Pivotal role for the ubiquitin Y59-E51 loop in lysine 48 polyubiquitination

Abstract

Lysine 48 (K48)-polyubiquitination is the predominant mechanism for mediating selective protein degradation, but the underlying molecular basis of selecting ubiquitin (Ub) K48 for linkage-specific chain synthesis remains elusive. Here, we present biochemical, structural, and cell-based evidence demonstrating a pivotal role for the Ub Y59-E51 loop in supporting K48-polyubiquitination. This loop is established by a hydrogen bond between Ub Y59's hydroxyl group and the backbone amide of Ub E51, as substantiated by NMR spectroscopic analysis. Loop residues Y59 and R54 are specifically required for the receptor activity enabling K48 to attack the donor Ub-E2 thiol ester in reconstituted ubiquitination catalyzed by Skp1-Cullin1-F-box (SCF) βTrCP E3 ligase and Cdc34 E2-conjugating enzyme. When introduced into mammalian cells, loop-disruptive mutant Ub R54A/Y59A diminished the production of K48-polyubiquitin chains. Importantly, conditional replacement of human endogenous Ub by Ub R54A/Y59A or Ub K48R yielded profound apoptosis at a similar extent, underscoring the global impact of the Ub Y59-E51 loop in cellular K48-polyubiquitination. Finally, disulfide cross-linking revealed interactions between the donor Ub-bound Cdc34 acidic loop and the Ub K48 site, as well as residues within the Y59-E51 loop, suggesting a mechanism in which the Ub Y59-E51 loop helps recruit the E2 acidic loop that aligns the receptor Ub K48 to the donor Ub for catalysis.receptor ubiquitin | E3 ubiquitin ligase | E2 ubiquitin-conjugating enzyme C entral to selective protein turnover by the 26S proteasome is the formation of homotypic lysine 48 (K48)-linked ubiquitin (Ub) chains that tag substrate proteins for degradation (1). Among the most extensively studied systems that produce K48-linked Ub chains is the SCF (Skp1-Cullin1-F-box) E3-directed ubiquitination. SCF is a member of the multisubunit Cullin-RING E3 Ub ligase (CRL) family, the largest of all E3s (2). CRL contains a tandem of a large scaffold protein [Cullin (CUL)] and a RING domain-containing protein (ROC1/Rbx1) that typically associates with an adaptor protein (such as Skp1) in complex with a substrate recognition protein (such as F-box protein). As such, the organization of CRL subunits positions the substrate receptor (such as the F-box protein) within the proximity of ROC1, which recruits an E2-conjugating enzyme that catalyzes the transfer of Ub to a bound substrate. In the SCF reconstitution system, K48-linked polyubiquitin chains on a substrate such as IκBα and β-catenin are produced in a two-step reaction. The E2 UbcH5c deposits the first Ub moiety, forming a substrate-Ub linkage, which is followed by repeated discharge of subsequent Ubs by E2 Cdc34 to form K48-specific Ub chains (3). Human Cdc34 contains a highly conserved charged acidic loop (residues 102-113) that participates in the elongation of K48 chains (4, 5). The current work addresses whether there are determinants on the Ub itself that dictate K48 linkage specificity and, moreover, how Cdc34 might recognize Ub K48. R...

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

“…In summary, these results are most consistent with a model where SCF controls the conformation of the Ube2R1/2 acidic loop which may serve to optimize stabilizing interactions between residues Glu 108 and Glu 112 and the donor ubiquitin. One notable exception may be Asp 103, for which some of the models predicted an interaction between this residue and acceptor ubiquitin, a result that is consistent with previous protein cross-linking experiments (26).…”

“…It is not surprising that Asp 143 may have an important role in Ube2R1/2 function, as it is invariably conserved in Ube2R1/2 ortholog sequences. Furthermore, there is evidence that Arg 54 on ubiquitin is also important, since the replacement of endogenous ubiquitin with a R54D mutant in S. cerevisiae resulted in a mild slow-growth phenotype (44), and, when introduced into mammalian cells, expression of the double R54A/Y59A ubiquitin mutant led to cellular apoptosis (26). Taking those findings in combination with the results presented here, the importance of ubiquitin residue Arg 54 has been demonstrated both in vivo and in vitro.…”

“…A previous study had shown that acceptor ubiquitin residue Arg 54 participated in Ube2R1-catalyzed Lys 48 polyubiquitin chain formation (26), and, consistent with this, the mutation of Arg 54 to an Asp residue in acceptor ubiquitin led to a 52-fold reduction in Ube2R2 activity compared with wild-type (WT) ubiquitin results (Fig. 2b and c and Table 1).…”

“…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).…”

“…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). Despite these advances, a detailed molecular characterization showing how an E2 achieves Lys 48 specificity during polyubiquitin chain synthesis is lacking.…”