Abstract
Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders. Yet the structural underpinnings of activity and specificity in this crucial ligase are incompletely understood. Here, we unravel the determinants of ubiquitin recognition by the catalytic domain of E6AP and assign them to particular steps in the catalytic cycle. We identify a functionally critical interface that is specifically required during the initial formation of a thioester-linked intermediate between the C terminus of ubiquitin and the ligase-active site. This interface resembles the one utilized by NEDD4-type enzymes, indicating that it is widely conserved across HECT ligases, independent of their linkage specificities. Moreover, we uncover surface regions in ubiquitin and E6AP, both in the N- and C-terminal portions of the catalytic domain, that are important for the subsequent reaction step of isopeptide bond formation between two ubiquitin molecules. We decipher key elements of linkage specificity, including the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site Lys-48. Intriguingly, mutation of Glu-51, a single residue within this region, permits formation of alternative chain types, thus pointing to a key role of ubiquitin in conferring linkage specificity to E6AP. We speculate that substrate-assisted catalysis, as described previously for certain RING-associated ubiquitin–conjugating enzymes, constitutes a common principle during linkage-specific ubiquitin chain assembly by diverse classes of ubiquitination enzymes, including HECT ligases.
Highlights
Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders
In line with the notion that donor ubiquitin recognition by E6AP resembles that of NEDD4-type ligases during thioester transfer, we found that the L814A and A842I HECT domain variants are impaired in thioester formation with ubiquitin compared with the wildtype (WT) (Fig. 3A)
To further decipher the requirements of acceptor ubiquitin recognition during E6AP-mediated ubiquitin linkage formation, we focused on an array of hydrophobic residues of ubiquitin (Leu-8, Ile-44, and Val-70), often referred to as the “hydrophobic patch” that is critical for E6AP activity [49]
Summary
E1, ubiquitin-activating enzyme; E2, ubiquitinconjugating enzyme; E3, ubiquitin ligase; FP, fluorescence polarization; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; -ME, -mercaptoethanol; DTNB, 5,5Ј-dithiobisnitrobenzoic acid; TCEP, tris(2-carboxyethyl)phosphine; CAPSO, 3-(cyclohexylamino)-2-hydroxy-1propanesulfonic acid; AQUA, absolute quantification; Ub, ubiquitin; Ubl, ubiquitin-like modifier; PDB, Protein Data Bank. During ubiquitin chain formation, the ubiquitin molecule that is thioester-linked to the catalytic cysteine of the ligase is referred to as the “donor”; the ubiquitin molecule that presents a primary amino group for the nucleophilic attack on the C terminus of the donor is known as the “acceptor.” The orientation of the acceptor toward the donor determines which primary amino group is modified and holds the key to specificity in linkage formation and associated signaling responses. How this mechanism is structurally implemented in HECT ligases is not entirely clear. We reveal that the N-lobe of E6AP interacts with ubiquitin and that the exosite region is required for isopeptide bond formation and influences ubiquitin binding, in a similar yet not identical manner as characterized for NEDD4 ligases
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