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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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Mechanism of RNF146 PAR-mediated RING activationa, (Top left) Apo-RNF146 RING domain solution structure ensemble (residues 30–85; PDB 2D8T). I38, L66, A71 and W65 form a hydrophobic cluster in all members of the ensemble. (Top center) The RING domain of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex (blue) adopts a canonical RING structure shown in the same orientation as the structure in the left panel. (Top right) Helix 1 of the RNF146 RING domain in our complex aligned with a representative NMR structure. Upon iso-ADPr binding, helix 1 is extended following G62 and W65 undergoes a dramatic relocation. b, (Left)1H-15N HSQC of 15N-UbcH5c (S22R/C85S) alone (black), with 0.25 molar equivalence (m.e.) RNF146(RING-WWE) (green), and with 0.25 m.e. RNF146(RING-WWE) plus 0.5 m.e. of iso-ADPr (red). (Right) Chemical shift perturbations of residues in left panel mapped to the surface of UbcH5c (PDB 2FUH) show the binding surface for RNF146(RING-WWE) (light blue, on green E2). c, E2~Ub/lysine reactivity assays of RNF146(RING-WWE) mutants with the E2 UbcH5c; full gels are shown in Extended Data Fig. 8a.
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Figure 3: Mechanism of RNF146 PAR-mediated RING activationa, (Top left) Apo-RNF146 RING domain solution structure ensemble (residues 30–85; PDB 2D8T). I38, L66, A71 and W65 form a hydrophobic cluster in all members of the ensemble. (Top center) The RING domain of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex (blue) adopts a canonical RING structure shown in the same orientation as the structure in the left panel. (Top right) Helix 1 of the RNF146 RING domain in our complex aligned with a representative NMR structure. Upon iso-ADPr binding, helix 1 is extended following G62 and W65 undergoes a dramatic relocation. b, (Left)1H-15N HSQC of 15N-UbcH5c (S22R/C85S) alone (black), with 0.25 molar equivalence (m.e.) RNF146(RING-WWE) (green), and with 0.25 m.e. RNF146(RING-WWE) plus 0.5 m.e. of iso-ADPr (red). (Right) Chemical shift perturbations of residues in left panel mapped to the surface of UbcH5c (PDB 2FUH) show the binding surface for RNF146(RING-WWE) (light blue, on green E2). c, E2~Ub/lysine reactivity assays of RNF146(RING-WWE) mutants with the E2 UbcH5c; full gels are shown in Extended Data Fig. 8a.

Mentions: Insight into the conformational changes that accompany iso-ADPr binding is provided by comparison to an NMR structure of the unliganded RNF146 RING domain (PDB 2D8T; RIKEN Structural Genomics/Proteomics Initiative). In the unliganded RING domain the central helix is one turn shorter, with residues 62–66 instead forming a loop that protrudes into the E2–E3 binding interface (Fig. 3a and Extended Data Fig. 7a). Trp 65 makes hydrophobic interactions with Ile 36, Leu 66, and Ala 71 and is in a position to block E2 binding. Residues 62–66 adopt the helical structure associated with active RING domains in the iso-ADPr-bound structure. Thus, the RNF146 RING can adopt two different conformations and binding of iso-ADPr stabilizes an active structure with a functional E2-binding surface.


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)

Mechanism of RNF146 PAR-mediated RING activationa, (Top left) Apo-RNF146 RING domain solution structure ensemble (residues 30–85; PDB 2D8T). I38, L66, A71 and W65 form a hydrophobic cluster in all members of the ensemble. (Top center) The RING domain of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex (blue) adopts a canonical RING structure shown in the same orientation as the structure in the left panel. (Top right) Helix 1 of the RNF146 RING domain in our complex aligned with a representative NMR structure. Upon iso-ADPr binding, helix 1 is extended following G62 and W65 undergoes a dramatic relocation. b, (Left)1H-15N HSQC of 15N-UbcH5c (S22R/C85S) alone (black), with 0.25 molar equivalence (m.e.) RNF146(RING-WWE) (green), and with 0.25 m.e. RNF146(RING-WWE) plus 0.5 m.e. of iso-ADPr (red). (Right) Chemical shift perturbations of residues in left panel mapped to the surface of UbcH5c (PDB 2FUH) show the binding surface for RNF146(RING-WWE) (light blue, on green E2). c, E2~Ub/lysine reactivity assays of RNF146(RING-WWE) mutants with the E2 UbcH5c; full gels are shown in Extended Data Fig. 8a.
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Figure 3: Mechanism of RNF146 PAR-mediated RING activationa, (Top left) Apo-RNF146 RING domain solution structure ensemble (residues 30–85; PDB 2D8T). I38, L66, A71 and W65 form a hydrophobic cluster in all members of the ensemble. (Top center) The RING domain of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex (blue) adopts a canonical RING structure shown in the same orientation as the structure in the left panel. (Top right) Helix 1 of the RNF146 RING domain in our complex aligned with a representative NMR structure. Upon iso-ADPr binding, helix 1 is extended following G62 and W65 undergoes a dramatic relocation. b, (Left)1H-15N HSQC of 15N-UbcH5c (S22R/C85S) alone (black), with 0.25 molar equivalence (m.e.) RNF146(RING-WWE) (green), and with 0.25 m.e. RNF146(RING-WWE) plus 0.5 m.e. of iso-ADPr (red). (Right) Chemical shift perturbations of residues in left panel mapped to the surface of UbcH5c (PDB 2FUH) show the binding surface for RNF146(RING-WWE) (light blue, on green E2). c, E2~Ub/lysine reactivity assays of RNF146(RING-WWE) mutants with the E2 UbcH5c; full gels are shown in Extended Data Fig. 8a.
Mentions: Insight into the conformational changes that accompany iso-ADPr binding is provided by comparison to an NMR structure of the unliganded RNF146 RING domain (PDB 2D8T; RIKEN Structural Genomics/Proteomics Initiative). In the unliganded RING domain the central helix is one turn shorter, with residues 62–66 instead forming a loop that protrudes into the E2–E3 binding interface (Fig. 3a and Extended Data Fig. 7a). Trp 65 makes hydrophobic interactions with Ile 36, Leu 66, and Ala 71 and is in a position to block E2 binding. Residues 62–66 adopt the helical structure associated with active RING domains in the iso-ADPr-bound structure. Thus, the RNF146 RING can adopt two different conformations and binding of iso-ADPr stabilizes an active structure with a functional E2-binding surface.

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