HUWE1 employs a giant substrate-binding ring to feed and regulate its HECT E3 domain

Abstract

HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin-proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure, where the C-terminal HECT domain heads an extended alpha-solenoid body that coils in on itself and houses various protein-protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins.

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Figure 1. HUWE1_N targets orphan proteins for degradation.

(a) Cartoon depicting the domain organization and functional roles of HUWE1. (b) HUWE1_N substrate screen. E1-E2-E3 ubiquitination assays were performed using 4 μM of the indicated substrates and either wild-type HUWE1_N or the inactive HUWE1_N-CA variant. The ubiquitinated products were visualized by using ubiquitin labeled with the DyLight488 fluorophore. (c) Linkage specificity of HUWE1_N as seen in autoubiquitination. Ubiquitin chains with the indicated linkage types were detected by mass spectrometry. The ratio of ubiquitinated to total detected peptides for each linkage type is shown as a percentage (d-e) Ubiquitination of an orphan ribosomal subunit and an orphan nucleosome subunit, respectively. Assays were performed with 500 nM substrate and only show the region of the gel containing ubiquitinated substrates, which were visualized with Ub-DyLight800 in (c) and Ub-DyLight488 in (d). The position of Rpl26 (colored red) within the ribosome (PDB 6T59) and histone H2B (colored red) within the nucleosome (PDB 3AFA) are displayed with nucleic acids colored yellow. (f) Histone H1 ubiquitination is blocked by 4WJ DNA. Assays were performed for 120 minutes. (g) An Rpl26 ubiquitination reaction was analyzed by mass spectrometry. Detected ubiquitinated lysines are indicated in the context of the ribosome (PDB 6T59). (h) Ubiquitination of orphan proteins by CeHUWE1, using 2 μM substrate and 4 μM 4WJ DNA in the last lane, and visualized with Ub-DyLight800.

Figure 2. Overall architecture of the giant E3 ligase HUWE1_N.

(a) Domain architecture of HUWE1_N. ARM repeats 1-34 are numbered, with the four insertions indicated. The position of human HUWE1 insertions, absent in HUWE1_N, are shown in square brackets. (b-c) Crystal structure of HUWE1_N. The structure is shown in cartoon representation from four different views, using the same color code as in a (catalytic Cys in red). A schematic cartoon illustrates the snake-like organization of the E3 ligase. (d) Negative stain EM analysis of CeHUWE1. The obtained EM density is shown from two viewpoints with approximate dimensions indicated. HECT and tail domains are labelled based on homology to HUWE1_N. (e) Organization and increasing complexity of HUWE1.

Figure 3. Functional insertions within the HUWE1_N ring.

(abc) Molecular details of insertions Ins1, Ins2, Ins3 and Ins4 within the alpha-solenoid. (d) Histone H1 ubiquitination assay of the HUWE1_N insertion deletion variants. Ubiquitination of 4 μM histone H1 was visualized by fluorescence with Ub-DyLight488. (e) Autoubiquitination activity of the HUWE1_N variants in the absence of a substrate. Only the region of the gel with HUWE1_N-Ub* band is shown.

Figure 4. Dynamic interfaces stabilize the HUWE1_N ring.

(a) Overview of the interface between the tail and neck regions of HUWE1_N. A cartoon schematic illustrates the three main interacting surfaces, highlighted in the right panel. (b) Detailed view of the molecular interactions at each of the three interfaces, which close the HUWE1_N ring. (c) HUWE1_N colored by B-factor, from low (blue) to high (red), with the three interfaces highlighted. The average B-factor of the HUWE1_N crystal structure is 105 Å.

Figure 5. Solution behavior of HUWE1_N.

(a) XL-MS analysis of HUWE1_N. The highest confidence detected cross-links are displayed on the primary structure of HUWE1_N. Cross-links between the neck and tail, supporting the closed ring structure, are shown in green, while cross-links between the HECT domain and the solenoid ring are shown in orange and all other cross-links in gray. (b) Sharpened electron density maps of the two CryoEM classes in two different views showing different degrees of ring opening. Maps are colored according to the schematic in Fig. 2a. (c) Breaking and shifting the ring-closure interfaces. The neck and tail contacts are compared between the crystal structure (cyan) and two cryo-EM classes (domain-based color code). The differences are summarized in a schematic cartoon (d).

Figure 6. Regulation of the HUWE1_N HECT domain.

(a) The HUWE1_N active site is spatially separated from the substrate binding ring. Catalytic cysteine and center of substrate binding ring are highlighted in green. (b) Binding of E2-Ub to the HECT domain is not impeded in the closed state of the full-length enzyme. The HUWE1_N HECT domain (N-lobe, orange; C-lobe, yellow) is superimposed with the crystal structure of the NEDD4L HECT domain (red) in complex with Ub (blue)-charged UbcH5b (magenta) (PDB 3JVZ) (c) Steric clashes between the HUWE1_N solenoid and a ubiquitin-charged HECT domain. The structure of the Rsp5 HECT domain (in red), charged with Ub (in blue) and in complex with a peptide substrate (PDB 4LCD), is superimposed on the HUWE1N HECT domain. Resultant clashes between the HECT C-lobe and ARM8-10 are highlighted in the zoomed-in drawing. (d) The isolated HUWE1_N HECT domain shows weak substrate ubiquitination. Ubiquitination of DyLight488-labelled Rpl26 was visualized by in-gel fluorescence. (e) Dysregulation of the isolated HECT domain. Autoubiquitination in the absence of a substrate was followed by using DyLight488-labelled ubiquitin. The autoubiquitinated products are indicated. (f) Single-turnover chase experiments of 1 μM E2 loaded with DyLight488-labelled ubiquitin by 2 μM HUWE1_N (Full-length and HECT), with or without 2 μM histone H1.