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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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Stabilizing Helix 1 of RNF146 activates the RING domaina, Complete images of gels shown in Figure 3c (Oriole-stained) for G62A, W65A, G62A/W65A (GAWA), K61A and K61D mutants of RNF146(RING-WWE) with and without iso-ADPr. G62A and GAWA mutants show reduced enhancement with iso-ADPr relative to wild type, likely due to a clash of the Ala side chain with a turn in the WWE domain at position 62 (data not shown). b, Alignment of RNF146(RING) solution structure (PDB 2D8T; white) and the crystal structure determined in this study (blue) shown in stereoview. Side chains are excluded for clarity; the backbone is represented by sticks. Comparison of the conformation of Gly 62 in the two structures suggests a need for a small side chain at position 62 to allow the structural transition from the inactive to active form of RNF146. c, Anti-HA western blot of the E2~Ub/lysine reactivity assay of RNF146(RING-WWE) compared with RNF146(RING) and RNF146(RING)-G62A showing enhanced reactivity for the G to A mutation. d, (Left) 1H-15N HSQC TROSY of 15N-UbcH5c(S22R/C85S) in the presence of 0.0 (black), 0.25 (red), 0.5 (green), and 1.0 (magenta) mol. equiv. of RNF146(RING) G62A. (Right) The same experiment performed with WT RNF146(RING). The most perturbed residues, indicated by letter and position (S100, etc.), show increased chemical shift perturbations for the RNF146(RING)-G62A mutant.
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Figure 12: Stabilizing Helix 1 of RNF146 activates the RING domaina, Complete images of gels shown in Figure 3c (Oriole-stained) for G62A, W65A, G62A/W65A (GAWA), K61A and K61D mutants of RNF146(RING-WWE) with and without iso-ADPr. G62A and GAWA mutants show reduced enhancement with iso-ADPr relative to wild type, likely due to a clash of the Ala side chain with a turn in the WWE domain at position 62 (data not shown). b, Alignment of RNF146(RING) solution structure (PDB 2D8T; white) and the crystal structure determined in this study (blue) shown in stereoview. Side chains are excluded for clarity; the backbone is represented by sticks. Comparison of the conformation of Gly 62 in the two structures suggests a need for a small side chain at position 62 to allow the structural transition from the inactive to active form of RNF146. c, Anti-HA western blot of the E2~Ub/lysine reactivity assay of RNF146(RING-WWE) compared with RNF146(RING) and RNF146(RING)-G62A showing enhanced reactivity for the G to A mutation. d, (Left) 1H-15N HSQC TROSY of 15N-UbcH5c(S22R/C85S) in the presence of 0.0 (black), 0.25 (red), 0.5 (green), and 1.0 (magenta) mol. equiv. of RNF146(RING) G62A. (Right) The same experiment performed with WT RNF146(RING). The most perturbed residues, indicated by letter and position (S100, etc.), show increased chemical shift perturbations for the RNF146(RING)-G62A mutant.

Mentions: Mutational analysis was performed to understand the function of key residues in the iso-ADPr-induced conformational switch. Mutation of RING Lys 61, which makes multiple contacts with iso-ADPr, to Ala or Asp increased the Kd for iso-ADPr to values comparable to that of the WWE domain alone (Kd of 214 nM (K61A-RING-WWE) or 457 nM (K61D-RING-WWE) vs. 372 nM (WWE); Extended Data Fig. 3a, b). Although the K61D mutant can still bind ligand, it is not activated by iso-ADPr (Fig. 3c and Extended Data Fig. 8a). Thus, Lys 61 serves to couple ligand binding to the activation of the RING domain. RING Gly 62 may serve to maintain the inactive RING conformation by disrupting the central helix (Extended Data Fig. 8b). Mutation of Gly 62 to Ala in the context of both the RNF146(RING) and RNF146(RING-WWE) constructs was performed. In the absence of ligand, the mutants promote E2~Ub lysine reactivity (Fig. 3c and Extended Data Fig. 8c). RNF146(RING)-G62A also shows increased E2 binding in NMR experiments (Extended Data Fig. 8d). Thus, Gly 62 may play a key role in the conformational transition of the central helix. Likewise, a W65A mutation in RNF146(RING-WWE) to disrupt Trp 65 interactions in the inactive state also increased basal E3 activity (Fig. 3c). The double mutant G62A/W65A of RNF146(RING-WWE) exhibits still greater activity than either of the single mutants(Fig. 3c). The mutational results are consistent with our model in which extension of the RING central helix and repositioning of Trp 65 from the E2–E3 binding site to the RING/iso-ADPr interface constitute the allosteric switch triggered by ligand binding.


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)

Stabilizing Helix 1 of RNF146 activates the RING domaina, Complete images of gels shown in Figure 3c (Oriole-stained) for G62A, W65A, G62A/W65A (GAWA), K61A and K61D mutants of RNF146(RING-WWE) with and without iso-ADPr. G62A and GAWA mutants show reduced enhancement with iso-ADPr relative to wild type, likely due to a clash of the Ala side chain with a turn in the WWE domain at position 62 (data not shown). b, Alignment of RNF146(RING) solution structure (PDB 2D8T; white) and the crystal structure determined in this study (blue) shown in stereoview. Side chains are excluded for clarity; the backbone is represented by sticks. Comparison of the conformation of Gly 62 in the two structures suggests a need for a small side chain at position 62 to allow the structural transition from the inactive to active form of RNF146. c, Anti-HA western blot of the E2~Ub/lysine reactivity assay of RNF146(RING-WWE) compared with RNF146(RING) and RNF146(RING)-G62A showing enhanced reactivity for the G to A mutation. d, (Left) 1H-15N HSQC TROSY of 15N-UbcH5c(S22R/C85S) in the presence of 0.0 (black), 0.25 (red), 0.5 (green), and 1.0 (magenta) mol. equiv. of RNF146(RING) G62A. (Right) The same experiment performed with WT RNF146(RING). The most perturbed residues, indicated by letter and position (S100, etc.), show increased chemical shift perturbations for the RNF146(RING)-G62A mutant.
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Figure 12: Stabilizing Helix 1 of RNF146 activates the RING domaina, Complete images of gels shown in Figure 3c (Oriole-stained) for G62A, W65A, G62A/W65A (GAWA), K61A and K61D mutants of RNF146(RING-WWE) with and without iso-ADPr. G62A and GAWA mutants show reduced enhancement with iso-ADPr relative to wild type, likely due to a clash of the Ala side chain with a turn in the WWE domain at position 62 (data not shown). b, Alignment of RNF146(RING) solution structure (PDB 2D8T; white) and the crystal structure determined in this study (blue) shown in stereoview. Side chains are excluded for clarity; the backbone is represented by sticks. Comparison of the conformation of Gly 62 in the two structures suggests a need for a small side chain at position 62 to allow the structural transition from the inactive to active form of RNF146. c, Anti-HA western blot of the E2~Ub/lysine reactivity assay of RNF146(RING-WWE) compared with RNF146(RING) and RNF146(RING)-G62A showing enhanced reactivity for the G to A mutation. d, (Left) 1H-15N HSQC TROSY of 15N-UbcH5c(S22R/C85S) in the presence of 0.0 (black), 0.25 (red), 0.5 (green), and 1.0 (magenta) mol. equiv. of RNF146(RING) G62A. (Right) The same experiment performed with WT RNF146(RING). The most perturbed residues, indicated by letter and position (S100, etc.), show increased chemical shift perturbations for the RNF146(RING)-G62A mutant.
Mentions: Mutational analysis was performed to understand the function of key residues in the iso-ADPr-induced conformational switch. Mutation of RING Lys 61, which makes multiple contacts with iso-ADPr, to Ala or Asp increased the Kd for iso-ADPr to values comparable to that of the WWE domain alone (Kd of 214 nM (K61A-RING-WWE) or 457 nM (K61D-RING-WWE) vs. 372 nM (WWE); Extended Data Fig. 3a, b). Although the K61D mutant can still bind ligand, it is not activated by iso-ADPr (Fig. 3c and Extended Data Fig. 8a). Thus, Lys 61 serves to couple ligand binding to the activation of the RING domain. RING Gly 62 may serve to maintain the inactive RING conformation by disrupting the central helix (Extended Data Fig. 8b). Mutation of Gly 62 to Ala in the context of both the RNF146(RING) and RNF146(RING-WWE) constructs was performed. In the absence of ligand, the mutants promote E2~Ub lysine reactivity (Fig. 3c and Extended Data Fig. 8c). RNF146(RING)-G62A also shows increased E2 binding in NMR experiments (Extended Data Fig. 8d). Thus, Gly 62 may play a key role in the conformational transition of the central helix. Likewise, a W65A mutation in RNF146(RING-WWE) to disrupt Trp 65 interactions in the inactive state also increased basal E3 activity (Fig. 3c). The double mutant G62A/W65A of RNF146(RING-WWE) exhibits still greater activity than either of the single mutants(Fig. 3c). The mutational results are consistent with our model in which extension of the RING central helix and repositioning of Trp 65 from the E2–E3 binding site to the RING/iso-ADPr interface constitute the allosteric switch triggered by ligand binding.

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