Structural basis for the RING-catalyzed synthesis of K63-linked ubiquitin chains

Abstract

RING E3 ligase-catalyzed formation of K63-linked ubiquitin chains by the Ube2V2-Ubc13 E2 complex is required in many important biological processes. Here we report the structure of the RING-domain dimer of rat RNF4 in complex with a human Ubc13∼Ub conjugate and Ube2V2. The structure has captured Ube2V2 bound to the acceptor (priming) ubiquitin with K63 in a position favorable for attack on the linkage between Ubc13 and the donor (second) ubiquitin held in the active 'folded back' conformation by the RING domain of RNF4. We verified the interfaces identified in the structure by in vitro ubiquitination assays of site-directed mutants. To our knowledge, this represents the first view of synthesis of K63-linked ubiquitin chains in which both substrate ubiquitin and ubiquitin-loaded E2 are juxtaposed to allow E3 ligase-mediated catalysis.

Figure 1. Biochemical analysis of RNF4 catalysed K63 linked polyubiquitination by Ube2V2–Ubc13 complex

(a) Cartoon representation of RNF4, 4xSUMO-2, and Ub~4xSUMO-2. RNF4 contains four N-terminal SIMs (yellow) followed by a C-terminal RING domain (dark pink). 4xSUMO-2 contains four linearly fused SUMO-2 molecules (blue). Ub~4xSUMO-2 has a ubiquitin molecule (orange) linearly fused to the N-terminus of 4xSUMO-2. (b) Coomassie-blue stained SDS-PAGE analysis of ubiquitination assays containing various combinations of Ube2V2, RNF4, Ub~4xSUMO-2, 4xSUMO-2 and ATP. Time points are denoted above the gels. The red asterisk indicates formation of di-ubiquitin.

Figure 2. Structure of ubiquitin loaded Ubc13 in complex with Ube2V2 and the RING domain of RNF4 showing specificity for K63 linked polyubiquitination

(a) Ribbon (top) and cartoon (bottom) representation of Ube2V2 (dark and pale blue) and the linearly fused RING domain dimer of RNF4 (dark and pale pink) in complex with ubiquitin loaded Ubc13. Ubc13 is shown in dark and pale green while the donor ubiquitin is yellow and Ub* is coloured orange. Ubc13 K87 and donor ubiquitin G76 are highlighted in magenta with the isopeptide linkage highlighted with a magenta sphere. Zn ions are shown as grey spheres. (b) Ribbon (top) and cartoon (bottom) representation of the ubiquitin-loaded Ubc13 in complex with the RING domain of RNF4. The colour scheme is the same as in a and the second donor ubiquitin is light orange. (c) Ribbon (left) and surface (right) representation of the same complex as in a where Ub* has been transformed in order to generate the canonical model. The colour scheme is the same as in a. Ub* K63 is coloured in yellow with the ε-amine highlighted with a yellow sphere. (d) Cartoon diagram of K63 linked chain formation based on the Ubc13~Ub– Ube2V2–RNF4 RING structure.

Figure 3. Mutational analysis of the Ubc13~Ub–Ube2V2–RNF4 RING complex

(a) Detailed molecular interface between Ub* (orange) and Ube2V2 (blue) from the canonical model. (b) Detail of the hydrophobic pocket in Ubc13 (green) for Ube2V2 (blue). (c) Interaction between the C-terminal tail of the donor ubiquitin (yellow) and the active site groove of Ubc13 (green). K63 of Ub* (orange) is shown and the distance between its ε–amino group and the carbonyl carbon of the donor ubiquitin G76 is indicated. Interactions between the D81 to R85 loop of Ubc13 and Ube2V2 (blue) are also shown. (d) The hydrophobic interface between Ubc13 (green) and ubiquitin (yellow) in the Ubc13~Ub conjugate bound to the RING domain of RNF4. (e) Molecular detail of the interaction surface between Ubc13 (green) and the RING domain of RNF4 (dark pink). (f) Coomassie-blue stained SDS-PAGE analysis of ubiquitination assays containing wild type (WT) or mutants of Ubc13 and Ube2V2. The red asterisk indicates formation of di-ubiquitin.

Figure 4. Both Ube2V2 and dimeric RNF4 are required for activation of the Ubc13~Ub thioester conjugate

(a) Cartoon representation of RNF4 and RNF4-RING. RNF4-RING has a second RING domain of RNF4 (pale pink) linearly fused to RNF4. The RING domain dimer, which was used in crystallography, is highlighted with a black dotted line. (b) SYPRO orange stained SDS-PAGE analysis of lysine discharge assays in the presence of RNF4, RNF4-RING, WT Ube2V2 and Ube2V2 S32A. Time points are denoted above each gel. Uncropped gels are available in Supplementary Data Set 1. (c) Reaction rates were obtained for lysine discharge assays shown in b. The reaction rates from two or three technical replicates are shown as black dots and the mean reaction rates are shown as black horizontal bars. Number of replicates (n) is denoted below the graph. Spreadsheet containing reaction rate calculations are available in Supplementary Data Set 2. (d) Schematic diagram of RNF4 catalysing the formation of K63 and K48 linked polyubiquitin chains in complex with Ube2V2–Ubc13 and UbcH5A respectively.