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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Coordinates of the HUWE1N crystal structure have been deposited at the Protein Data Bank (PDB) under accession code 7BII. Cryo-EM maps and atomic coordinates have been deposited in the Electron Microscopy Data Bank (EMDB) with accession codes EMD-12318 and EMD-12319, and in the PDB under 7NH1 and 7NH3. Source data are provided with this paper.
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We thank all members of the Clausen group for remarks on the manuscript and discussions, and the Mass Spectrometry and ProTech services from the Vienna BioCenter Core Facilities for their support. We thank the scientific staff at beamline X10SA, Paul Scherrer Institute (Villigen, Switzerland), as well as those at beamline P11, DESY (Hamburg, Germany) for support in crystallographic data collection. We thank the following cryo-EM facilities for their access and support: CEITEC MU of CIISB, Instruct-CZ Centrer (proposal no. LM2018127); the UK national Electron Bio-imaging Center (Diamond Lightsource, proposal no. EM BI25222); the EM facility at the Institute of Science and Technology (IST) Austria; and the EM facility at the Vienna BioCenter Core Facilities. We thank members of the Protein Science laboratory at Boehringer Ingelheim for support in connection with the expression and purification of HUWE1N, and V.-V. Hodirnau for help with cryo-EM analysis. This project received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (no. AdG 694978), a Marie Skłodowska-Curie grant (agreement no. 847548), an FFG Headquarter grant (no. 852936) and the Austrian Science Fund (no. FWF, SFB F 79). P.M. and O.A.P are members of the Boehringer Ingelheim Discovery Research global postdoctorate program. IMP is supported by Boehringer Ingelheim.
G.B., J.B., P.S.-B., G.F., B.W. and D. Kessler are currently employees of Boehringer Ingelheim.
Peer review information Nature Chemical Biology thanks Ronald Hay and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, The anomalous difference Fourier map at 2.5 σ is shown over representative ARM repeats from the structure of SeMet HUWE1N. Selenomethionines are colored in cyan. b, The asymmetric unit is shown with the two copies of HUWE1N colored in green and cyan, in cartoon representation. The 2FO-FC map at 1 σ is displayed. c, Representative 2FO-FC map at 1 σ of the ARM repeats.
a, The primary and tertiary structure of HUWE1N is shown as in Fig. 2a, but with every ARM repeat labelled. b, The predicted positions of the previously annotated human HUWE1 domains in the context of the HUWE1N structure. The D/E-rich region is highly conserved in character and corresponds to HUWE1N IDR1. c, The predicted positions of X-linked intellectual disability mutations on the structure of HUWE1N. Mutated residues are shown in sphere representation, with HECT domain mutations in orange and all other mutations in blue.
a, Comparison of the HUWE1N ARM repeats with peptide-binding ARM repeat proteins. HUWE1N is shown in gray, bound peptides in red, and in the upper panel p120 catenin (PDB 3L6X) in light blue, and in the lower panel, a designed ARM repeat protein (PDB 6S9O) in dark blue. b, The surface conservation of the ring-closing interface is shown as a spectrum with magenta indicating strong conservation, and cyan low conservation. The score was calculated using AACon1 from an alignment of 49 HUWE1 orthologs. The protein was separated to show the surface of the interface on the side of the neck (upper panel) and tail (lower panel). c, Comparison of the two chains in the asymmetric unit superimposed by their first 300 amino acids.
a, Overall fit between the model and map for Class 1. b, Overall fit between the model and map for Class 2. c,d, Density for ARM repeat helices in regions of the structure with high local resolution.
a, Relief of potential clashes during catalysis. 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 from the crystal structure in the upper panel, and CryoEM Class 2 in the lower panel. b, ARM34 as a molecular hinge. The H1, H2, H3 helices of ARM34 and the N-terminal HECT helix are shown in the leftmost panel together with an overview and cartoon presentation. The two copies present in the crystal structure of an extended HUWE1 HECT domain (PDB 5LP8) are overlaid on our structure and shown in cyan. Structural rearrangements of ARM34 helices H2 and H3 are indicated. c, The position of F2105. d, Autoubiquitination activity of full-length CeHUWE1 compared to the isolated HECT domain (residues 3793-4180) followed using Ub-DyLight800.
Supplementary Tables 1 and 2 and Fig. 1.
Electron density quality of the HUWE1N crystal structure. The 2FO-FC map at 1 σ is shown with the structure colored as in Fig. 2.
Heterogeneity of the Cryo-EM data analyzed using CryoDRGN.
Heterogeneity of the Cryo-EM data analyzed using CryoDRGN, second viewpoint.
Unprocessed and Coomassie-stained gels, and mass spectrometry datasets.
Unprocessed and Coomassie-stained gels.
Mass spectrometry data used for Fig. 5a and Extended Data Fig. 4.
Unprocessed and Coomassie-stained gels.
Unprocessed and Coomassie-stained gels.
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Grabarczyk, D.B., Petrova, O.A., Deszcz, L. et al. HUWE1 employs a giant substrate-binding ring to feed and regulate its HECT E3 domain. Nat Chem Biol 17, 1084–1092 (2021). https://doi.org/10.1038/s41589-021-00831-5