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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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Crystal structure of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complexa. Cartoon representation of the RNF146/UbcH5a complex with RING domain colored blue, WWE domain colored purple, and UbcH5a colored green. Zn2+ ions are shown as grey spheres, and the iso-ADPr ligand is represented as sticks. b. The RNF146/iso-ADPr interface. (Left) Surface electrostatic view of RNF146(RING-WWE), showing the iso-ADPr/PAR binding pocket; (Center) same view, cartoon representation; (Right) close-up view of iso-ADPr pocket. Polar contacts between protein and the ligand, iso-ADPr, are indicated by dashed lines; RING residues K61 (magenta) and W65 (orange) are highlighted.
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Figure 2: Crystal structure of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complexa. Cartoon representation of the RNF146/UbcH5a complex with RING domain colored blue, WWE domain colored purple, and UbcH5a colored green. Zn2+ ions are shown as grey spheres, and the iso-ADPr ligand is represented as sticks. b. The RNF146/iso-ADPr interface. (Left) Surface electrostatic view of RNF146(RING-WWE), showing the iso-ADPr/PAR binding pocket; (Center) same view, cartoon representation; (Right) close-up view of iso-ADPr pocket. Polar contacts between protein and the ligand, iso-ADPr, are indicated by dashed lines; RING residues K61 (magenta) and W65 (orange) are highlighted.

Mentions: The structural basis of RNF146 activation by iso-ADPr/PAR is evident in a 1.9Å crystal structure of a RNF146(RING-WWE)/UbcH5a/iso-ADPr complex (Fig. 2 and Extended Data Table 1). The RING domain structure is largely similar to other structurally characterized RINGs (see below), and the RNF146 WWE domain structure is almost identical to an existing crystal structure13 (Extended Data Fig. 5a). The most notable feature is the location of iso-ADPr, which contacts both the RING and WWE domains (Fig. 2b and Extended Data Fig. 5b, c). Contacts between the WWE domain and iso-ADPr are similar to those previously described13. Lys 61 from the RING is within hydrogen-bond distance to hydroxyl groups on both ribose moieties of iso-ADPr and to a water molecule that can mediate a hydrogen bond with the adenine ring. In addition, RING residue Trp 65 forms a hydrogen bond with a main chain carbonyl in the WWE domain and has van der Waals contacts with the ligand. Although iso-ADPr is buried in a valley between the RING and WWE domains, the phosphate groups on either end of iso-ADPr are exposed. Thus, the observed ligand orientation is consistent with the notion that RNF146 binds an internal unit of a PAR polymer.


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)

Crystal structure of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complexa. Cartoon representation of the RNF146/UbcH5a complex with RING domain colored blue, WWE domain colored purple, and UbcH5a colored green. Zn2+ ions are shown as grey spheres, and the iso-ADPr ligand is represented as sticks. b. The RNF146/iso-ADPr interface. (Left) Surface electrostatic view of RNF146(RING-WWE), showing the iso-ADPr/PAR binding pocket; (Center) same view, cartoon representation; (Right) close-up view of iso-ADPr pocket. Polar contacts between protein and the ligand, iso-ADPr, are indicated by dashed lines; RING residues K61 (magenta) and W65 (orange) are highlighted.
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Related In: Results  -  Collection

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Figure 2: Crystal structure of the RNF146(RING-WWE)/UbcH5a/iso-ADPr complexa. Cartoon representation of the RNF146/UbcH5a complex with RING domain colored blue, WWE domain colored purple, and UbcH5a colored green. Zn2+ ions are shown as grey spheres, and the iso-ADPr ligand is represented as sticks. b. The RNF146/iso-ADPr interface. (Left) Surface electrostatic view of RNF146(RING-WWE), showing the iso-ADPr/PAR binding pocket; (Center) same view, cartoon representation; (Right) close-up view of iso-ADPr pocket. Polar contacts between protein and the ligand, iso-ADPr, are indicated by dashed lines; RING residues K61 (magenta) and W65 (orange) are highlighted.
Mentions: The structural basis of RNF146 activation by iso-ADPr/PAR is evident in a 1.9Å crystal structure of a RNF146(RING-WWE)/UbcH5a/iso-ADPr complex (Fig. 2 and Extended Data Table 1). The RING domain structure is largely similar to other structurally characterized RINGs (see below), and the RNF146 WWE domain structure is almost identical to an existing crystal structure13 (Extended Data Fig. 5a). The most notable feature is the location of iso-ADPr, which contacts both the RING and WWE domains (Fig. 2b and Extended Data Fig. 5b, c). Contacts between the WWE domain and iso-ADPr are similar to those previously described13. Lys 61 from the RING is within hydrogen-bond distance to hydroxyl groups on both ribose moieties of iso-ADPr and to a water molecule that can mediate a hydrogen bond with the adenine ring. In addition, RING residue Trp 65 forms a hydrogen bond with a main chain carbonyl in the WWE domain and has van der Waals contacts with the ligand. Although iso-ADPr is buried in a valley between the RING and WWE domains, the phosphate groups on either end of iso-ADPr are exposed. Thus, the observed ligand orientation is consistent with the notion that RNF146 binds an internal unit of a PAR polymer.

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