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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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Multiple sequence alignment (MSA) of RNF146 orthologsThe colored bars above the sequence alignment indicate regions of interest in human RNF146: RING domain (blue), WWE domain (purple), and potential TNKS binding motifs, numbered I through V (orange). While there are no apparent RXXGDG TNKS binding motifs, the five potential binding motifs indicated here are based on the TNKS-substrate interface plasticity demonstrated by a recent crystal structure of the Axin-TNKS complex26.
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Figure 5: Multiple sequence alignment (MSA) of RNF146 orthologsThe colored bars above the sequence alignment indicate regions of interest in human RNF146: RING domain (blue), WWE domain (purple), and potential TNKS binding motifs, numbered I through V (orange). While there are no apparent RXXGDG TNKS binding motifs, the five potential binding motifs indicated here are based on the TNKS-substrate interface plasticity demonstrated by a recent crystal structure of the Axin-TNKS complex26.

Mentions: Two domains can be identified within the 358-residue sequence of RNF146: an N-terminal RING domain followed by a WWE domain that binds iso-ADPr13 (Fig. 1a and Extended Data Fig. 1). RING E3 ligases activate a ubiquitin-conjugating enzyme (E2) to transfer ubiquitin directly from the E2 active site to a lysine residue of a substrate. The intrinsic ability of RING E3s to stimulate Ub transfer can be assayed by following Ub transfer from E2~Ub to free amino acid lysine14. Unexpectedly, neither purified RNF146 RING domain nor full-length RNF146 significantly enhance the rate of Ub transfer from UbcH5~Ub to lysine (Fig. 1b and Extended Data Fig. 2a). However, addition of the WWE ligands iso-ADPr or PAR to the full-length RNF146 or a fragment containing the RING and WWE domains (RING-WWE), but not a RING-only construct, results in robust activation (Fig. 1 and Extended Data Fig. 2), consistent with reports that RNF146 is a PARylation-dependent E3 (refs 10–12). Isothermal titration calorimetry (ITC) analysis shows that iso-ADPr binds to RNF146(RING-WWE) ten times tighter than to the WWE domain alone (Kd 39 nM and 372 nM, respectively; Extended Data Fig. 3a, b), suggesting that the presence of the RING domain contributes to ligand binding. Furthermore, RNF146(RING-WWE) is sensitive to subtilisin digestion in the absence of iso-ADPr, but is more resistant to proteolysis in the presence of iso-ADPr (Extended Data Fig. 3c), and NMR experiments indicate structural changes within the RING domain of the RING-WWE fragment upon iso-ADPr binding (Extended Data Fig. 4). Altogether, the data indicate that both the RING and WWE domains are involved in iso-ADPr binding and that ligand binding affects the conformation and/or stability of the RING domain, leading to increased E3 ligase activity.


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)

Multiple sequence alignment (MSA) of RNF146 orthologsThe colored bars above the sequence alignment indicate regions of interest in human RNF146: RING domain (blue), WWE domain (purple), and potential TNKS binding motifs, numbered I through V (orange). While there are no apparent RXXGDG TNKS binding motifs, the five potential binding motifs indicated here are based on the TNKS-substrate interface plasticity demonstrated by a recent crystal structure of the Axin-TNKS complex26.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4289021&req=5

Figure 5: Multiple sequence alignment (MSA) of RNF146 orthologsThe colored bars above the sequence alignment indicate regions of interest in human RNF146: RING domain (blue), WWE domain (purple), and potential TNKS binding motifs, numbered I through V (orange). While there are no apparent RXXGDG TNKS binding motifs, the five potential binding motifs indicated here are based on the TNKS-substrate interface plasticity demonstrated by a recent crystal structure of the Axin-TNKS complex26.
Mentions: Two domains can be identified within the 358-residue sequence of RNF146: an N-terminal RING domain followed by a WWE domain that binds iso-ADPr13 (Fig. 1a and Extended Data Fig. 1). RING E3 ligases activate a ubiquitin-conjugating enzyme (E2) to transfer ubiquitin directly from the E2 active site to a lysine residue of a substrate. The intrinsic ability of RING E3s to stimulate Ub transfer can be assayed by following Ub transfer from E2~Ub to free amino acid lysine14. Unexpectedly, neither purified RNF146 RING domain nor full-length RNF146 significantly enhance the rate of Ub transfer from UbcH5~Ub to lysine (Fig. 1b and Extended Data Fig. 2a). However, addition of the WWE ligands iso-ADPr or PAR to the full-length RNF146 or a fragment containing the RING and WWE domains (RING-WWE), but not a RING-only construct, results in robust activation (Fig. 1 and Extended Data Fig. 2), consistent with reports that RNF146 is a PARylation-dependent E3 (refs 10–12). Isothermal titration calorimetry (ITC) analysis shows that iso-ADPr binds to RNF146(RING-WWE) ten times tighter than to the WWE domain alone (Kd 39 nM and 372 nM, respectively; Extended Data Fig. 3a, b), suggesting that the presence of the RING domain contributes to ligand binding. Furthermore, RNF146(RING-WWE) is sensitive to subtilisin digestion in the absence of iso-ADPr, but is more resistant to proteolysis in the presence of iso-ADPr (Extended Data Fig. 3c), and NMR experiments indicate structural changes within the RING domain of the RING-WWE fragment upon iso-ADPr binding (Extended Data Fig. 4). Altogether, the data indicate that both the RING and WWE domains are involved in iso-ADPr binding and that ligand binding affects the conformation and/or stability of the RING domain, leading to increased E3 ligase activity.

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