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Allosteric activation of the RNF146 ubiquitin ligase by a poly(ADP-ribosyl)ation signal.

DaRosa PA, Wang Z, Jiang X, Pruneda JN, Cong F, Klevit RE, Xu W - Nature (2014)

Bottom Line: Disruption of the RNF146-TNKS interaction inhibits turnover of the substrate Axin in cells.Thus, both substrate PARylation and PARdU are catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction.We propose that the maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146-TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA [2] Department of Biological Structure, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
Protein poly(ADP-ribosyl)ation (PARylation) has a role in diverse cellular processes such as DNA repair, transcription, Wnt signalling, and cell death. Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several crucial regulatory proteins, including Axin and 3BP2 (refs 7, 8, 9). The RING-type E3 ubiquitin ligase RNF146 (also known as Iduna) is responsible for PARylation-dependent ubiquitination (PARdU). Here we provide a structural basis for RNF146-catalysed PARdU and how PARdU specificity is achieved. First, we show that iso-ADP-ribose (iso-ADPr), the smallest internal poly(ADP-ribose) (PAR) structural unit, binds between the WWE and RING domains of RNF146 and functions as an allosteric signal that switches the RING domain from a catalytically inactive state to an active one. In the absence of PAR, the RING domain is unable to bind and activate a ubiquitin-conjugating enzyme (E2) efficiently. Binding of PAR or iso-ADPr induces a major conformational change that creates a functional RING structure. Thus, RNF146 represents a new mechanistic class of RING E3 ligases, the activities of which are regulated by non-covalent ligand binding, and that may provide a template for designing inducible protein-degradation systems. Second, we find that RNF146 directly interacts with the PAR polymerase tankyrase (TNKS). Disruption of the RNF146-TNKS interaction inhibits turnover of the substrate Axin in cells. Thus, both substrate PARylation and PARdU are catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction. We propose that the maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146-TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell.

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Rotation and crystal packing at the E2-E3 binding interface of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complexa, Superposition of the E2 in the RNF146(RING-WWE)/UbcH5a/iso-ADPr (colored as in Figure 2a) with a representative RING E3-E2 structure, the Bmi1/Ring1b-UbcH5c complex (grey) (PDB 3RPG)18. The WWE domain is excluded for clarity. Boxes show close-up views of the RING domains revealing a rotation of the RING domain relative to the E2. (Bottom right) RING domains rotated 90° to show the E2 binding surface of the E3s. The RING of the RNF146(RING-WWE)/UbcH5a/iso-ADPr structure is rotated relative to Ring1b-UbcH5c and other E3-E2 complexes15–20,23 (indicated by red arrow) when the E2s are aligned. b, A close-up view of the E2-E3 interface of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex shows that RING residue R74 (yellow) is too far (~7.7 Å) from the E2 Q92 (magenta) carbonyl to make the hydrogen bond observed in activated E3-E2~Ub structures17,21,22,24. The side chain of R74 in the RING domain packs against F128 (orange), a WWE domain residue of a symmetry-related molecule in this crystal form. It is likely that crystal packing interferes with the formation of the “allosteric” hydrogen bond. c, E2~Ub/lysine reactivity with RNF146(RING-WWE)-R74A shows a dependence of RNF146 activity on the allosteric arginine17,21,22,24 with or without ligand. Because RNF146 activation requires R74, which does not make contacts with the E2 in our structure, and because RNF146 shows canonical E2 binding in solution (see Extended Data Fig. 7), we conclude that the orientation of RNF146 in the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex is likely an unproductive E2-E3 association. The observed rotation is likely a crystallographic artifact.
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Figure 10: Rotation and crystal packing at the E2-E3 binding interface of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complexa, Superposition of the E2 in the RNF146(RING-WWE)/UbcH5a/iso-ADPr (colored as in Figure 2a) with a representative RING E3-E2 structure, the Bmi1/Ring1b-UbcH5c complex (grey) (PDB 3RPG)18. The WWE domain is excluded for clarity. Boxes show close-up views of the RING domains revealing a rotation of the RING domain relative to the E2. (Bottom right) RING domains rotated 90° to show the E2 binding surface of the E3s. The RING of the RNF146(RING-WWE)/UbcH5a/iso-ADPr structure is rotated relative to Ring1b-UbcH5c and other E3-E2 complexes15–20,23 (indicated by red arrow) when the E2s are aligned. b, A close-up view of the E2-E3 interface of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex shows that RING residue R74 (yellow) is too far (~7.7 Å) from the E2 Q92 (magenta) carbonyl to make the hydrogen bond observed in activated E3-E2~Ub structures17,21,22,24. The side chain of R74 in the RING domain packs against F128 (orange), a WWE domain residue of a symmetry-related molecule in this crystal form. It is likely that crystal packing interferes with the formation of the “allosteric” hydrogen bond. c, E2~Ub/lysine reactivity with RNF146(RING-WWE)-R74A shows a dependence of RNF146 activity on the allosteric arginine17,21,22,24 with or without ligand. Because RNF146 activation requires R74, which does not make contacts with the E2 in our structure, and because RNF146 shows canonical E2 binding in solution (see Extended Data Fig. 7), we conclude that the orientation of RNF146 in the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex is likely an unproductive E2-E3 association. The observed rotation is likely a crystallographic artifact.

Mentions: In the crystal, the E2, UbcH5a, binds to the RING domain at the canonical E2–E3 interface, away from the iso-ADPr binding site (Fig. 2a). Similar to other RING E3–E2 complex structures15–24, two Zn2+-binding loops and the central helix of the RING bind E2 Loops 4 and 7. A slight difference in the orientation of the RING and E2 relative to other E2–E3 complex structures is likely due to crystal packing (Extended Data Fig. 6), as the E2/E3 interactions observed in solution by NMR are similar to other well-characterized systems (see below).


Allosteric activation of the RNF146 ubiquitin ligase by a poly(ADP-ribosyl)ation signal.

DaRosa PA, Wang Z, Jiang X, Pruneda JN, Cong F, Klevit RE, Xu W - Nature (2014)

Rotation and crystal packing at the E2-E3 binding interface of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complexa, Superposition of the E2 in the RNF146(RING-WWE)/UbcH5a/iso-ADPr (colored as in Figure 2a) with a representative RING E3-E2 structure, the Bmi1/Ring1b-UbcH5c complex (grey) (PDB 3RPG)18. The WWE domain is excluded for clarity. Boxes show close-up views of the RING domains revealing a rotation of the RING domain relative to the E2. (Bottom right) RING domains rotated 90° to show the E2 binding surface of the E3s. The RING of the RNF146(RING-WWE)/UbcH5a/iso-ADPr structure is rotated relative to Ring1b-UbcH5c and other E3-E2 complexes15–20,23 (indicated by red arrow) when the E2s are aligned. b, A close-up view of the E2-E3 interface of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex shows that RING residue R74 (yellow) is too far (~7.7 Å) from the E2 Q92 (magenta) carbonyl to make the hydrogen bond observed in activated E3-E2~Ub structures17,21,22,24. The side chain of R74 in the RING domain packs against F128 (orange), a WWE domain residue of a symmetry-related molecule in this crystal form. It is likely that crystal packing interferes with the formation of the “allosteric” hydrogen bond. c, E2~Ub/lysine reactivity with RNF146(RING-WWE)-R74A shows a dependence of RNF146 activity on the allosteric arginine17,21,22,24 with or without ligand. Because RNF146 activation requires R74, which does not make contacts with the E2 in our structure, and because RNF146 shows canonical E2 binding in solution (see Extended Data Fig. 7), we conclude that the orientation of RNF146 in the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex is likely an unproductive E2-E3 association. The observed rotation is likely a crystallographic artifact.
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Figure 10: Rotation and crystal packing at the E2-E3 binding interface of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complexa, Superposition of the E2 in the RNF146(RING-WWE)/UbcH5a/iso-ADPr (colored as in Figure 2a) with a representative RING E3-E2 structure, the Bmi1/Ring1b-UbcH5c complex (grey) (PDB 3RPG)18. The WWE domain is excluded for clarity. Boxes show close-up views of the RING domains revealing a rotation of the RING domain relative to the E2. (Bottom right) RING domains rotated 90° to show the E2 binding surface of the E3s. The RING of the RNF146(RING-WWE)/UbcH5a/iso-ADPr structure is rotated relative to Ring1b-UbcH5c and other E3-E2 complexes15–20,23 (indicated by red arrow) when the E2s are aligned. b, A close-up view of the E2-E3 interface of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex shows that RING residue R74 (yellow) is too far (~7.7 Å) from the E2 Q92 (magenta) carbonyl to make the hydrogen bond observed in activated E3-E2~Ub structures17,21,22,24. The side chain of R74 in the RING domain packs against F128 (orange), a WWE domain residue of a symmetry-related molecule in this crystal form. It is likely that crystal packing interferes with the formation of the “allosteric” hydrogen bond. c, E2~Ub/lysine reactivity with RNF146(RING-WWE)-R74A shows a dependence of RNF146 activity on the allosteric arginine17,21,22,24 with or without ligand. Because RNF146 activation requires R74, which does not make contacts with the E2 in our structure, and because RNF146 shows canonical E2 binding in solution (see Extended Data Fig. 7), we conclude that the orientation of RNF146 in the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex is likely an unproductive E2-E3 association. The observed rotation is likely a crystallographic artifact.
Mentions: In the crystal, the E2, UbcH5a, binds to the RING domain at the canonical E2–E3 interface, away from the iso-ADPr binding site (Fig. 2a). Similar to other RING E3–E2 complex structures15–24, two Zn2+-binding loops and the central helix of the RING bind E2 Loops 4 and 7. A slight difference in the orientation of the RING and E2 relative to other E2–E3 complex structures is likely due to crystal packing (Extended Data Fig. 6), as the E2/E3 interactions observed in solution by NMR are similar to other well-characterized systems (see below).

Bottom Line: Disruption of the RNF146-TNKS interaction inhibits turnover of the substrate Axin in cells.Thus, both substrate PARylation and PARdU are catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction.We propose that the maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146-TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA [2] Department of Biological Structure, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
Protein poly(ADP-ribosyl)ation (PARylation) has a role in diverse cellular processes such as DNA repair, transcription, Wnt signalling, and cell death. Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several crucial regulatory proteins, including Axin and 3BP2 (refs 7, 8, 9). The RING-type E3 ubiquitin ligase RNF146 (also known as Iduna) is responsible for PARylation-dependent ubiquitination (PARdU). Here we provide a structural basis for RNF146-catalysed PARdU and how PARdU specificity is achieved. First, we show that iso-ADP-ribose (iso-ADPr), the smallest internal poly(ADP-ribose) (PAR) structural unit, binds between the WWE and RING domains of RNF146 and functions as an allosteric signal that switches the RING domain from a catalytically inactive state to an active one. In the absence of PAR, the RING domain is unable to bind and activate a ubiquitin-conjugating enzyme (E2) efficiently. Binding of PAR or iso-ADPr induces a major conformational change that creates a functional RING structure. Thus, RNF146 represents a new mechanistic class of RING E3 ligases, the activities of which are regulated by non-covalent ligand binding, and that may provide a template for designing inducible protein-degradation systems. Second, we find that RNF146 directly interacts with the PAR polymerase tankyrase (TNKS). Disruption of the RNF146-TNKS interaction inhibits turnover of the substrate Axin in cells. Thus, both substrate PARylation and PARdU are catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction. We propose that the maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146-TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell.

Show MeSH
Related in: MedlinePlus