Ubiquitylation occurs as the result of a hierarchical multi-enzyme process. Ubiquitin-activating enzyme (E1) activates ubiquitin, forming a thiolester linkage between the active site Cys of E1 and the C-terminus of ubiquitin. Ubiquitin is transferred to the conserved active site Cys of ubiquitin-conjugating enzymes (E2), of which there are over 30 in mammals. E2s bind to specific ubiquitin-protein ligases (E3s), which mediate the transfer of ubiquitin to primary amines on substrates or to growing chains of ubiquitin (polyubiquitin or multiubiquitin). In many cases the E3s also undergo auto- or self-ubiquitylation.
There are more than 500 E3s in mammals that can be divided into two major classes. The HECT E3s include ~30 E3s and are characterized by a conserved 350 amino acid catalytic domain. HECT E3s are catalytic intermediates in substrate ubiquitylation as a consequence of the transthiolation of ubiquitin from bound E2 to their conserved catalytic Cys (Fang and Weissman, 2004
RING finger and RING finger-like E3s collectively represent the large majority of E3s. The RING finger is a compact Zn-binding domain of 40 to 100 amino acids. These domains generally bind E2s with low affinity and do not form catalytic intermediates with ubiquitin. It is generally believed that RING fingers either position E2~Ub to facilitate transfer to substrates or function as allosteric activators of E2~Ub (Lorick et al., 2006
; Ozkan et al., 2005
). Binding sites on E2s for RING fingers, HECT domains and E1 all overlap. Therefore, E2s must dissociate from ligase domains to reload with ubiquitin (Huang et al., 2005
; Eletr et al., 2005
). Interestingly, there are a few examples where other regions, either within a multi-subunit ubiquitin ligase complex or a single subunit E3, bind specific E2s through generally uncharacterized interactions (Madura et al., 1993
; Hatakeyama et al., 1997
; Wu et al., 2002
; Biederer et al., 1997
; Chen et al., 2006
). This could increase the availability of E2~Ub and theoretically allow for reloading of E2 with ubiquitin without dissociation from the E3.
Ubiquitylation and proteasomal degradation perform critical functions in degradation of misfolded, unassembled and highly regulated proteins from the endoplasmic reticulum (ER). ER-associated degradation (ERAD) is a multi-step, highly coordinated process (Nakatsukasa and Brodsky, 2008
). In mammals there are at least five known ER membrane-spanning ERAD E3s. Among these is gp78, also known as the human tumor autocrine motility factor receptor (AMFR). gp78 is implicated in degradation of T cell antigen receptor subunits, regulatory proteins in lipid metabolism (Kostova et al., 2007
), CFTRΔ508 (Morito et al., 2008
) and the metastasis suppressor KAI1 (CD82) (Tsai et al., 2007
gp78 has a complex domain structure for a single subunit E3. In addition to its RING finger, it has at least three more C-terminal domains in its extended cytoplasmic tail (). Each of these is implicated in ubiquitylation and degradation of ERAD substrates. They include a ubiquitin-binding CUE (c
oupling of u
biquitin conjugation to E
RAD) domain and a C-terminal binding site for p97. Unique to gp78 is a high affinity binding site for its cognate E2, Ube2g2. This Ube2g2 binding region (G2BR) is required for the function of gp78 in cells (Chen et al., 2006
Figure 1 The G2BR and NMR-Determined Interactions with Ube2g2. (A) Schematic representation of gp78 in the ER membrane (left). To the right is a linear representation of gp78 cytoplasmic tail with amino acids (corresponding to the entire human gp78) indicated. (more ...)
We now report that the G2BR binds Ube2g2 through an extended interface distinct from sites of RING finger and E1 binding. This results in subtle changes in the Ube2g2 core that are manifested in functional alterations in loading with ubiquitin and a marked increase in affinity for the gp78 RING finger, which is reflected in enhanced ubiquitylation.