The ubiquitin-associated domain of cellular inhibitor of apoptosis proteins facilitates ubiquitylation

Abstract

The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases.

Keywords: Apoptosis; Protein-Protein Interaction; RING; Ubiquitin; Ubiquitin Ligase; Ubiquitin-conjugating Enzyme (E2 Enzyme); cIAP.

FIGURE 1.

The UBA domain. A, superposition of structures of the UBA domains from cIAP1 (blue, PDB code 3T6P), XIAP (green, PDB code 2KNA), Dsk2p (magenta, PDB code 1WR1), and EDE1 (yellow, PDB code 2G3Q), highlighting the relative orientations of α1–α3. B, multiple sequence alignment of representative UBA domains highlighting the conserved MGF and LL motifs (red). Elements of secondary structures are indicated below the alignment. C, the solution structure of Dsk2p UBA domain (magenta) in complex with ubiquitin (gray) (PDB code 1WR1). Residues from the Dsk2p UBA domain that interact with ubiquitin are shown as yellow sticks. Ile⁴⁴-centered hydrophobic patch residues on ubiquitin are shown as blue sticks. The positions of α1–α3 of the UBA domain are indicated. D, ribbon diagram of the cIAP1 UBA domain (PDB code 3T6P) (15) showing residues mutated in this study: M402A/F404A (MF/AA), L435A (L/A), M402A/L435A (ML/AA), E401R (E/R), N428R (N/R), and E401R/428R (EN/RR).

FIGURE 2.

Mutation of the conserved UBA domain motifs in cIAP1 destabilizes the protein. A, schematic representation of the cIAP1 constructs used in this study. Full-length cIAP1 is shown for comparison, and residue numbers are indicated (human cIAP1 numbering). B, CD spectra of WT cIAP1 B3UCR and its L/A, ML/AA, and MF/AA variants. C, trypsin-limited proteolysis comparing WT cIAP1 B3UCR with the MF/AA mutant (top) or with the ML/AA mutant (bottom). Reaction products were resolved using a 4–12% gradient NuPAGE gel (top) or 12.5% SDS-PAGE gel (bottom) and visualized by Coomassie Blue staining. The sizes of molecular mass markers (in kDa) are shown.

FIGURE 3.

Mutation of the conserved motifs perturbs the structure of several UBA domains. A, CD spectra (left) and Superdex-75 HiLoad 16/60 size exclusion profiles (right) of the UBA domain variants from cIAP1. B and C, CD spectra (left) and Superdex-75 10/300 GL size exclusion profiles of the N-terminal UBA domain (UBA1) from human HHR23A (B) and the UBA domain of S. cerevisiae EDE1 (C) as well as their MGF motif mutants: HHR23A UBA1-MY/AA and EDE1 UBA-MF/AA.

FIGURE 4.

Identification of a folded cIAP1 UBA domain mutant. A, CD spectra of WT and EN/RR cIAP1 UBA domains. B, SEC-MALLS analyses of WT, MF/AA, and EN/RR cIAP1 UBA domains. Samples were injected into a Superdex-75 10/300 GL connected in-line with a MALLS detector. The refractive index traces and the measured masses for all samples are shown. The dashed line indicates the expected masses for monomeric and dimeric species. C, CD spectra of WT and EN/RR cIAP1 B3UCR proteins. D, trypsin-limited proteolysis of cIAP1 B3UCR variants comparing the WT species with the MF/AA and EN/RR mutants. Parent bands are indicated.

FIGURE 5.

The affinity of the EN/RR UBA domain mutant for ubiquitin is significantly reduced. A, binding of ubiquitin to WT, E/R, N/R, or EN/RR cIAP1 UBA domain (as indicated) was measured using ITC. Because the ITC curve generated from injections of ubiquitin into UBA-EN/RR did not yield an accurate K_d estimation, SPR analyses were carried out (B). Equilibrium binding analyses was carried out. The sensorgrams resulting from injections of 0–450 μm ubiquitin onto chip-immobilized WT or EN/RR GST-UBA are shown to the right. Dissociation constants and stoichiometry determined from ITC and SPR measurements are shown in Table 1.

FIGURE 6.

The structure and ubiquitin binding properties of the UBA domain influence cIAP1 autoubiquitylation. In vitro assays comparing autoubiquitylation of WT and EN/RR cIAP1 B3UCR proteins (A) or WT and MF/AA proteins (C). Purified cIAP1 B3UCR proteins were incubated with Uba1 (E1), UbcH5b, Mg²⁺-ATP, and either WT ubiquitin (left) or Lys-less (K0) ubiquitin (right) for the indicated times. Reactions were stopped by adding reducing SDS-PAGE sample buffer and were resolved. Coomassie Blue staining was used to visualize the reaction products. Reactions containing no E1 were included as negative controls. Ub-cIAP1, monoubiquitylated B3UCR species. The bands just below UbcH5b indicate the formation of diubiquitin, a side product that is often observed in this condition. B, the disappearance of parent bands in A was quantified using densitometry. The mean band intensity values from triplicate measurements were plotted as a function of time. Error bars, S.D. D, plot profiles from line scanning of assay gels after 5 min incubation for WT, EN/RR, and MF/AA cIAP1 B3UCR proteins. The peaks corresponding to the parent band, monoubiquitylated (Ub-cIAP1) and diubiquitylated cIAP1 (Ub2-cIAP1), and E1 are indicated. The WT sample that was incubated without E1 is included as a reference.

FIGURE 7.

The UBA domain alone is not sufficient to promote ubiquitin transfer or E2∼Ub discharge. A, purified GST-fused cIAP1 UBA domain was incubated with UbcH5b, Mg²⁺-ATP, and WT ubiquitin at 37 °C for 1 h in the absence (−) or presence (+) of an E1 enzyme. Reactions were resolved by SDS-PAGE and visualized by staining with Coomassie Blue. B, purified untagged WT and MF/AA cIAP1 UBA-CARD fragments were incubated with assay component as in A. C, purified cIAP1 UBA domain was added in molar excess to UbcH5b-C85S∼Ub conjugate for up to 120 min.

FIGURE 8.

Ubiquitin binding by the UBA domain promotes autoubiquitylation of cIAP B2B3UCR proteins and SMAC ubiquitylation. A, in vitro assays comparing autoubiquitylation of WT and EN/RR cIAP1 B2B3UCR proteins. The assays were carried out in the same manner as in Fig. 6. Reactions were resolved by SDS-PAGE and visualized by staining with Coomassie Blue. B, autoubiquitylation assays comparing WT and EN/RR cIAP2 B2B3UCR proteins in the presence of WT or K0 ubiquitin. Reaction products were visualized with immunoblotting using anti-cIAP2 antibody. C, ubiquitylation of SMAC by WT or EN/RR cIAP1 B2B3UCR proteins was monitored over time by immunoblotting using anti-SMAC antibody (left). The intensity of monoubiquitylated SMAC (Ub-SMAC) was quantified and normalized against the intensity of SMAC parent band at t = 0 min. The mean average intensity values ± S.D. (error bars) from duplicate measurements were plotted against time (right). D, ubiquitylation of SMAC by WT or EN/RR cIAP2 B2B3UCR proteins. SMAC was probed using anti-His antibody. The intensity of Ub-SMAC was quantified as in C from triplicate measurements.

FIGURE 9.

Introduction of EN/RR mutation impairs activity in single-turnover and discharge assays. A, the time-dependent charging of UbcH5b with either WT or K0 ubiquitin was visualized using non-reducing SDS-PAGE (left). The disappearance of the UbcH5b band was quantified using densitometry. The UbcH5b band intensity at t = 0 min was set as 100%, and the mean normalized intensity values ± S.D. (error bars) from triplicate assays were plotted as a function of time (right). B, following quenching of UbcH5b∼Ub thioester conjugate formation by the addition of apyrase, WT or EN/RR cIAP1, B2B3UCR protein was added. The extent of cIAP1 autoubiquitylation over time was monitored by immunoblotting using anti-cIAP1 antibody (left). The intensity of monoubiquitylated cIAP1 (Ub-cIAP1) was quantified and normalized against the intensity of the cIAP1 parent band at t = 0 min. The mean average intensity values ± S.D. from triplicate assays were plotted against time (right). C, the ability of WT or EN/RR cIAP1 B2B3UCR protein to promote ubiquitin discharge from UbcH5b-C85S∼Ub conjugate was monitored by SDS-PAGE and Coomassie Blue staining (left). The progress of discharge was evaluated by monitoring the increase in UbcH5b band intensity over time. The mean average intensity values from triplicate measurements were normalized against the E2 band intensity at t = 0 min and were plotted as a function of time. Error bars, S.D. (right).

FIGURE 10.

Introduction of EN/RR mutation does not alter the monomer-dimer equilibria of cIAP1 B3UCR. A, SEC-MALLS analyses of cIAP1 B3UCR proteins in the absence or presence of a bivalent SMAC-mimetic compound, Compound A. A sample of WT (gray, without Compound A; black, with Compound A) or EN/RR (pink, without Compound A; red, with Compound A) cIAP1 B3UCR protein was injected into a Superdex-200 10/300 GL column connected in-line with a MALLS detector and differential refractometer. The refractive index traces and the measured molecular masses are shown. The horizontal dashed lines that appear across the trace indicate the theoretical masses for monomeric and dimeric species. B, autoubiquitylation assays comparing the activity of WT and EN/RR cIAP1 B3UCR proteins in the presence of Compound A and either WT ubiquitin (left) or K0 ubiquitin (right). C, the disappearance of parent bands was quantified using densitometry as in Fig. 6B. Error bars, S.D.

FIGURE 11.

The EN/RR mutation impairs ubiquitylation at low UbcH5b∼Ub concentrations. A, autoubiquitylation assays of WT and EN/RR cIAP1 B3UCR proteins with an increasing concentration of UbcH5b (indicated in μm below the gel). E1, UbcH5b, and Mg²⁺-ATP were incubated for 1 h at 37 °C with either WT (top) or K0 (bottom) ubiquitin prior to the addition of cIAP1 proteins and further incubation for 1 h. B, SMAC ubiquitylation in the presence of WT or EN/RR cIAP2 B2B3UCR proteins with different UbcH5b concentrations. Unmodified SMAC and ubiquitylated SMAC species were detected using anti-His antibody (top).

FIGURE 12.

The UBA domain binds to UbcH5b∼Ub conjugate. A, UbcH5b-C85S∼Ub conjugate pull-down assays using GST-tagged WT or various mutants of cIAP1 B3UCR proteins immobilized on glutathione-Sepharose resin. The mutants included in the pull-down were EN/RR, VR/AA (mutations of E2-binding residues on the RING domain: V573A/R606A), EN/RR + VR/AA, and MF/AA. The extent of conjugate binding was detected by immunoblotting with anti-UbcH5b antibody. B, GST-cIAP1 UBA variants were used to pull down conjugate (top) or UbcH5b (bottom). The amount of bound conjugate or UbcH5b was visualized as in A. C, SPR measurement of conjugate binding to chip-immobilized WT GST-cIAP1 UBA. The K_d value for the interaction was found to be 79 μm.