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. 2012 Jul 6;287(28):23900-10.
doi: 10.1074/jbc.M112.359653. Epub 2012 May 15.

Molecular insights into the function of RING finger (RNF)-containing proteins hRNF8 and hRNF168 in Ubc13/Mms2-dependent ubiquitylation

Stephen J Campbell  1 , Ross A Edwards, Charles C Y Leung, Dante Neculai, Curtis D Hodge, Sirano Dhe-Paganon, J N Mark Glover
Affiliations

Molecular insights into the function of RING finger (RNF)-containing proteins hRNF8 and hRNF168 in Ubc13/Mms2-dependent ubiquitylation

Stephen J Campbell et al. J Biol Chem. .

Abstract

The repair of DNA double strand breaks by homologous recombination relies on the unique topology of the chains formed by Lys-63 ubiquitylation of chromatin to recruit repair factors such as breast cancer 1 (BRCA1) to sites of DNA damage. The human RING finger (RNF) E3 ubiquitin ligases, RNF8 and RNF168, with the E2 ubiquitin-conjugating complex Ubc13/Mms2, perform the majority of Lys-63 ubiquitylation in homologous recombination. Here, we show that RNF8 dimerizes and binds to Ubc13/Mms2, thereby stimulating formation of Lys-63 ubiquitin chains, whereas the related RNF168 RING domain is a monomer and does not catalyze Lys-63 polyubiquitylation. The crystal structure of the RNF8/Ubc13/Mms2 ternary complex reveals the structural basis for the interaction between Ubc13 and the RNF8 RING and that an extended RNF8 coiled-coil is responsible for its dimerization. Mutations that disrupt the RNF8/Ubc13 binding surfaces, or that truncate the RNF8 coiled-coil, reduce RNF8-catalyzed ubiquitylation. These findings support the hypothesis that RNF8 is responsible for the initiation of Lys-63-linked ubiquitylation in the DNA damage response, which is subsequently amplified by RNF168.

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Figures

FIGURE 1.
FIGURE 1.
RNF8345–485 binds Ubc13/Mms2 to catalyze Lys-63-linked polyubiquitylation. A, RNF8345–485 enhances Ubc13/Mms2 polyubiquitylation. B, RNF8345–485 forms a complex with Ubc13 as determined by SEC. Peak elution volume is marked by a dotted line, and composition and molecular mass is labeled above. The elution volumes of size standards are shown above.
FIGURE 2.
FIGURE 2.
Crystal structure of RNF8345–485/Ubc13/Mms2. A, two orientations of the crystal structure of the RNF8/Ubc12/Mms2 ternary complex. The RNF8 protomer interacting with Ubc13 is shown in salmon, and the unbound RNF8 protomer is shown in red. Ubc13 is shown in dark blue, and Mms2 is shown in light blue. The catalytic Cys-87 in Ubc13 is shown as yellow spheres, and the Zn atoms coordinated by the ZnFs of RNF8 are shown as green spheres. B, RNF8/Ubc13 binding interface. Important residues in complex formation are shown as spheres on RNF8 and sticks on Ubc13. RNF8 is shown in salmon, and Ubc13 is shown in dark blue. The Ser-Pro-Ala motif is highlighted with dots. C, alignment of a single RNF8 protomer with the crystal structure of RNF4 (PDB ID code 2XEU). RNF8 is shown in red, and RNF4 is shown in gray. The coiled-coil is shown as a line, and important structural features are labeled. The truncated construct of RNF8 lacks the coiled-coil as indicated by the transition between lines and scheme.
FIGURE 3.
FIGURE 3.
Stoichiometry of the RNF8/Ubc13 interaction. A, titration of increasing Ubc13 (20–500 μm) into a constant concentration of RNF8 (200 μm). Peak elution volumes are indicated by dotted lines, and the composition of each peak is labeled above. B, MALLS of purified samples of RNF8/Ubc13 complexes. The Gaussian curves indicate protein elution based on refractive index, and the horizontal curves indicated predicted molecular mass. C, scheme showing the possible complexes forming in solution. A dimer of RNF8 interacting with a two Ubc13 molecules fits the molecular mass predictions determined by MALLS.
FIGURE 4.
FIGURE 4.
RNF8 interacts with Ubc13 through its ZnFs. A, alignment of the RNF8 RING domain with the crystal structure of the TRAF6 RING domain bound to Ubc13 (PDB ID code 3HCU) showing important residues involved in E2/E3 complex formation. RNF8 is shown in red, TRAF6 RING in gray, and Ubc13 in blue. Aspartic acid side chain rotomers were predicted based on the relative side chain positions in the high resolution TRAF6 RING structure. B, gel filtration of mutant Ubc13 (upper) and RNF8 (lower). Composition is shown above each peak, and peak elution volume is indicated by a dotted line. C, ubiquitylation assays of mutant Ubc13 and RNF8 carried out for the indicated times.
FIGURE 5.
FIGURE 5.
RNF8392–485 does not interact tightly with Ubc13 and is deficient in Lys-63-Ub chain formation. A, gel filtration of RNF8392–485 free and with Ubc13. Peak elution volume is indicated with a dotted line, and composition and molecular mass are shown above the peaks. Elution volumes of standards are shown above the graph. B, ubiquitylation time course indicating that RNF8392–485 catalyzes polyubiquitylation less efficiently than RNF345–485.
FIGURE 6.
FIGURE 6.
Complex formation and ubiquitylation activity of RNF1681–113 and RNF1681–200. A, gel filtration of purified RNF168 constructs. Peak elution volume is indicated by a dotted line. Size and composition is labeled above each peak. Oligomerization is determined by elution volume relative to the standard elution volumes, shown above. B, ubiquitylation assays of RNF1681–113 and RNF1681–200.
FIGURE 7.
FIGURE 7.
Crystal structure of RNF1681–113 shows partial occlusion of the Ubc13 binding site. A, crystal structure of RNF1681–113 (cyan) aligned with a single protomer of RNF8345–485 (red) in schematic representation. Important structural features are labeled. Zn atoms are shown as green spheres. B, potential binding interface of RNF168 and Ubc13 created by a structural alignment of RNF1681–113 with RNF8345–485. Conserved residues in the ZnFs of RNF168 are shown as sticks. The C-terminal tail of RNF1681–113 occluding the Ubc13 binding site is shown in dark green. RNF168 is shown in cyan, and Ubc13 is shown in semitransparent dark blue. C, ubiquitylation assay using RNF1681–113 L110E.
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