Conjugation of ubiquitin to target proteins involves three groups of enzymes, the E1, E2, and E3 enzymes, which function cooperatively in a cascade of ubiquitin transfer reactions [1
]. The E3 enzymes, or ubiquitin ligases, interact with both an upstream E2 enzyme and specific target proteins, facilitating protein ubiquitination. RING type E3s, which include both monomeric proteins (e.g
., Mdm2, Cbl) and multimeric protein complexes (e.g
., cullin-based E3s, the APC), function primarily as scaffolds, orienting the E2~ubiquitin thioester complex and target protein for ubiquitin transfer. HECT domain E3s are unique among the several classes of E3s in that ubiquitin is transferred from the E2 to an active-site cysteine within the HECT domain, forming an E3~ubiquitin thioester complex [2
]. Ubiquitin is then transferred to target proteins that are bound to the substrate recognition determinants of the E3.
The HECT domain, itself, is an approximately 350 amino acid domain that is always found at the C-terminus of the ligase, and structural information is available for three HECT domains and one HECT domain – E2 complex [3
]. Briefly, the HECT domain consists of a larger N lobe that contains the E2 binding site, and a smaller C lobe that contains the active-site cysteine. The lobes are connected by a short flexible linker, and conformational flexibility about this linker appears to be critical for juxtaposing the active site cysteines of the E2 and E3 in order to facilitate the transthiolation reaction. Most aspects of isopeptide bond catalysis are uncharacterized, particularly with respect to how polyubiquitin chains are formed, although one key requirement for ubiquitin transfer from the active-site cysteine to the target protein is a phenylalanine residue located, in most HECT E3s, four amino acids from the end of the protein [6
]. This may function to properly orient the ubiquitin molecule that is tethered to the active-site cysteine.
The smallest HECT E3s are approximately 90 kD and the largest are over 500 kD. The region upstream of the HECT domain contains determinants of substrate specificity, intracellular localization, and in some cases, as discussed below, regulation. E6AP was the first HECT E3 identified, and this protein mediates the ubiquitination of p53 in cells that express the human papillomavirus (HPV) E6 oncoprotein. This appears to be an unregulated reaction in that cells that express E6 constitutively ubiquinate p53, causing it to be constantly degraded. There are approximately 50 HECT E3s in humans and five in S. cerevisiae
. Only a few of these have been characterized in detail, although the best characterized subgroup of HECT E3s, with respect to both substrate identification and regulation, are the C2-WW-HECT E3s [7
]. These proteins contain an N-terminal C2 phospholipid binding domain and multiple (from two to four) WW domains in the central portion of the protein that mediate enzyme-substrate interactions. Yeast Rsp5 belong to this group, as well as seven human proteins, including Nedd4, Smurf1, Smurf2, and Itch. The C2-WW-HECT E3s have been more amenable to analysis than other HECT E3s, in part because of genetic tools in yeast and the fact that it has been relatively simple to isolate proteins (substrates) that bind specifically to WW domains. The regulation of substrate recognition by C2-WW-HECT E3s, as well as the physiologic functions of these enzymes, has been reviewed recently [7
]. We will focus here on mechanisms of regulation of the catalytic activity of HECT E3s.