Multimodal Mechanism of Action for the Cdc34 Acidic Loop

Abstract

Background: Ubiquitin-mediated proteolysis is the principal mechanism for regulating protein half-lives in cells. Results: Cdc34 promotes ubiquitin chain assembly onto protein substrates and contains an essential acidic loop near the active site. Conclusion: The Cdc34 acidic loop promotes both protein-protein interactions and catalysis of ubiquitin chain formation. Significance: These results uncover specific biochemical activities for the Cdc34 acidic loop.

Structure of a RING E3 Trapped in Action Reveals Ligation Mechanism for the Ubiquitin-like Protein NEDD8

“…Although future studies will be required to overcome challenges of neddylating the RBX1 R46A mutant to test its effects on SCF ubiquitination, this RBX1 region was implicated by NMR as binding to CDC34~UB (Spratt et al, 2012). Furthermore, modeling an RBX1 complex with CDC34 in place of UBC12 shows potential for an acidic loop essential for CDC34-mediated UB ligation to clash with a canonical linchpin (Duda et al, 2012; Huang et al, 2014; Petroski and Deshaies, 2005; Spratt et al, 2012; Ziemba et al, 2013). Accordingly, despite its role in activating UBCH5~UB, the N98R substitution in RBX1 impairs UB ligation from CDC34 (Figures S2C–S2E).…”

“…Although a “lever” directing RBX1 RING-E2~UB to ubiquitination targets remains to be identified, we speculate that NEDD8, CUL1, an F-box protein-bound substrate, or other E3 or E2 features could help steer the active site toward a ubiquitination target. For example, the E2 CDC34 has distinctive acidic loops and a C-terminal tail that may coordinate the donor or acceptor UB and anchor the RBX1-CDC34-UB orientation for target ubiquitination, much like DCN1 restrains the orientation of RBX1 RING-activated UBC12~NEDD8 toward CUL1 (Choi et al, 2010; Kleiger et al, 2009a; Petroski and Deshaies, 2005; Spratt and Shaw, 2011; Ziemba et al, 2013). …”

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

“…Rather than forming direct interactions with the acceptor ubiquitin, it appears that the acidic-loop residues interact either with SCF or with the donor ubiquitin. In support of this, note that we had previously generated a human Ube2R2 mutant protein in which all four acidic residues were mutated to alanine, and while the activity of this mutant was severely defective, it was still capable of forming polyubiquitin chains on a SCF-bound substrate with Lys 48 specificity (25). Also, models of the Ube2R1-donor ubiquitin/acceptor ubiquitin-Rbx1 complex predicted an interaction between Ube2R1 residue Asp 102 and SCF subunit Rbx1 residue Arg 91 that was supported by functional binding experiments.…”

“…Another topic of interest involves an essential step during catalysis, the deprotonation of Lys 48 on acceptor ubiquitin. We had previously speculated that a conserved histidine (His 98 in Ube2R1/2) may participate in deprotonation (25), and while our models show that His 98 is in proximity to Lys 48 on acceptor ubiquitin and may therefore play some role in lysine activation, it is likely that the mechanism also involves Ube2R1/2 residue Ser 138, which is structurally equivalent to an aspartic acid residue present in most E2s that has been shown to be critical in promoting lysine deprotonation on the substrate (50).…”

“…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).…”