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Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis for catalytic priming

Nature Structural & Molecular Biology volume 20, pages 696–701 (2013)Cite this article

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Abstract

Homologous to E6-AP C terminus (HECT) E3 ligases recognize and directly catalyze ligation of ubiquitin (Ub) to their substrates. Molecular details of this process remain unknown. We report the first structure, to our knowledge, of a Ub-loaded E3, the human neural precursor cell–expressed developmentally downregulated protein 4 (Nedd4). The HECTNedd4~Ub transitory intermediate provides a structural basis for the proposed sequential addition mechanism. The donor Ub, transferred from the E2, is bound to the Nedd4 C lobe with its C-terminal tail locked in an extended conformation, primed for catalysis. We provide evidence that the Nedd4-family members are Lys63-specific enzymes whose catalysis is mediated by an essential C-terminal acidic residue.

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Figure 1: Structure of the Ub-loaded Nedd4 HECT in complex with Ub.
Figure 2: UbD is primed for catalysis by C-terminal tail-locking.
Figure 3: The C-terminal acidic residue is critical for the catalysis of Nedd4 family members.
Figure 4: Ub chain specificity of Nedd4 family members.
Figure 5: Critical role of the helix α14 for HECT C-terminal positioning.

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Acknowledgements

We thank P. Romano for critically reading the manuscript and for helpful discussions and advice and the staff at the European Synchrotron Radiation Facility and Swiss Light Source for assistance in data collection. This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC, IG11627), the European Community (Network of Excellence FP6, 100601–201012) and the European Molecular Biology Organization Young Investigator Program to S. Polo. Access to the High Throughput Crystallization Laboratory of the European Molecular Biology Laboratory Grenoble outstation was funded by the European Community's Seventh Framework Programme (FP7/2007-2013) PCUBE (grant agreement 227764).

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Authors and Affiliations

  1. Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy

    Elena Maspero, Eleonora Valentini, Sara Mari, Paolo Soffientini & Simona Polo

  2. Department of Experimental Oncology, Crystallography Unit, European Institute of Oncology, Milan, Italy

    Valentina Cecatiello & Sebastiano Pasqualato

  3. Dipartimento di Scienze della Salute, Università degli Studi di Milano, Milan, Italy

    Simona Polo

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  1. Elena Maspero
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Contributions

E.M. conducted the experiments with the help of E.V. and S.M.; V.C. assisted in crystallization; P.S. carried out MS analysis; S. Pasqualato performed structure determination and description; S. Pasqualato and E.M. participated in experimental design and data analysis; S. Polo conceived of the project, interpreted the results and wrote the paper.

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Correspondence to Sebastiano Pasqualato or Simona Polo.

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Maspero, E., Valentini, E., Mari, S. et al. Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis for catalytic priming. Nat Struct Mol Biol 20, 696–701 (2013). https://doi.org/10.1038/nsmb.2566

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