Human E6-AP consists of 865 amino acids, while the murine homolog is slightly longer with 885 amino acids, and these two proteins are 99% similar. E6-AP possesses five well-characterized functional domains: (i) a hect
domain, (ii) an E6 binding domain, (iii) a p53 binding domain, (iv) three nuclear receptor interaction domains and (v) an activation domain [Huibregtse et al., 1993
; Nawaz et al., 1999b
The carboxy terminal 350 amino acids form the conserved hect
domain of E6-AP, a region of homology shared by several proteins structurally and functionally related to E6-AP [Huibregtse et al., 1995
]. The hect
domain of E6-AP has a 40 kDa conserved carboxy-terminal catalytic domain that has at least four biochemical functions: 1) it binds to specific E2 ubiquitin-conjugating enzymes like UbcH7, UbcH5B and UbcH8; 2) it accepts ubiquitin from E2 enzyme, forming a ubiquitin-thioester intermediate with its active site cysteine [Kumar et al., 1997
]. A conserved cysteine residue at position 833 in the hect
domain confers the ubiquitin ligase activity of E6-AP. In vitro
studies have shown that the mutation of cysteine 833 residue to alanine or serine renders it unable to form a thioester bond with ubiquitin; 3) it transfers ubiquitin to the ε-amino groups of lysine side chain on the substrate by catalyzing the formation of an isopeptide bond; and 4) it transfers additional ubiquitin molecules to the growing end of the multi-ubiquitin chain [Huang et al., 1999
; Scheffner et al., 1995
E6-AP has been shown to function as an E3 ligase in the ubiquitination of p53 in cooperation with HPV E6 protein [Cooper et al., 2003
]. The viral E6 protein acts as an adaptor between p53 and E6-AP [Huibregtse et al., 1993
]. This E6-dependent binding of p53 to E6-AP involves amino acids 280-781 of E6-AP, which form the central leucine-rich core that is crucial for the biological function and structural stability of E6-AP. An 18 amino acid region of E6-AP, from 391-408, has been shown to be necessary and sufficient for binding to E6 [Zanier et al., 2005
]. This sequence was later characterized as a leucine-rich peptide, LQELL, which is a signature LXXLL motif (a receptor interacting motif) [Cooper et al., 2003
; Kishino et al., 1997
; Scheffner et al., 1993
; Talis et al., 1998
]. An essential intermediate step in E6-AP-dependent ubiquitination is the formation of a thioester complex between E6-AP and ubiquitin-conjugating enzymes. In the case of ubiquitin-conjugating enzyme UbcH5B, the implicated binding site corresponding to amino acids 521-679 of E6-AP, falls within the central leucine-rich core of E6-AP (). The region of E6-AP involved in complex formation with UbcH7 and UbcH8 was mapped to its hect
domain [Huang et al., 1999
; Scheffner et al., 1994
]. Recently, Mani et al.
identified AIB1 (amplified in breast cancer 1)/SRC-3 (steroid receptor coactivator-3) as a substrate for E6-AP-mediated ubiquitination and established that the steady-state level of AIB1 is regulated by E6-AP. The interaction between E6-AP and AIB1 is mediated through the carboxy terminus of AIB1 [Mani et al., 2006
Schematic representation of the functional domains of E6-AP.
Since its identification as a coactivator of SHRs, E6-AP has been shown to interact with a variety of other proteins, which includes SHRs and other coregulators. E6-AP contains 3 consensus receptor-interacting LXXLL motifs. Two of these motifs are located in the amino terminus and the third one is located within the carboxy terminus of the protein. After its characterization as a PR-interacting protein, E6-AP has also been shown to interact with other SHRs, such as AR and ER, and these interactions are enhanced in the presence of appropriate hormone [Dhananjayan et al., 2006
; Khan et al., 2006
]. Nawaz et al.
showed that E6-AP not only interacts with different SHRs, it also contains an intrinsic, transferable activation domain that extends from amino acids 170 to 680 [Nawaz et al., 1999b
]. In addition to SHRs, Dhananjayan et al.
have recently shown that E6-AP also interacts with a WW-domain binding protein -2 (WBP-2), and further investigation revealed that E6-AP and WBP-2 had additive effect on ER and PR coactivation when both of them were coexpressed [Dhananjayan et al., 2006