Allosteric regulation of E2:E3 interactions promote a processive ubiquitination machine

Abstract

RING finger proteins constitute the large majority of ubiquitin ligases (E3s) and function by interacting with ubiquitin-conjugating enzymes (E2s) charged with ubiquitin. How low-affinity RING-E2 interactions result in highly processive substrate ubiquitination is largely unknown. The RING E3, gp78, represents an excellent model to study this process. gp78 includes a high-affinity secondary binding region for its cognate E2, Ube2g2, the G2BR. The G2BR allosterically enhances RING:Ube2g2 binding and ubiquitination. Structural analysis of the RING: Ube2g2:G2BR complex reveals that a G2BR-induced conformational effect at the RING:Ube2g2 interface is necessary for enhanced binding of RING to Ube2g2 or Ube2g2 conjugated to Ub. This conformational effect and a key ternary interaction with conjugated ubiquitin are required for ubiquitin transfer. Moreover, RING:Ube2g2 binding induces a second allosteric effect, disrupting Ube2g2: G2BR contacts, decreasing affinity and facilitating E2 exchange. Thus, gp78 is a ubiquitination machine where multiple E2-binding sites coordinately facilitate processive ubiquitination.

The Ubiquitination Machinery of the Ubiquitin System

“…After the E2~Ub binds to the RING domain, reactivity of the E2~Ub thioester bond is further de-stabilized, thus promoting attack by an amino group (Das et al, 2009;Das et al, 2013). The one exception is a subgroup of RING proteins, the RBR proteins, described below.…”

Ubiquitin chain diversity at a glance

“…Subsequently, AMFR catalyzes K27-linked polyubiquitylation of STING, which serves as a scaffold for the recruitment and activation of TBK1 (Wang et al, 2014). Of note, it has been shown that AMFR interacts with the E2 enzyme UBE2G2 through a specialized binding region on AMFR, and that this interaction is a prerequisite for processive assembly of K48-linked ubiquitin chains on ER-associated degradation (ERAD) substrates (Das et al, 2013(Das et al, , 2009). It would be exciting to uncover the molecular mechanisms of how AMFR can also assemble K27-linked ubiquitin chains.…”

Human Liver Cytochrome P450 3A4 Ubiquitination

“…The crystal structure of Ube2g2 (UBC7) complexed with the isolated G2BR domain of gp78 reveals extensive highly distributed contacts between the UBC7 "backside" and the helical G2BR region composed of residues Ser-574 to Lys-600 that include hydrogen bonds, salt bridges, and hydrophobic interactions that facilitate "frontside" interactions with gp78-RING finger domain and/or UBA1 (69,70). The UBC7-interacting residues in this G2BR region include both Lys-586, a residue we found to be cross-linked to CYP3A4Lys-257, and Lys-600 that was cross-linked to UBC7Lys-156.…”