotting. (B) PolyUb chains from HEK293 extracts were captured using indicated bacterially expressed Halo NZF domains of TRABID, followed by DUB treatment as in (A). (C) PolyUb chains from untransfected (control) or Flag-TRABID NZF1-3 (NZF1-3)-expressing HEK293 cells were captured using Halo-TRABID NZF1-3, TRABID NZF1, or Ubiquilin-1 (UBQLN1) UBA domain. The isolated polyUb chains were either left untreated or treated with vOTU. (D) Immunobloting analysis of polyUb chains captured from HEK293 extracts using the minimal NZF1 that is selective for K29 and K33 linkages. Presence of K29 linkages was assayed by incubation with vOTU, which does not hydrolyze K29 linkages. The vOTU-resistant polyUb chains that bound to Halo-TRABID NZF1 were separated from monoUb, washed, and the linkage-types of these chains were assayed by incubation with a panel of DUBs that hydrolyze different linkage types. See also Figures S6 and S7.

Figure 7

K29-Linked Chains Are Present within Mixed or Branched Heterotypic Chains (A) Cartoon illustrating the different possibilities of how K29 linkages may be present in the heterotypic chains captured by TRABID NZF1. (B) A schematic illustrates the experimental procedure for determining the linkage composition of the mixed or branched heterotypic chains isolated by the linkage-selective UBDs. (C) PolyUb chains from the HEK293 were first captured using Halo-tagged RAD23B (K48-selective binder), TRABID NZF1 (K29- and K33-selective binder), or UBQLN1 (non-selective binder). Subsequently, samples in the indicated lanes were treated with vOTU. The total capture (T), supernatant (S), and beads (B) fractions after the DUB reaction were analyzed separately as shown in (B). (D) Recapture assay using linkage-selective UBDs to determine the linkage type of the chains remaining after removing K29 linkages. PolyUb chains captured from HEK293 cell extracts using Halo-NZF1 were incubated with TRABID that preferentially hydrolyzes K29 and K33 linkages. The polyUb chains that were released into the supernatant fraction after removing K29 linkages were recaptured using either the K48-selective Halo-RAD23B or the K29 and K33 selective Halo-TRABID NZF1. See also Figure S8.