Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis

Abstract

Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING domain of rat RNF4 in complex with E2 (UbcH5A) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The carboxy-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilize the consequent tetrahedral transition-state intermediate.

Figure 1. Structure of the RNF4 RING bound to ubiquitin-loaded UbcH5a

a, Surface representation of the complex. Individual RING protomers are coloured cyan and blue, UbcH5a is green, ubiquitin is orange and the isopeptide linkage between the C-terminus of ubiquitin and K85 of UbcH5a is shown in yellow. b, Ribbon diagram of the complex with the same orientation and colour scheme as in a. Zinc atoms are indicated as grey spheres. c, As in b, but the complex is rotated by 90^o as indicated.

Figure 2. Molecular interfaces in the RNF4 RING–UbcH5a~Ub complex

a, Detail of the interaction between ubiquitin (orange) and the α2 helix of UbcH5a (green). b, Detail of the interaction interface between ubiquitin (orange) and UbcH5a (green) in the E2 active site groove. The side chain of K85 in UbcH5a that forms the isopeptide bond with ubiquitin is coloured violet. c, The hydrophobic cluster at the centre of the ubiquitin (orange), UbcH5a (green), RING (cyan) heterotrimer. d, Stacking interaction between the main chain of ubiquitin (orange) in one heterotrimer and Y193 of the RING (blue) from the other heterotrimer.

Figure 3. Mutational analysis of the RNF4 RING–UbcH5a~Ub complex

a, Side chains of altered residues in ubiquitin contacting RNF4 (blue), UbcH5a (green), or both RNF4 and UbcH5a (yellow). b, Side chains of altered residues in UbcH5a contacting RNF4 (blue), ubiquitin (orange), both RNF4 and ubiquitin (yellow), or neither (green). c, Reaction rates were determined (mean ± s.d. of duplicates) for single-turnover, RNF4-dependent substrate ubiquitylation assays with mutant forms of ubiquitin. Wild-type ubiquitin is in grey and mutants are colour coded as in a. d, Assays with UbcH5a mutants quantified as in c and colour coded as in b. e, RNF4-mediated hydrolysis of UbcH5a^C85S~Ub oxyesters with mutations in ubiquitin. Rates are mean ± s.d. of duplicates. f, As in e, with mutations in UbcH5a.

Figure 4. The same interfaces in E2 and ubiquitin are important for CHIP and RNF4 activity

a, Autoubiquitylation activity of CHIP (upper panel) and RNF4 (lower panel) with ubiquitin mutants. Western blots probed with anti-ubiquitin antibody are shown. Longer exposure is shown for I36A and L71A ubiquitin, as binding of the antibody is affected by these mutations. b, Autoubiquitylation activity as in a, but with UbcH5a mutants.

Figure 5. E3-mediated structural changes associated with the catalytically primed form of UbcH5a~Ub

a, Model of UbcH5a~ubiquitin thioester (grey) with UbcH5a K85 (violet). b, Comparison of modeled thioester with isopeptide linkage. Hydrogen bonds are black (isopeptide) or grey (modeled thioester) dashes. c, Comparison of position of ubiquitin relative to E2 in UbcH5a~ubiquitin–RING complex reported here with UbcH5b~ubiquitin–HECT(NEDD4L) complex (PDB 3JW0), and UbcH5b~Ub oxyester (PDB 3A33). d, RING mediated remodeling of UbcH5a active site. The position of the C-terminus of ubiquitin linked to the active site cysteine/serine of the E2 is shown relative to residues N77 and D117 in the three structures shown in c. e, Model for nucleophilic attack by substrate lysine (pink) on the E2~Ub thioester bond, based on the SUMO-RanGAP1–Ubc9–RanBP2 structure.