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Crystal Structure of a Tankyrase-Axin Complex and Its Implications for Axin Turnover and Tankyrase Substrate Recruitment

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Crystal Structure of a Tankyrase-Axin Complex and Its Implications for Axin Turnover and Tankyrase Substrate Recruitment Crystal structure of a Tankyrase-Axin complex and its implications for Axin turnover and Tankyrase substrate recruitment Seamus Morronea,1, Zhihong Chenga,1, Randall T. Moonb, Feng Congc, and Wenqing Xua,2 aDepartment of Biological Structure, University of Washington School of Medicine, Seattle, WA 98195; bDepartment of Pharmacology, Howard Hughes Medical Institute, and Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, WA 98195; and cNovartis Institutes for Biomedical Research, Cambridge, MA 02139 Edited by* Stephen C. Harrison, Children's Hospital, Harvard Medical School, and Howard Hughes Medical Institute, Boston, MA, and approved December8, 2011 (received for review October 9, 2011) Axin is a tumor suppressor and a key negative regulator of the Ubiquitination of Axin, which leads to its subsequent turnover, Wnt/β-catenin signaling pathway. Axin turnover is controlled by its requires its poly-ADP-ribosylation (PARylation) (11). PARylation poly-ADP-ribosylation catalyzed by tankyrase (TNKS), which re- of proteins is catalyzed by a family of poly-ADP-ribose poly- quires the direct interaction of Axin with TNKS. This interaction merases (PARPs), with 18 known members in humans, which re- is thus an attractive drug target for treating cancers, brain injuries, gulate many aspects of biology including genomic stability, cell and other diseases where β-catenin is involved. Here we report the cycle, and energy metabolism (15, 16). Axin PARylation is speci- crystal structure of a mouse TNKS1 fragment containing ankyrin- fically catalyzed by tankyrases 1 and 2 (a.k.a. TNKS1/PARP5a repeat clusters 2 and 3 (ARC2-3) in a complex with the TNKS-bind- and TNKS2/PARP5b, respectively). Indeed, Wnt pathway inhibi- ing domain of mouse Axin1. Surprisingly, we found that Axin con- tors discovered in cell-based assays stabilize cytosolic Axin by tains two discrete TNKS-binding segments, both of which bind blocking the active sites of TNKS catalytic PARP domains (17). simultaneously to the two ARC2 domains in the ARC2-3 homodi- Since TNKS1 and TNKS2 catalyze PARylation of many different mer. Our crystal structure shows that in each TNKS-binding seg- protein substrates (18), TNKS active-site inhibitors are not ment of Axin there is a conserved glycine residue that lies in the specific for the Wnt/β-catenin pathway. It may therefore be useful bottom of a narrow “gate” formed by two parallel tyrosine side to develop specific Wnt/β-catenin pathway inhibitors that prevent chains on the TNKS surface. This glycine-selection gate is crucial for Axin recognition by TNKS (8). TNKS-Axin interactions, as mutation of the TNKS gate-forming re- TNKS1 and TNKS2 share three conserved domains: the sidues, or mutation of either glycine residue in the two Axin seg- ankyrin-repeat domain responsible for substrate recruitment, a ments, completely abolishes the binding of the corresponding Axin SAM domain that mediates homo- and hetero-oligomerization of segment to TNKS. The bivalent binding of Axin to TNKS is required TNKS, and a C-terminal catalytic PARP domain (Fig. 1A) (18– for Axin turnover, since mutations in either gate-binding glycine 24). TNKS1 also contains a unique N-terminal HPS-rich domain residue in Axin lead to Axin stabilization in the cell. In addition, with unknown function. The ankyrin-repeat domain is composed our analyses also reveal the structural basis for TNKS substrate of five conserved subdomains, or ankyrin-repeat clusters (ARCs) – recruitment, and shed light on the overall structure of TNKS that (19, 20, 22 24). There are conserved linkers between neighboring should help in developing specific inhibitors of Wnt/β-catenin sig- ARCs. It is presently unclear what structure the linker adopts naling. and how these five ARCs relate with one another. Several TNKS binding partners or substrate proteins contain a consensus TNKS cancer ∣ drug target ∣ poly-ADP-ribosylation ∣ tumor suppressor ∣ Wnt binding motif (TBM) with a sequence of RXXPDG, which ap- pears to be sufficient for binding to each of the five subdomains he Wnt/β-catenin signaling pathway plays critical roles in (19, 20, 22, 23). It is noteworthy that Axin uses its N-terminal Tembryonic development, stem cell biology and tissue home- domain (residues 1-80), which does not contain an apparent ostasis in adults, and in disease. Deregulation of the canonical TBM. Previous work shows that Axin residues 19-30, the most β conserved region of Axin, is required for TNKS binding (11). For Wnt/ -catenin signaling pathway is tightly associated with several β cancers, particularly colon cancer (1, 2). Development of Wnt/β- understanding Wnt/ -catenin signaling mechanisms and for developing Wnt/β-catenin pathway inhibitors, it would be useful catenin pathway inhibitors is of great interest for the treatment of to know how Axin interacts with TNKS, which lead us to our multiple diseases, including cancers and brain injuries (3–6). One present investigation. potential target for small molecule inhibitors of Wnt/β-catenin Here we report the crystal structure of an Axin-TNKS complex, signaling is Axin (a.k.a. Axin1), which is a scaffold of the β-cate- which provides a unique view of structural organization of multi- nin destruction complex and a known tumor suppressor (2, 7, 8). The assembly or disassembly of the destruction complex in the cytosol is a central regulatory step of the Wnt/β-catenin pathway Author contributions: S.M., Z.C., R.T.M., F.C., and W.X. designed research; S.M., Z.C., F.C., (2, 9). Since Axin levels are much lower than most other key com- and W.X. performed research; S.M., Z.C., R.T.M., F.C., and W.X. analyzed data; and S.M., ponents of the destruction complex in the cell, Axin is believed Z.C., R.T.M., F.C., and W.X. wrote the paper. β The authors declare a conflict of interest. R.T.M. is a cofounder of, and consultant with, to be the rate-limiting factor for the assembly of the -catenin FATE Therapeutics. The other authors declare no competing financial interests. destruction complex (10). Recently, various cell-based screenings *This Direct Submission article had a prearranged editor. of Wnt pathway inhibitors led to the discovery of compounds that Data deposition: Diffraction data and coordinates of the mouse TNKS1(306-655)-Axin1 inhibit Wnt signaling by inhibiting Axin turnover, demonstrating (1-80) complex structure have been deposited in the Protein Data Bank, www.pdb.org that Axin turnover is a crucial target for developing Wnt pathway (PDB ID code 3UTM). inhibitors (11, 12). In addition to the Wnt/β-catenin pathway, pre- 1S.M. and Z.C. contributed equally to this work. vention of Axin degradation may also suppress tumor growth by 2To whom correspondence should be addressed. E-mail: [email protected] promoting the turnover of the c-myc oncoprotein and stimulation This article contains supporting information online at www.pnas.org/lookup/suppl/ of p53 function (13, 14). doi:10.1073/pnas.1116618109/-/DCSupplemental. 1500–1505 ∣ PNAS ∣ January 31, 2012 ∣ vol. 109 ∣ no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1116618109 Downloaded by guest on September 30, 2021 Fig. 1. Overall structure of the TNKS(308-655)/Axin1(1-80) complex. (A) Schematic representation of the domain organization of mouse Tankyrase and Axin1. “ARC” stands for ankyrin-repeat cluster. Five ARCs are colored in gray. Linkers between them are colored dark blue. Domains SAM and PARP are also labeled. “GBD” stands for GSK3 binding domain; “BD” stands for β-catenin binding domain. RGS and DIX domains are also shown. Segment-N and –C of mAxin1(1-80) observed in the crystal structure are colored in magenta and cyan, respectively. (B) Overall structure of a complex between mTNKS ARC2-3 and mAxin1(1-80). Two copies of ARC2 and ARC3 in the dimer are shown in cartoon with light blue and light orange, respectively. Linker helices before and after ARC2 and ARC3in two monomers are shown in blue and orange, respectively. The two Axin1 segments are shown in solid surface with magenta and cyan, respectively. ple ankyrin-repeat clusters in TNKS. Combined with our bio- agreement with our domain structure model (Fig. 1A), derived chemical and functional data, the structure reveals two discrete from our crystal structure shown in Fig. 1B. We then overex- Axin segments involved in TNKS interaction, which are also re- pressed and purified each of the five ARCs separately and tested quired for Axin turnover. Interestingly, our studies also reveal that their Axin1 binding activities. Pulldown experiments using GST- the Axin-TNKS interaction involves a unique structural mechanism ARCs and untagged Axin1(1-80) (Fig. S1) demonstrated that for Gly selectivity. Together, our work provides an enhanced foun- ARC2, ARC4, and ARC5, but not ARC1 and ARC3, can interact dation for understanding the molecular mechanism of Axin turn- with Axin1(1-80), respectively. over and for TNKS substrate recruitment, and should facilitate drug discovery that targets the TNKS-Axin interface. Crystal Structure of an Axin-TNKS Complex Numerous combinations of mouse TNKS-Axin complexes were tested for crystallization. Results High-quality crystals were obtained with the mouse TNKS1(308- Mapping of the TNKS-Axin Binding Domains The N-terminal domain 655)-Axin1(1-80) complex. The crystal structure was determined of mouse Axin1, which contains residues 1-80, was shown to by molecular replacement and refined at 2.0 Å resolution. This be responsible for TNKS binding, whereas residues 19-30, which TNKS fragment contains ARC2 and ARC3, as well as three inter- is fully conserved in all Axin1 analogues, is necessary for this ARC linkers: linkers 1∕2, 2∕3, and 3∕4 (linker 1∕2 represents interaction (11).
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