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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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RNF146/iso-ADPr binding allows the RING domain to bind and activate a ubiquitin conjugating enzyme (E2)a, (Left) Superposition of the RING domain of unliganded RNF146 (PDB 2D8T; grey, Trp 65 is shown as orange spheres) with the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex (colored as in Fig 2a), shows a clash of Trp 65 with UbcH5a at the E2-E3 binding interface. This clash is observed when the RNF146 RING structure (2D8T) is aligned with all other E2-E3 structures15–20,23. b, Peak broadening (Top; intensity relative to free E2) and chemical shift perturbations (CSPs; bottom) of 15N-UbcH5c(S22R/C85S) resonances (data are from the spectra shown in Figure 3b). Histograms shown in blue compare the spectral properties of free E2 to E2 plus RNF146(RING-WWE); histograms shown in red compare free E2 to E2 plus RNF146(RING-WWE) and iso-ADPr. Dashed lines indicate one standard deviation from the mean value of the liganded (red) plots. Values below and above the dashed lines for the relative intensities and CSPs respectively are plotted on the E2 surface shown in panel c and Figure 3b. c, (Left) The RNF146(RING-WWE) binding surface inferred from data in panel b (light blue, on green E2), is compared with (right) the BRCA1/BARD1 binding surface on E2 (yellow, on green E2; residues 1–112 and residues 26–115, respectively) previously inferred by an analogous experiment25. When the NMR perturbations are mapped to the surface of UbcH5c, the revealed binding sites are very similar, and are consistent with previously reported binding surfaces for RING E3s on free ubiquitin conjugating enzymes15–20,23. d, Chemical shift perturbations and broadening of resonances from 15N-E2~Ub conjugate (UbcH5c(S22R/C85S)-O-Ub) upon RNF146(RING-WWE)/iso-ADPr binding (determined by the same method as shown in panel b, but with only 0.125 mol. equiv. E3 added to minimize hydrolysis of the E2~Ub oxyester during NMR data collection). (Left) Perturbed residues are mapped onto UbcH5b (magenta on green E2) and ubiquitin (yellow on red ubiquitin). (Center and right) Perturbed residues mapped onto the structure of E2~Ub as it appears in the E3/E2~Ub complex of BIRC7/UbcH5b-Ub (PDB 4AUQ; BIRC7 not shown for clarity)24 show that the surfaces highlighted in the left panel are buried in the “closed” state. The data show that RNF146 activates the E2~Ub conjugate by inducing the closed conformation17,21,22,24. Because only the most perturbed residues are mapped to the E2~Ub surface, the E3 binding surface is not highlighted on the E2 in panel d.
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Figure 11: RNF146/iso-ADPr binding allows the RING domain to bind and activate a ubiquitin conjugating enzyme (E2)a, (Left) Superposition of the RING domain of unliganded RNF146 (PDB 2D8T; grey, Trp 65 is shown as orange spheres) with the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex (colored as in Fig 2a), shows a clash of Trp 65 with UbcH5a at the E2-E3 binding interface. This clash is observed when the RNF146 RING structure (2D8T) is aligned with all other E2-E3 structures15–20,23. b, Peak broadening (Top; intensity relative to free E2) and chemical shift perturbations (CSPs; bottom) of 15N-UbcH5c(S22R/C85S) resonances (data are from the spectra shown in Figure 3b). Histograms shown in blue compare the spectral properties of free E2 to E2 plus RNF146(RING-WWE); histograms shown in red compare free E2 to E2 plus RNF146(RING-WWE) and iso-ADPr. Dashed lines indicate one standard deviation from the mean value of the liganded (red) plots. Values below and above the dashed lines for the relative intensities and CSPs respectively are plotted on the E2 surface shown in panel c and Figure 3b. c, (Left) The RNF146(RING-WWE) binding surface inferred from data in panel b (light blue, on green E2), is compared with (right) the BRCA1/BARD1 binding surface on E2 (yellow, on green E2; residues 1–112 and residues 26–115, respectively) previously inferred by an analogous experiment25. When the NMR perturbations are mapped to the surface of UbcH5c, the revealed binding sites are very similar, and are consistent with previously reported binding surfaces for RING E3s on free ubiquitin conjugating enzymes15–20,23. d, Chemical shift perturbations and broadening of resonances from 15N-E2~Ub conjugate (UbcH5c(S22R/C85S)-O-Ub) upon RNF146(RING-WWE)/iso-ADPr binding (determined by the same method as shown in panel b, but with only 0.125 mol. equiv. E3 added to minimize hydrolysis of the E2~Ub oxyester during NMR data collection). (Left) Perturbed residues are mapped onto UbcH5b (magenta on green E2) and ubiquitin (yellow on red ubiquitin). (Center and right) Perturbed residues mapped onto the structure of E2~Ub as it appears in the E3/E2~Ub complex of BIRC7/UbcH5b-Ub (PDB 4AUQ; BIRC7 not shown for clarity)24 show that the surfaces highlighted in the left panel are buried in the “closed” state. The data show that RNF146 activates the E2~Ub conjugate by inducing the closed conformation17,21,22,24. Because only the most perturbed residues are mapped to the E2~Ub surface, the E3 binding surface is not highlighted on the E2 in panel d.

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)

RNF146/iso-ADPr binding allows the RING domain to bind and activate a ubiquitin conjugating enzyme (E2)a, (Left) Superposition of the RING domain of unliganded RNF146 (PDB 2D8T; grey, Trp 65 is shown as orange spheres) with the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex (colored as in Fig 2a), shows a clash of Trp 65 with UbcH5a at the E2-E3 binding interface. This clash is observed when the RNF146 RING structure (2D8T) is aligned with all other E2-E3 structures15–20,23. b, Peak broadening (Top; intensity relative to free E2) and chemical shift perturbations (CSPs; bottom) of 15N-UbcH5c(S22R/C85S) resonances (data are from the spectra shown in Figure 3b). Histograms shown in blue compare the spectral properties of free E2 to E2 plus RNF146(RING-WWE); histograms shown in red compare free E2 to E2 plus RNF146(RING-WWE) and iso-ADPr. Dashed lines indicate one standard deviation from the mean value of the liganded (red) plots. Values below and above the dashed lines for the relative intensities and CSPs respectively are plotted on the E2 surface shown in panel c and Figure 3b. c, (Left) The RNF146(RING-WWE) binding surface inferred from data in panel b (light blue, on green E2), is compared with (right) the BRCA1/BARD1 binding surface on E2 (yellow, on green E2; residues 1–112 and residues 26–115, respectively) previously inferred by an analogous experiment25. When the NMR perturbations are mapped to the surface of UbcH5c, the revealed binding sites are very similar, and are consistent with previously reported binding surfaces for RING E3s on free ubiquitin conjugating enzymes15–20,23. d, Chemical shift perturbations and broadening of resonances from 15N-E2~Ub conjugate (UbcH5c(S22R/C85S)-O-Ub) upon RNF146(RING-WWE)/iso-ADPr binding (determined by the same method as shown in panel b, but with only 0.125 mol. equiv. E3 added to minimize hydrolysis of the E2~Ub oxyester during NMR data collection). (Left) Perturbed residues are mapped onto UbcH5b (magenta on green E2) and ubiquitin (yellow on red ubiquitin). (Center and right) Perturbed residues mapped onto the structure of E2~Ub as it appears in the E3/E2~Ub complex of BIRC7/UbcH5b-Ub (PDB 4AUQ; BIRC7 not shown for clarity)24 show that the surfaces highlighted in the left panel are buried in the “closed” state. The data show that RNF146 activates the E2~Ub conjugate by inducing the closed conformation17,21,22,24. Because only the most perturbed residues are mapped to the E2~Ub surface, the E3 binding surface is not highlighted on the E2 in panel d.
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Figure 11: RNF146/iso-ADPr binding allows the RING domain to bind and activate a ubiquitin conjugating enzyme (E2)a, (Left) Superposition of the RING domain of unliganded RNF146 (PDB 2D8T; grey, Trp 65 is shown as orange spheres) with the RNF146(RING-WWE)/UbcH5a/iso-ADPr complex (colored as in Fig 2a), shows a clash of Trp 65 with UbcH5a at the E2-E3 binding interface. This clash is observed when the RNF146 RING structure (2D8T) is aligned with all other E2-E3 structures15–20,23. b, Peak broadening (Top; intensity relative to free E2) and chemical shift perturbations (CSPs; bottom) of 15N-UbcH5c(S22R/C85S) resonances (data are from the spectra shown in Figure 3b). Histograms shown in blue compare the spectral properties of free E2 to E2 plus RNF146(RING-WWE); histograms shown in red compare free E2 to E2 plus RNF146(RING-WWE) and iso-ADPr. Dashed lines indicate one standard deviation from the mean value of the liganded (red) plots. Values below and above the dashed lines for the relative intensities and CSPs respectively are plotted on the E2 surface shown in panel c and Figure 3b. c, (Left) The RNF146(RING-WWE) binding surface inferred from data in panel b (light blue, on green E2), is compared with (right) the BRCA1/BARD1 binding surface on E2 (yellow, on green E2; residues 1–112 and residues 26–115, respectively) previously inferred by an analogous experiment25. When the NMR perturbations are mapped to the surface of UbcH5c, the revealed binding sites are very similar, and are consistent with previously reported binding surfaces for RING E3s on free ubiquitin conjugating enzymes15–20,23. d, Chemical shift perturbations and broadening of resonances from 15N-E2~Ub conjugate (UbcH5c(S22R/C85S)-O-Ub) upon RNF146(RING-WWE)/iso-ADPr binding (determined by the same method as shown in panel b, but with only 0.125 mol. equiv. E3 added to minimize hydrolysis of the E2~Ub oxyester during NMR data collection). (Left) Perturbed residues are mapped onto UbcH5b (magenta on green E2) and ubiquitin (yellow on red ubiquitin). (Center and right) Perturbed residues mapped onto the structure of E2~Ub as it appears in the E3/E2~Ub complex of BIRC7/UbcH5b-Ub (PDB 4AUQ; BIRC7 not shown for clarity)24 show that the surfaces highlighted in the left panel are buried in the “closed” state. The data show that RNF146 activates the E2~Ub conjugate by inducing the closed conformation17,21,22,24. Because only the most perturbed residues are mapped to the E2~Ub surface, the E3 binding surface is not highlighted on the E2 in panel d.
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