Structure of the HECT:ubiquitin complex and its role in ubiquitin chain elongation - PubMed (original) (raw)

Structure of the HECT:ubiquitin complex and its role in ubiquitin chain elongation

Elena Maspero et al. EMBO Rep. 2011 Apr.

Abstract

Several mechanisms have been proposed for the synthesis of substrate-linked ubiquitin chains. HECT ligases directly catalyse protein ubiquitination and have been found to non-covalently interact with ubiquitin. We report crystal structures of the Nedd4 HECT domain, alone and in complex with ubiquitin, which show a new binding mode involving two surfaces on ubiquitin and both subdomains of the HECT N-lobe. The structures suggest a model for HECT-to-substrate ubiquitin transfer, in which the growing chain on the substrate is kept close to the catalytic cysteine to promote processivity. Mutational analysis highlights differences between the processes of substrate polyubiquitination and self-ubiquitination.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

Structure of the HECTNedd4 domain in apo form and in complex with ubiquitin. (A) GST pull-down assay with the HECT domains of various Nedd4 family HECT E3 ligases. GST-fusion proteins were incubated for 2 h at 4°C in YY buffer with synthetic Lys 63-polyubiquitin chains and analysed by IB as indicated. Coomassie staining shows comparable loading of GST proteins. Similar results were obtained with linear and Lys 48-polyubiquitin chains (not shown). (B) Overall structure of HECTNedd4 (N-lobe, blue; C-lobe, green). The red dotted line indicates the boundary between the large and small subdomains of the N-lobe. (C) Overall structure of HECTNedd4 in complex with ubiquitin (yellow). The HECT structure is represented in the same orientation as in B; N-lobe, light blue; C-lobe, dark green. (D) Superposition on the large subdomain of the N-lobe of HECTNedd4 and HECTNedd4:ubiquitin. In the complex (light blue), the β5–β6 hairpin of the small subdomain of the N-lobe is closer to the large subdomain, with respect to the isolated HECT (dark blue). (E) Sequence alignment of the HECTNedd4 domain with other crystallized HECT domains. Secondary structure elements are depicted. Dotted line indicates that the residues were not visible in the electron density maps. Yellow circles indicate residues in contact with ubiquitin in the structure of HECTNedd4:ubiquitin (according to PISA; Krissinel & Henrick, 2007). Numbering refers to Nedd4 sequence. GST, glutathione _S_-transferase; IB, immunoblotting; Ub, ubiquitin.

Figure 2

Figure 2

HECTNedd4:ubiquitin interaction and mutant validation. (A) Close-up view of HECTNedd4 N-lobe:ubiquitin interaction. (B) GST pull-down assay with the indicated Nedd4 constructs and Lys 63-linked polyubiquitin chains was performed as described in Fig 1A. (C) Fluorescence-polarization assay with the indicated Nedd4 constructs and monomeric ubiquitin was performed. The HECTNedd4:ubiquitin interaction displays a moderate affinity with a _K_D of 11 μM, F707A mutant displays a thirty times lower affinity. Details are described in the supplementary Methods online and similar results for the Y605A mutant obtained by SPR assay are in supplementary Fig S1 online. IB, immmunoblotting; GST, glutathione _S_-transferase; SPR, surface plasmon resonance; Ub, ubiquitin; WT, wild type.

Figure 3

Figure 3

Disruption of HECTNedd4:ubiquitin interaction impairs substrate polyubiquitination. (A) Mutations do not affect E2 binding. Left panel: GST pull-down assay with the indicated HECT mutants and the E2 enzyme Ube2D3. IB was performed as indicated. Coomassie staining shows comparable loading of GST proteins. Right panel: the HECTNedd4:Ube2D3 interaction displays a modest affinity, that is not perturbed by the F707A and Y605A mutations. SPR assay was performed as described in the supplementary methods online. (B) Mutations do not affect the kinetics of the E2-to-HECT transthiolation process. The transfer of ubiquitin was monitored by quenching the reaction at different time points, with the addition of Laemmli buffer with or without the reducing agent (100 mM DTT). Arrow indicates thioesther HECT∼ubiquitin (−DTT) or monoubiquitinated HECT (+DTT) running at the same position. DTT-resistant higher molecular bands represent self-ubiquitinated HECT. Similar results were obtained with Y605A mutant. (C) Mutations impair substrate polyubiquitination. Upper panel: GST-γENaC ubiquitination kinetics with WT HECT and F707A mutant (ubiquitin (pellet)). Middle panel: Coomassie staining showing comparable loading of GST proteins. Lower panel: kinetics of free ubiquitin chain formation (ubiquitin (supernatant)) during the reaction. IB was performed as indicated. (D) Self-ubiquitination kinetics with WT HECT and Y605A and F707A mutants. IB was performed as indicated. Coomassie staining shows comparable loading of HECT proteins. Similar results were obtained with full-length Nedd4 mutants. DTT, dithiothreitol; ENaC, epithelial Na+ channel; GST, glutathione _S_-transferase; IB, immunoblotting; SPR, surface plasmon resonance; Ub, ubiquitin; WT, wild type.

Figure 4

Figure 4

Ubiquitin binding to the HECTNedd4 domain is compatible with HECTNedd4-like:E2∼ubiquitin complex and does not dictate chain specificity. (A) Substrate ubiquitination assay with the indicated ubiquitin KR mutants was performed. The reaction was quenched after 30 min for the WT HECT and after 60 min for the F707A mutant. Upper panel: GST-γENaC ubiquitination with the indicated constructs. Middle panel: Coomassie staining showing comparable loading of GST proteins. Lower panel: free ubiquitin chain formation during the reaction. IB was performed as indicated. Lower panel: 1 μg of ubiquitin KR mutants were loaded for comparison and visualized by Coomassie staining. (B) Position of ubiquitin lysines in the Nedd4 HECT/ubiquitin complex. HECT N-lobe is shown as surface representation, whereas the C-lobe and ubiquitin are shown as cartoon representations. Ubiquitin lysine side chains are indicated in sticks. Six of the seven ubiquitin lysines are shown, K 6 being in the back. (C) Model of Ubch5B∼Ub:C-lobe complex (Kamadurai et al, 2009) binding to the N-lobe:ubiquitin complex. Details are in the supplementary Methods online. C 867 on the HECT and K 63 on the Ub are shown. ENaC, epithelial Na+ channel; GST, glutathione _S_-transferase; IB, immunoblotting; KR, lysine-to-arginine mutation; Ub, ubiquitin; WT, wild type.

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