Loop 7 of E2 Enzymes: An Ancestral Conserved Functional Motif Involved in the E2-Mediated Steps of the Ubiquitination Cascade

Abstract

The ubiquitin (Ub) system controls almost every aspect of eukaryotic cell biology. Protein ubiquitination depends on the sequential action of three classes of enzymes (E1, E2 and E3). E2 Ub-conjugating enzymes have a central role in the ubiquitination pathway, interacting with both E1 and E3, and influencing the ultimate fate of the substrates. Several E2s are characterized by an extended acidic insertion in loop 7 (L7), which if mutated is known to impair the proper E2-related functions. In the present contribution, we show that acidic loop is a conserved ancestral motif in E2s, relying on the presence of alternate hydrophobic and acidic residues. Moreover, the dynamic properties of a subset of family 3 E2s, as well as their binary and ternary complexes with Ub and the cognate E3, have been investigated. Here we provide a model of L7 role in the different steps of the ubiquitination cascade of family 3 E2s. The L7 hydrophobic residues turned out to be the main determinant for the stabilization of the E2 inactive conformations by a tight network of interactions in the catalytic cleft. Moreover, phosphorylation is known from previous studies to promote E2 competent conformations for Ub charging, inducing electrostatic repulsion and acting on the L7 acidic residues. Here we show that these active conformations are stabilized by a network of hydrophobic interactions between L7 and L4, the latter being a conserved interface for E3-recruitment in several E2s. In the successive steps, L7 conserved acidic residues also provide an interaction interface for both Ub and the Rbx1 RING subdomain of the cognate E3. Our data therefore suggest a crucial role for L7 of family 3 E2s in all the E2-mediated steps of the ubiquitination cascade. Its different functions are exploited thank to its conserved hydrophobic and acidic residues in a finely orchestrate mechanism.

Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

“…To rationalize these results at a structural level, a ternary complex of Cdc34~Ub donor complex 30 in association with the Sic1 48-52 was modeled by comparative modeling using as template the known structure of Ube2I and its substrate (pdb code: 2GRN) 31 by Modeler v.9.11. 32 Twenty different models were generated for each variant.…”

“…Previous studies have provided models of Sic1 bound to Cdc34/SCF Cdc4 , 33 and of the Cdc34~Ub thioester complex. 30 Our molecular modeling analysis provides a rationale for the importance of particular amino acids proximal to Sic1-K50. The native complex is rich in salt-bridge interactions, which are well-organized in several networks (Fig.…”

“…A ternary complex of Cdc34~Ub donor complex 30 in association with the Sic1 48-52 was modeled by comparative modeling using as template the known experimental structure of Ube2I and its substrate (pdb entry 2GRN) 31 by Modeler v.9.11. 32 The Sic1 48-52 was oriented within the catalytic site of the Cdc34~Ub donor complex, in agreement with the model of Sic1 bound to Cdc34/SCF Cdc4 .…”

Multimodal Mechanism of Action for the Cdc34 Acidic Loop

“…Recently, Papaleo et al (42) used molecular dynamics simulations to model conformations of the Cdc34 acidic loop in the presence of E3. Interestingly, their results predict that several acidic loop residues may form intermolecular interactions with the Rbx1 subunit of the E3.…”

Mechanism of Lysine 48 Selectivity during Polyubiquitin Chain Formation by the Ube2R1/2 Ubiquitin-Conjugating Enzyme

“…Ube2R1/2 is a critical E2 that functions with the cullin-RING ubiquitin ligases (1,18), and, together, these enzymes may be responsible for 20% of all proteasome-dependent degradation in human cells (19). Ube2R1/2 contains an atypical insertion distal to its active site that contains several conserved acidic residues (20)(21)(22)(23)(24)(25), and this acidic loop has also been shown to have an important role in Lys 48 selectivity (26,27). More recent work has identified a loop on acceptor ubiquitin that contains residues that interact with the E2 in order to help place Lys 48 in the E2 active site (26).…”