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. 2016 Nov 20;428(23):4639-4650.
doi: 10.1016/j.jmb.2016.09.018. Epub 2016 Sep 24.

Role of E2-RING Interactions in Governing RNF4-Mediated Substrate Ubiquitination

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Free PMC article

Role of E2-RING Interactions in Governing RNF4-Mediated Substrate Ubiquitination

Anthony DiBello et al. J Mol Biol. .
Free PMC article

Abstract

Members of the really interesting new gene (RING) E3 ubiquitin ligase family bind to both substrate and ubiquitin-charged E2 enzyme, promoting the transfer of ubiquitin from the E2 to substrate. Either a single ubiquitin or one of the several types of polyubiquitin chains can be conjugated to substrate proteins, with different types of ubiquitin modifications signaling the distinct outcomes. E2 enzymes play a central role in governing the nature of the ubiquitin modification, although the essential features of the E2 that differentiate mono- versus polyubiquitinating E2 enzymes remain unclear. RNF4 is a compact RING E3 ligase that directs the ubiquitination of polySUMO chains in concert with several different E2 enzymes. RNF4 monoubiquitinates polySUMO substrates in concert with RAD6B and polyubiquitinates substrates together with UBCH5B, a promiscuous E2 that can function with a broad range of E3 ligases. We find that the divergent ubiquitination activities of RAD6B and UBCH5B are governed by differences at the RING-binding surface of the E2. By mutating the RAD6B RING-binding surface to resemble that of UBCH5B, RAD6B can be transformed into a highly active UBCH5B-like E2 that polyubiquitinates SUMO chains in concert with RNF4. The switch in RAD6B activity correlates with increased affinity of the E2 for RNF4. These results point to an important role of the affinity between an E3 and its cognate E2 in governing the multiplicity of substrate ubiquitination.

Keywords: E2 ubiquitin-conjugating enzyme; E3 ubiquitin ligase; RAD6B; RING finger protein 4 (RNF4); ubiquitin.

Figures

Figure 1
Figure 1. RNF4-catalyzed ubiquitination of a polySUMO substrate by UBCH5B and RAD6B enzymes
(a) Time course comparing the in vitro ubiquitination of 4xSUMO2, using both native and methylated Ub, by RNF4 in the presence of UBCH5B. Reactions contained 200 nM E1, 5 μM E2, 1 μM RNF4, 15 μM 4xSUMO-2 and 100 μM of native or methylated Ub, and were incubated at 37°C. Samples were analyzed by SDS-PAGE, followed by Coomassie Brilliant Blue stain. (b) Time course comparing the in vitro ubiquitination of 4xSUMO2, using both native and methylated Ub, by RNF4 in the presence of RAD6B. Reactions contained 200 nM E1, 5 μM E2, 1 μM RNF4, 15 μM 4xSUMO-2 and 100 μM of native or methylated Ub, and were incubated at 37°C. Samples were analyzed by SDS-PAGE, followed by Coomassie Brilliant Blue stain.
Figure 2
Figure 2. Mutating the RAD6B RING-binding surface to resemble UBCH5B
(a) Sequence alignment of human UBCH5B and RAD6B. RING-binding surface regions are highlighted in blue. RAD6B residues targeted for mutagenesis shown in red. (b) Docking of the structure of RAD6B (cyan, PDB ID: 2YB6) and UBCH5B (yellow, PDB ID: 2ESK) on the crystal structure of UBCH5A~Ub in complex with an RNF4 RING dimer (PDB ID: 4AP4). The donor ubiquitin is shown in orange and the RNF4 RING dimer is shown in blue and red. (c) Residues at the RING-binding surfaces of RAD6B, the RAD6Bs5B variants, UBCH5Bs6B, and UBCH5B. RAD6B is shown in cyan, UBCH5B is shown in yellow, substituted residues in the RAD6Bs5B conversion mutants are shown as yellow spheres, and substituted residues in the UBCH5Bs6B conversion mutant are shown as cyan spheres.
Figure 3
Figure 3. RNF4-catalyzed ubiquitination of polySUMO by RAD6B and UBCH5B conversion mutants
(a) Time course comparing the in vitro ubiquitination of 4xSUMO2, using both native and methylated Ub, by RNF4 in the presence of the RAD6Bs5B variants. Reactions contained 200 nM E1, 5 μM E2, 1 μM RNF4, 15 μM 4xSUMO-2 and 100 μM of native or methylated Ub, and were incubated at 37°C. Samples were analyzed by SDS-PAGE, followed by Coomassie Brilliant Blue stain. (b) Time course comparing the in vitro ubiquitination of 4xSUMO2, using both native and methylated Ub, by RNF4 in the presence of UBCH5Bs6B. Reactions contained 200 nM E1, 5 μM E2, 1 μM RNF4, 15 μM 4xSUMO-2 and 100 μM of native or methylated Ub, and were incubated at 37°C. Samples were analyzed by SDS-PAGE, followed by Coomassie Brilliant Blue stain.
Figure 4
Figure 4. Affinity of RNF4 for E2 enzymes
Sedimentation equilibrium analytical ultracentrifugation (AUC) was used to monitor E2 association with the RNF4 homodimer. Absorbance (300 nm) profiles at three different rotor speeds are shown. Data were fit to heteroassociation model of the interaction between E2 and the RNF4 homodimer, except where noted as otherwise. (a) UBCH5B (50 μM) with RNF4 (75 μM), (b) RAD6B (50 μM) with RNF4 (75 μM), (c) RAD6B (50 μM) with RNF4 (75 μM) fit to a noninteracting species model for two species, RAD6B and the RNF4 homodimer, (d) RAD6Bs5B-7 (50 μM) with RNF4 (50 μM), (e) and RAD6Bs5B-12 (50 μM) with RNF4 (50 μM). (f) Table summarizing E2-RNF4 affinities measured by sedimentation equilibrium AUC.
Figure 5
Figure 5. Reactivity of wild type and mutant E2~Ub thioester in the presence and absence of RNF4
The rates of ubiquitin discharge from the E2~Ub to lysine in the presence (black columns) and absence (grey columns) of RNF4, with native RAD6B and UBCH5B, UBCH5Bs6B, as well as with the RAD6Bs5B conversion constructs. E2 was pre-charged with fluorescein-tagged ubiquitin, UbFL, and then added to the E2~UbFL discharge reaction. E2~UbFL discharge reactions contained 50 mM lysine, 10 μM RNF4, and 5 μM E2~UbFL. Ub-discharge was monitored by SDS-PAGE, followed by fluorescent imaging, and fluorescence signal densitometry.

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