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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.

The HECT-type ubiquitin ligase E6AP (UBE3A) is critically involved in several neurodevelopmental disorders and human papilloma virus-induced cervical tumorigenesis; the structural mechanisms underlying the activity of this crucial ligase, however, are incompletely understood. Here, we report a crystal structure of the C-terminal lobe (“C-lobe”) of the catalytic domain of E6AP that reveals two molecules in a domain-swapped, dimeric arrangement. Interestingly, the molecular hinge that enables this structural reorganization with respect to the monomeric fold coincides with the active-site region. While such dimerization is unlikely to occur in the context of full-length E6AP, we noticed a similar domain swap in a crystal structure of the isolated C-lobe of another HECT-type ubiquitin ligase, HERC6. This may point to conformational strain in the active-site region of HECT-type ligases with possible implications for catalysis.Significance Statement:The HECT-type ubiquitin ligase E6AP has key roles in human papilloma virus-induced cervical tumorigenesis and certain neurodevelopmental disorders. Here, we present a crystal structure of the C-terminal, catalytic lobe of E6AP, providing basic insight into the conformational properties of this functionally critical region of HECT-type ligases.

Ubiquitin regulates countless physiological and pathophysiological processes and, as a result, ubiquitination enzymes have emerged as key targets on the drug discovery arena. Here we discuss recent progress in the development of small-molecule inhibitors directed at ubiquitin ligases and highlight novel strategies that may pave the way for rational drug design approaches.

The biophysical analysis of multidomain proteins often is difficult because of overlapping signals from the individual domains. Previously, the fluorescent unnatural amino acid p-cyanophenylalanine has been used to study the folding of small single-domain proteins. Here we extend its use to a two-domain protein to selectively analyze the folding of a specific domain within a multidomain protein.

The signal adapter protein c-CrkII from chicken but not from human uses isomerization at Pro238 in the SH3C domain to regulate the activity of the SH3N domain. The different behavior of human and chicken c-CrkII originates from only two differences in sequence, at positions 239 after Pro238 and 272 in the N-Src loop of SH3C. We analyzed the kinetics of substrate binding to SH3N and an assay for its coupling with Pro238 isomerization in SH3C to identify the molecular path from Pro238 to the substrate binding site of SH3N. The trans→cis isomerization at Pro238 and a relocation of Phe239 re-organize the energetics of a hydrophobic cluster in the N-Src loop of SH3C and re-shape this region to optimize its interactions with SH3N. Concomitantly, the backbone becomes strained at Met272. We suggest that, in human c-CrkII, movement at position 239 and strain at position 272 are not tolerated because the β-branched residues Ile239 and Val272 restrain the backbone mobility and thus destabilize the cis Pro238 form.