Protein ubiquitination is an essential eukaryotic pathway that influences nearly all cellular processes1
. The conjugation of ubiquitin to a protein substrate requires a cascade of enzymatic reactions beginning with the ubiquitin activating enzyme (E1). The E1 uses ATP to form a thioester bond between the carboxyl-terminus of ubiquitin and the E1 active site cysteine. The E1 then binds a ubiquitin conjugating enzyme (E2) and transfers ubiquitin to the E2 active site cysteine. The next step in the pathway requires a ubiquitin ligase (E3) which utilizes distinct domains to bind both E2 and substrate. The two major classes of E3s bind E2s using either a RING (r
ene) domain or a HECT (h
omology to E
erminus) domain. E3s containing a HECT domain form a third thioester intermediate with ubiquitin prior to transfer to substrate2
. E3s that contain a RING domain do not form a covalent intermediate with ubiquitin, but rather bring the substrate in proximity of the E2 whereby transfer of ubiquitin proceeds directly from E2 to substrate.
The importance of specific E3-substrate interactions is apparent as aberrations can lead to improper substrate regulation and severe physiological consequences3; 4
. Often overlooked is the specificity of E2-E3 interactions as similar outcomes could potentially arise. The hierarchical nature of the ubiquitin pathway presents a formidable task in dissecting all E2-E3 and E3-substrate interactions. For example in humans there is a single E1, ~30 E2s and hundreds of potential E3s. In general, a given E2 will interact with multiple E3s while E3s only function with a limited subset of E2s4
. Additionally, a given E3 may have more than one substrate and some substrates can be recognized by multiple E3s4
. One possible method to dissect the network of interactions is to design altered specificity E2-E3 pairs that will function together but not with their wild type partners. Such an approach has been taken by Marc Timmers and colleagues who used a charge swap interaction across the UbcH5b–cNOT4 interface to create an altered specificity E2-RING pair5
. The Timmers study, as well as others, have helped map the determinants of E2-RING specificity5; 6; 7; 8; 9
. Here, we examine the sequence determinants of E2-HECT specificity. In particular, we focus on the HECT domain protein, E6AP.
E6AP is the founding member of the HECT domain class of E3s. It was first identified as a protein that cooperates with the E6 protein from oncogenic forms of the human papillomavirus to down regulate the p53 tumor suppressor10
. The conserved ~350 amino acid HECT domain adopts a bilobal structure and is always found at the C-terminus of E3s. A three residue hinge connects the N-terminal, E2 binding lobe with the catalytic C-terminal lobe and the interface created by the two lobes forms a highly conserved cleft containing the catalytic cysteine11
(). Three crystal structures of HECT domains have revealed significantly different orientations of the two lobes implying conformational changes are necessary for catalysis11; 12; 13
. Hinge mutations to proline that restrict the conformational flexibility of the HECT domain result in impaired E3 activity12
. Also, some patients with Angelman syndrome (AS), a severe neurological disorder linked to E6AP, have acquired mutations within the conserved cleft which have been shown to reduce E6AP ligase activity14; 15
. Despite the significant progress on AS associated E6AP mutations, none of the identified E6AP substrates have been directly linked to the disorder16
. A better understanding of HECT domains as well as their E2s and substrates may help combat such E3 associated diseases.
Figure 1 Cartoon representation of the UbcH7–E6AP crystal structure11. (a) The E6AP N-terminal lobe (blue) and C-terminal lobe (cyan) are connected by a 3 residue hinge (red). E6AP uses a ~80 amino acid subdomain in the N-terminal lobe to bind (more ...)
All E2 enzymes possess a conserved catalytic core domain of approximately 150 amino acids though some E2s also contain N- and C-terminal extensions which serve diverse functions16
. The E2 core domain contains the residues responsible for catalysis as well as binding E1 and E3s. Crystal structures of the E2 UbcH7 bound to a HECT E3, E6AP11
, and a RING E3, c-Cbl17
, revealed that E2s utilize similar residues to bind both classes of E3s. The majority of E3 binding residues are contained within the N-terminal helix (helix 1) as well as loops 4 and 7 11; 17
(). In both E2-E3 structures, the primary contact at the interface arises from F63 on loop 4 of UbcH7 which is buried in a hydrophobic groove created by the E311; 17
. This phenylalanine is present in all E2s that have been shown to function with HECT E3s11; 18
. The sequences of helix 1 and loop 7 of HECT binding E2s are more varied, and it has been proposed that these regions of the interface determine which HECT domains an E2 will bind11
. HECT E3s have been shown to form selective interactions with at least 3 distinct E2 subfamilies19; 20; 21; 22
. To decipher the sequence determinants of E2-E6AP specificity, we have performed a variety of mutagenesis experiments to map out which E2 and E6AP residues are important for E2-E6AP affinity, and have used this information along with multiple sequence alignments to rationally perturb E2-E6AP binding preferences.