Mechanism of ubiquitin chain synthesis employed by a HECT domain ubiquitin ligase

Abstract

Homologous to E6AP C-terminal (HECT) ubiquitin (Ub) ligases (E3s) are a large class of enzymes that bind to their substrates and catalyze ubiquitination through the formation of a Ub thioester intermediate. The mechanisms by which these E3s assemble polyubiquitin chains on their substrates remain poorly defined. We report here that the Nedd4 family HECT E3, WWP1, assembles substrate-linked Ub chains containing Lys-63, Lys-48, and Lys-11 linkages (Lys-63 > Lys-48 > Lys-11). Our results demonstrate that WWP1 catalyzes the formation of Ub chains through a sequential addition mechanism, in which Ub monomers are transferred in a successive fashion to the substrate, and that ubiquitination by WWP1 requires the presence of a low-affinity, noncovalent Ub-binding site within the HECT domain. Unexpectedly, we find that the formation of Ub chains by WWP1 occurs in two distinct phases. In the first phase, chains are synthesized in a unidirectional manner and are linked exclusively through Lys-63 of Ub. In the second phase, chains are elongated in a multidirectional fashion characterized by the formation of mixed Ub linkages and branched structures. Our results provide new insight into the mechanism of Ub chain formation employed by Nedd4 family HECT E3s and suggest a framework for understanding how this family of E3s generates Ub signals that function in proteasome-independent and proteasome-dependent pathways.

Keywords: E3 ubiquitin ligase; HECT; polyubiquitin chain; protein degradation; ubiquitin; ubiquitylation (ubiquitination).

Figure 1.

Identification of E2s that cooperate with WWP1. A, Coomassie-stained gels showing purified protein preparations used for ubiquitination assays with WWP1. The KLF5^1–350 and WBP2-CT substrates were His₆-tagged. All other constructs were untagged full-length proteins. B, time-course ubiquitination assay carried out with E1, UbcH7, WWP1, Ub, and His₆-KLF5^1–350. Reactions were carried out in the presence of wild-type Ub (WT Ub) or a lysine-less Ub mutant (0K Ub). Detection was by anti-His₆ immunoblotting. C, ubiquitination of KLF5^1–350 was assayed as described in B in the presence of the indicated E2 and wild-type Ub. Reactions were carried out for 3 h at 37 °C. The asterisk represents a cross-reacting band corresponding to WWP1. D, KLF5^1–350 ubiquitination assays were conducted as described above in the presence of UbcH7, UbcH5a, or UbcH6. E, results of two independent experiments conducted as shown in D were quantified by measuring the disappearance of unmodified KLF5^1–350 via densitometry. The rate of KLF5^1–350 ubiquitination, calculated from initial data points in the linear phase of the reaction, was 38.9 ± 1.9 fmol/min for UbcH7, 10.8 ± 0.2 fmol/min for UbcH5a, and 6.4 ± 0.5 fmol/min for UbcH6. Error bars represent S.D. of the mean.

Figure 2.

Linkage specificity of Ub chains synthesized by WWP1. A, diubiquitin chain synthesis assays were conducted in the absence or presence of the WWP1 HECT domain (top panel) and in the absence or presence of full-length WWP1 (bottom panel). Reactions were quenched at the indicated times and resolved by Tris-Tricine SDS-PAGE, and the reaction products were analyzed by anti-ubiquitin immunoblotting. 2-fold higher concentrations of UbcH7 and WWP1 were required for robust detection of diubiquitin by full-length WWP1. B, diubiquitin chain synthesis assays were carried out with the WWP1 HECT domain as described in A, except the indicated lysine to arginine Ub mutant was substituted into the reaction. C, anti-His₆ western blots showing the starting material used for quantification of Ub chain linkages by mass spectrometry. Ubiquitinated KLF5^1–350 and WBP2-CT substrates were purified from all other reaction components under denaturing conditions. D, quantification of Ub chain linkages synthesized by WWP1 using the ubiquitinated material shown in C. Lys-63, Lys-48, and Lys-11 linkages were the only linkages detected (see “Experimental procedures” for details). Data are represented as a percentage of the sum of all linkages detected. Error bars represent triplicate measurements (± S.D. of the mean).

Figure 3.

Linkage analysis of Ub chains assembled by WWP1 on substrates. A, WBP2-CT ubiquitination was assayed in the presence of wild-type Ub (WT Ub) or a lysine-less Ub mutant (0K Ub). Reactions were quenched in 8 m urea at the indicated times, and ubiquitinated products were purified under denaturing conditions prior to SDS-PAGE. Detection was by anti-HA immunoblotting. B, ubiquitination of KLF5^1–350 was assayed as described in Fig. 1, but the reactions were carried out for 60 min in the presence of wild-type Ub or the indicated lysine to arginine Ub mutant. C, WBP2-CT was ubiquitinated by WWP1 in the presence of wild-type Ub or the indicated lysine to arginine Ub mutant. Reactions were carried out for 3 min and quenched directly in sample buffer, and the reaction products were detected by anti-T7 immunoblotting. D, time-course ubiquitination assays were carried out with WBP2-CT as the substrate in the presence of wild-type, K63R, or K48R Ub. Time points were withdrawn at the indicated times and analyzed as described in C. The asterisk represents a cross-reacting band corresponding to WWP1. E, results shown in D were quantified via densitometry by measuring the percentage of Ub chains >125 kDa in mass present in the reaction. The cross-reacting band denoted by the asterisk was used to approximate the position of 125 kDa for quantification purposes. Note that the 15-min time point has been omitted from the quantification.

Figure 4.

WWP1 assembles Ub chains on WBP2-CT^K222 via a sequential addition mechanism. A, schematic of full-length WBP2, WBP2-CT (C-terminal fragment of WBP2), and the WBP2-CT^K222 single-lysine substrate. The position of each lysine and PY motif is indicated. Lys-222 is highlighted in red. B, ubiquitination of WBP2-CT^K222 was assayed in the presence of wild-type Ub (WT Ub) or a lysine-less Ub mutant (0K Ub) to confirm modification on a single site. Reactions were quenched at the indicated times, and the reaction products were purified under denaturing conditions prior to SDS-PAGE. Detection was by anti-T7 immunoblotting. C, time-course ubiquitination assay carried out with WBP2-CT^K222 and full-length WWP1 in the presence of wild-type Ub to evaluate the mechanism of Ub chain synthesis. The reaction was quenched directly in sample buffer at the indicated times, and the reaction products were analyzed by anti-T7 immunoblotting. The asterisk denotes a cross-reacting band corresponding to WWP1. D, quantification of the results shown in C. Band intensities were quantified by infrared fluorescent scanning on the Odyssey imager (LI-COR). Data points represent the percentage of each WBP2-CT^K222 species present in the reaction at each time point.

Figure 5.

Sequential ubiquitination on WBP2-CT^K222 under different reaction conditions. A, ubiquitination of WBP2-CT^K222 was assayed in the presence of full-length WWP1 or the WWP1 HECT domain. Time points were quenched in 8 m urea, and the reaction products were purified under denaturing conditions prior to SDS-PAGE. Detection was by anti-T7 immunoblotting. B, time-course ubiquitination assay was carried out with WBP2-CT^K222 as described in Fig. 4C, except UbcH7 was first charged with Ub and then added to the preformed WWP1·WBP2-CT^K222 complex to initiate the reaction. The asterisk represents a cross-reacting band corresponding to WWP1. C, results shown in B were quantified by infrared fluorescent scanning on the Odyssey imager (LI-COR). Data points were plotted as described in Fig. 4D. D, ubiquitination of WBP2-CT^K222 was assayed as described in Fig. 4C, but in the presence of the Lys-63-only Ub mutant. The absence of high molecular weight polyubiquitinated species suggests the presence of branched structures and mixed linkages in chains longer than four subunits. Note that a fraction of the total signal in each lane was lost over time due to aggregation of the Lys-63-only Ub mutant.

Figure 6.

The WWP1 HECT domain contains a low-affinity, noncovalent Ub-binding site. A, binding of GST-Ub to the WWP1 and Rsp5 HECT domains was assayed by incubating immobilized His₆-tagged HECT domains with an E. coli lysate containing GST-Ub or GST alone. Input and bound proteins (top panels) were detected by anti-GST immunoblotting. HECT domains were visualized by Coomassie staining (bottom panel). B, fluorescence anisotropy assay was performed with fluorescein-labeled ubiquitin and increasing concentrations of the WWP1 or Rsp5 HECT domains. Normalized anisotropy is plotted as a function of HECT domain concentration. The K_d value for the Rsp5 HECT-Ub interaction was determined to be 70.3 ± 8.9 μm. It was not possible to determine the K_d value for the WWP1 HECT-Ub interaction from this experiment. C, structural alignment of the WWP1 HECT domain N-lobe (PDB 1ND7) with the Ub-binding Rsp5 N-lobe (PDB 3OLM). The root-mean-square deviation was 2.9 Å over 240 Cα. Residues present at the interface between the Rsp5 N-lobe and Ub are shown. D, sequence alignment of the HECT domain N-lobes from yeast Rsp5 and human Nedd4 family members (constructed in Clustal Omega). Residues highlighted in purple make direct contacts with Ub in the structure of Rsp5 HECT·Ub (PDB 3OLM). The conserved Phe present in three of the four Ub-binding N-lobes (red circle) is absent in WWP1, WWP2, and Itch. A colon is used to represent a gap in sequences that are not part of the Ub-binding site. E, ubiquitination of KLF5^1–350 was assayed as described in Fig. 1, except the indicated WWP1 protein (wild-type or mutant) was added to the reaction. F, results of two independent experiments conducted as shown in E were quantified as described in Fig. 1E. The rate of KLF5^1–350 ubiquitination was 38.8 ± 1.9 fmol/min for wild-type WWP1, 11.3 ± 0.4 fmol/min for the Y633A mutant, and 11.8 ± 0.3 fmol/min for the I649D mutant. Error bars represent S.D. of the mean.

Figure 7.

Specificity for Lys-63-linked Ub chain synthesis by WWP1 is a function of chain length. A, anti-T7 western blots showing the starting material used for quantification of Ub chain linkages formed on WBP2-CT^K222 over time by mass spectrometry. The reaction was quenched in 8 m urea at the indicated times, and the ubiquitinated products were purified under denaturing conditions prior to SDS-PAGE. B, quantification of Ub chain linkages using the ubiquitinated material shown in A. Results are represented as a percentage of the sum of all linkages detected. Error bars represent triplicate measurements (±S.D. of the mean). C, ubiquitination of WBP2-CT^K222 was assayed in the presence of wild-type Ub as described in Fig. 4B. The reaction products were probed with linkage-specific antibodies to detect Lys-63-, Lys-48-, and Lys-11-linked chains. D, ubiquitination of WBP2-CT^K222 was assayed in two rounds of reaction to detect a branching activity by WWP1. WBP2-CT^K222 was first modified with a Lys-63-linked Ub₄ chain, such that the substrate was fully converted to its tetraubiquitinated form (lane 2). A molar excess of a lysine-less Ub (0K Ub) was then added to the reaction. The reaction products were quenched at the indicated times as described in A and detected by anti-T7 immunoblotting. The asterisk denotes a cross-reacting species that originates from an aggregate present in the Lys-63-linked Ub₄ preparation.

Figure 8.

Two-phase model for substrate-linked Ub chain synthesis by WWP1. Once the initiator Ub has been conjugated to the substrate, the formation of Ub chains by WWP1 occurs in two distinct phases. In phase 1, Ub monomers are transferred from the active-site cysteine of WWP1 to the distal Ub on the end of the growing chain. This phase of Ub chain synthesis is unidirectional and occurs with strict specificity for Lys-63 linkages. In phase 2, Ub chain assembly proceeds in a manner that consists of mixed Lys-48, Lys-11, and Lys-63 Ub linkages and is characterized by formation of branched structures. Ub chain synthesis in this phase is multidirectional, resulting in an increase in the density of Ub subunits surrounding the substrate. Substrates that dissociate from the E3 rapidly and therefore do no enter phase 2 are modified with monoubiquitin or short Lys-63-linked chains (2–4 subunits), which typically mark proteins for non-proteasomal pathways. Substrates that remain more stably associated with the E3 and therefore enter into phase 2 have the potential to be modified with larger, more complex Ub chains containing mixed linkages, which are likely to serve as efficient proteasome targeting signals in vivo.