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). To test if the most conserved region of Axin1 the linker between ARC1 and ARC2, and so on; Fig. 1 A and B). (residues 19–30) is sufficient for TNKS binding, we compared the The electron densities for the entire TNKS linker 1∕2 and 2∕3 are binding of recombinant Axin1(1-80) and Axin1(1-43) with the 3∕4 entire TNKS ankyrin-repeat domain (ARC1-5, residues 173-961; visible, although the density for the linker is only visible for Fig. 1A). While Axin1(1-80) forms a stable complex with ARC1-5 the N-terminal portion. There are two TNKS subunits and one that can be copurified using size-exclusion chromatography Axin molecule in each asymmetric unit in the crystal lattice (see (SEC) and ion-exchange chromatography (IEC), Axin1(1-43)/ below). Each ARC consists of four ankyrin repeats, and each β TNKS complexes cannot be copurified using SEC or IEC, indi- ankyrin repeat contains two helices and a -hairpin linking the cating that Axin1 residues 44-80 are important for TNKS binding. two. Each of the conserved inter-ARC linkers forms a single long Therefore, we used Axin1(1-80) for our structural analysis. helix and packs on the ends of neighboring ARCs (Fig. 1B). The It has been proposed that the ankyrin-repeat domain of TNKS two TNKS copies form a swapped dimer with the swap occurring contains five ankyrin-repeat clusters (ARCs) that are separated behind the first helix of ARC3 (Fig. 1B and Fig. S2). In solution, by four conserved linkers, with each ARC containing four ankyrin TNKS(308-655) has a tendency to form a dimer, whereas the repeats (23). While other models with distinct ankyrin-repeat binding of Axin1(1-80) promotes homogeneous dimerization of BIOPHYSICS AND

assignments have also been proposed, this model is in general TNKS(308-655) (Fig. S3). COMPUTATIONAL BIOLOGY

Morrone et al. PNAS ∣ January 31, 2012 ∣ vol. 109 ∣ no. 5 ∣ 1501 Downloaded by guest on September 30, 2021 Fig. 2. Structural details of the mouse TNKS1(308-655)/Axin1(1-80) interactions. (A) TNKS1-Axin1 segment-N interface. One copy of ARC2 of TNKS is shown in gray solid surface. Segment-N of Axin1 is shown in stick model in magenta. Residues involved in the interface are labeled. Regular font is used for labeling TNKS1 residues and bold font for Axin1 residues. The critical glycine is shown in sphere mode. (B) TNKS1-Axin1 segment-C interface. Another copy of ARC2 of TNKS is shown in gray solid surface as in A. Segment-C of Axin1 is shown in stick model in cyan. Residues are labeled in the same way as in A. (C) Superpositon of two TNKS1 ARC2-Axin1 interfaces. Two ARC2s are shown in cylindrical cartoon in light pink (interacting with Segment-N) and light gray (interacting with Segment-C). Residues of TNKS in the interface are labeled. The distance between the two parallel tyrosine side chains is approximately 7.3 Å. (D) Superposition of the two Axin1 segments. Color coding is the same as in (A) and (B). Sequences of two segments are shown in vertical text with critical residues colored in magenta and cyan, respectively. (E) Sequence alignment of five ARCs in mouse TNKS1. Individual ankyrin repeats as well as the linker helices between ARCs are boxed and labeled. Critical residues for the interaction are highlighted with black stars. The two gate-forming tyrosines (Tyr-372 and Tyr-405) are highlighted with green stars. Tyr-405 side chain also forms a hydrogen bond with the Axin mainchain. (F) Sequence alignment of the TNKS-binding domains of human and mouse Axin1/Axin2. Segment-N, Segment-C as well as the disordered region in the structure are boxed and labeled. Key residues from Segment-N and Seg- ment-C are highlighted with red and cyan stars, respectively.

Consistent with the results of the mapping experiments, Axin1 chain forms two salt bridges with TNKS Asp-425 and Glu-434, (1-80) interacts with TNKS ARC2 but not ARC3 in the crystal respectively. After Pro-23 and Pro-24 that maintain the Axin1 lattice. Electron densities of two Axin1 segments, Axin1(18-30) mainchain conformation in the groove, the side chain of Axin1 and Axin1(60-79), can be clearly visualized on the two ARC2 sur- Val-25 is buried in a pocket in the bottom of the groove. The faces in the swapped TNKS dimers (Fig. S4). While the remaining main-chain carbonyl groups of Val-25 and Pro-26 interact with parts of Axin1 cannot be seen in the electron density maps, these TNKS Tyr-405 and Asn-401 side chains, respectively. These inter- two Axin1 segments are likely from the same Axin1(1-80) mole- actions lead Gly-27 into a narrow valley formed by two parallel cule, since SEC results indicate that Axin1(1-80) stabilizes TNKS tyrosine residues on the surface of TNKS (Tyr-372 and Tyr-405). (308-655) dimerization in solution (Fig. S3), most likely by inter- Past the valley, the Gly-27 mainchain carbonyl interacts with acting with both copies of the swapped dimer. In addition, the TNKS His-407, and the side chains of Glu-28 and Glu-29 interact distance between Axin1 G30 and A60 Cα atoms in our structure with TNKS Arg-374 and Arg-440, respectively. is 49.4 Å, which can be covered by the 29 missing residues in For the Axin1(60-79) segment (Fig. 2B and Fig. S5), Axin1 between. The sequences of Axin1(18-30) and Axin1(60-79) are residues 60-71 form loose interactions with the TNKS surface. EDAPRPPVPGEEG and TPRRSDLDLGYEPEGSASPT, re- Among them, Arg62 is roughly in the same spatial position as spectively. It is interesting that two Axin1 segments with distinct Arg-22 in the N-terminal segment, and forms two salt bridges with sequences bind to identical TNKS surfaces in the TNKS dimer. TNKS Asp-425 and Glu-434, respectively. Pro-72 is positioned in the bottom of the TNKS groove. Like the Axin1 N-terminal seg- The TNKS-Axin Interfaces: Both Axin segments Bind to the Same TNKS ment, main-chain carbonyl groups of Pro-72 and Glu-73 form hy- Surface Both Axin segments interact with a groove formed by drogen bonds with the side chains of TNKS Tyr-405 and Asn-401, the second helices and the β-hairpins of TNKS ARC2. For the respectively. The side chain of Glu-73 forms a salt bridge with N-terminal segment, Axin1(18-30) adopts an extended conforma- TNKS Arg-337. These interactions place Gly-74 inside the narrow tion and runs roughly perpendicular with the groove (Fig. 2A and valley formed by TNKS Tyr-372 and Tyr-405. Finally, like the Axin1 Fig. S5). Inside the groove, the side chains of Axin1 Arg-22 side N-terminal segment, the other end of Gly-74 is stabilized by a

1502 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1116618109 Morrone et al. Downloaded by guest on September 30, 2021 Fig. 4. Both TNKS-binding segments are important for Axin turnover in vivo. Gly-27 and Gly-74 are required for XAV939-induced Axin1 protein accumulation in SW480 cells. The expression of GFP-Axin1 was under the control of the metallothionein promoter to ensure low transcription.

ing mode, mutations in the Axin1(1-80) fragment, R22A, V25F, G27V had greatly reduced their affinities with TNKS-ARC2, but did not completely disrupt the binding. Mutations in the Axin1 C-terminal segment, such as R62A or P72A, had even less effect (Fig. S6). In contrast, when we made corresponding mutations in Axin1(1-43) and Axin1(44-80), R22A, G27A and G74A all completely disrupted their interaction with TNKS-ARC2 (Fig. 3 C and D). These results demonstrate that the docking of glycine residues with the gates is crucial for the TNKS-Axin interaction. For TNKS mutants, mutations in the “gate” residue Y405A or in the “after-gate-anchor” residue H407A abolished any obser- vable binding; D425A lost much of the affinity, whereas mutations L396A, N401A, E434A, and R440A had only minor effects Fig. 3. Biochemical characterization of TNKS-Axin interaction and the (Fig. 3E). The gate-forming residues, Tyr-372 and Tyr-405 in critical role of a Glycine-selection gate. Isothermal titration calorimetry ARC2, are only conserved in ARC4 and ARC5 in corresponding (ITC) analysis of the interactions between TNKS1 ARC2 and (A) Axin1(1-43); positions (Fig. 2E). This is in agreement with our observation that – μ (B) Axin1(44 80). The Kd value for Axin1(1-43) is 0.92 M, whereas the Kd ARC2, ARC4, and ARC5, but not ARC1 and ARC3, interact with for Axin1(44-80) is in the range of 3–5 μM. (C and D) Mutagenesis of Axin1-N and Axin1-C segments and in vitro GST-pulldown assays define key Axin (Fig. S1). interactions in each Axin1 segment. (E) Mutagenesis of Axin1-binding resi- Mutation of a corresponding residue in Axin2, V26D, also canp dues of TNKS1 and in vitro Axin1(1-80) binding assay using GST-pulldown. known as the mouse Axin2 mutant, leads to phenotypes char- It is clear that the Gly-gate interaction region is most crucial for the Axin- acteristic for decreased Wnt/β-catenin signaling in most tissues TNKS interaction. and a loss of Axin2 turnover (26). In our in vitro binding assay, the Axin1(V25D) mutation in the Axin1(1-43) fragment comple- hydrogen bond between the Gly-74 main-chain carbonyl and the tely disrupted its interaction with the TNKS ARC2 domain side chain of TNKS His-407. The interaction between Axin1 resi- (Fig. 3D). due 75-79 and the TNKS surface are mostly weak van der Waals interactions. The folded Axin1 RGS domain starts with Pro-80 and Both Axin Segments are Required for Axin Turnover in the Cell Are is responsible for interacting with SAMP repeats of APC in the both Axin-TNKS-binding segments in the crystal structure re- β -catenin destruction complex (25). quired for Axin turnover in vivo? We examined this possibility Superposition of the two Axin1 segments on ARC2 demon- by overexpressing WT Axin1 and Axin1 G27Vand G74V mutants strates that ARC2 structures in the dimer are mostly identical in SW480 cells. In our in vitro pulldown assay, both G27V and (Fig. 2C). There are variations on side chain arrangements for G74V mutants disrupted the TNKS-Axin interaction in the cor- residues involved in Axin1 binding. The two Axin1 segments responding TNKS-binding segment (Fig. 3C). Either mutation take almost identical paths on TNKS ARC2 surface; i.e., roughly abolished Axin1 degradation in the cell as well (Fig. 4), demon- perpendicular to the groove formed by the four loops and the top- ends of second helices of the four ankyrin-repeats in ARC2 strating that both Axin1 segments are crucial for regulating Axin1 (Fig. 2 A–D). To demonstrate that both Axin1 segments can bind turnover. to the same ARC surface area in solution, we performed a competition assay, in which Axin1(44-80) competes with GST-tagged Axin Binding Promotes Dimerization of the TNKS Ankyrin-Repeat Domain We observed a 2∶1 molar ratio TNKS-Axin complex in Axin1(1-43) for TNKS binding in a dosage-dependent manner, our crystal lattice, in which TNKS1 forms a swapped dimer in the thereby confirming the overlapping core binding sites (Fig. S6). first ankyrin repeat of ARC3 (Fig. S2). To examine if this dimer- The binding affinities for the ARC2-Axin1(1-43) and ARC2- ization also happens in solution, we analyzed the molecular Axin1(44-80) interactions are 0.92 and 3–5 μM, respectively, as measured by ITC (Fig. 3 A and B). weight of TNKS and TNKS-Axin complexes in solution, using a combination of SEC and dynamic light scattering. Our results Mutagenesis Analysis and Structural Comparison Reveals a Crucial indicate that the ARC4-5 fragment forms a monomer indepen- Glycine-Selection Gate To examine the importance of individual dent of Axin1(1-80) in solution (Fig. S3). In contrast, a fraction residues on the TNKS-Axin interface in TNKS-Axin interaction, of ARC2-3 forms dimer in solution, and the presence of Axin1 we analyzed the TNKS-Axin interaction using either purified (1-80), or a high TNKS concentration, stabilizes TNKS in the

TNKS1(308-485) mutant proteins or Axin1(1-80) mutant pro- dimeric form (Fig. S3). This supports the physiological relevance BIOPHYSICS AND

teins in a GST pulldown assay. Consistent with the bivalent bind- of our crystal structure and Axin-mediated TNKS dimerization. COMPUTATIONAL BIOLOGY

Morrone et al. PNAS ∣ January 31, 2012 ∣ vol. 109 ∣ no. 5 ∣ 1503 Downloaded by guest on September 30, 2021 Discussion PARylation of many substrates, Axin is the only known substrate Axin is a crucial regulator of the Wnt/β-catenin signaling path- (other than TNKS per se) that binds to TNKS bivalently and is way, and also plays a role in myc degradation and p53 regulation. subsequently degraded. It will be interesting to pursue the poten- Inhibition of Axin degradation, which would enhance the degra- tial correlation between bivalent TNKS binding and subsequent dation of β-catenin and thereby attenuate the Wnt/β-catenin ubiquitination and degradation. pathway, is expected to be beneficial to the treatment of cancer and other clinical conditions. Structural information regarding Implications for TNKS Structure and TNKS Substrate Recruitment The how TNKS interacts with Axin is important not only for under- ankyrin-repeat domain of TNKS contains five ARCs, each con- standing Wnt/β-catenin signaling mechanisms and understanding sisting of four ankyrin repeats, which are connected by four lin- TNKS substrate recognition, but also for potential drug devel- kers (approximately 25 residues). The structures of these linkers opment. were previously not known. Our TNKS structure contains three linkers and two ARCs. Each of the three linkers forms a long Structural Basis of TNKS-Axin Interaction and the Implication for Axin helix and interacts with the helices in the neighboring ARCs. Turnover Our work provides a first snapshot of the TNKS-Axin The C-terminal part of these linkers is conserved and has a con- complex at a high resolution. Our biochemical data are fully consensus sequence of LLEAARXG (Fig. 2F). This region accounts sistent with the structure, and together they define the overall for the C-terminal part of the long helix for both linker 1∕2 and binding mode and critical residues of this interaction. Conserva- 2∕3 in our structure and forms a four-helix bundle with the first tion of all key residues of both TNKS-binding segments in the three helices of the following ARC (Fig. 1B). We predict that any Axin family indicates that the interactions we observed for this of the four linkers forms a long helix and uses its N-terminal and mouse TNKS1-Axin1 complex would be preserved for most, if C-terminal half to interact with helices in the neighboring ARCs, not all, Axin and TNKS proteins (Fig. 2F). Our work thus pro- respectively. The linker 3∕4 is six residues longer than the other vides a structural basis for interpreting the V26D mutation found three (Fig. 2E). Since we were only able to observe the electron in Axin2canp mouse, which is associated with Axin2(V26D) stabi- density before Glu-635 of this linker, it is possible that this linker lization and decreased Wnt/β-catenin signaling in most tissues contains a bended long helix or even two helices and thus may (26). In our structure, Axin2 V26 (V25 in Axin1) is a key residue generate a pivot point for overall conformational flexibility or binding to a TNKS hydrophobic pocket, and the V25D mutation conformational change in TNKS. completely disrupted the interaction between TNKS ARC2 Most known TNKS binding proteins share a conserved and Axin1(1–43). Conversely, mouse genetic work supports the sequence motif (TBM). Axin is the only known TNKS binding physiological relevance of our structural model. protein that does not contain an apparent sequence homology Our crystal structure and biochemical analysis demonstrate with TBM. However, the core binding sites may be preserved that two discrete segments of Axin1 can individually interact with amongst Axin and TBM peptides. Previous work showed that the the TNKS. While the first segment, Axin1(18-30), was previously Gly residue in RXXPDG motif is crucial for the TNKS-TBM in- characterized (11), the discovery of the second segment, as well teraction (24). The PDG tripeptide motif may correspond to a as the bivalent binding mode, is unexpected. Importantly, both PEG tripeptide region in Axin1 segment-C, Axin1(72-74), while Axin1 segments are required for TNKS-dependent Axin1 degra- the missing Arg residue may be compensated by Arg-62 further dation, since the missense mutation of either glycine in the Axin1 away in the primary sequence (Fig. 2 B–D). Our data also show segments stabilizes Axin1 (Fig. 4). The identification of two that the TBM peptide and the TNKS-binding domain containing segments will be important for understanding the regulation of the TBM competes with Axin for TNKS binding in a dosage-de- Wnt/β-catenin signaling and Axin1 function. For example, the pendent manner (Fig. S7). Therefore, we predict that the PDG second TNKS-binding segment is immediately N-terminal to the part of the RXXPDG motif interacts with TNKS in a way quite Axin1 RGS domain (residues 80-211), which is responsible for similar to Axin1(72-74), and the conserved arginine in this motif APC binding. It is interesting that APC SAMP repeats on the interacts with TNKS Asp425 and Glu434, which form salt bridges RGS domain surface is near Axin1 residue Pro-80 (25) and thus with arginine residues in both Axin binding segments (Fig. S8). may be in close proximity to TNKS when both bind to Axin1. In summary, our structural and biochemical analysis allowed us It is intriguing that Axin regulates its stability using two seg- to reveal the unexpected bivalent binding mode of the Axin- ments and a bivalent TNKS-binding mode. One simple mechan- TNKS interaction, identify crucial interactions in the Axin-TNKS istic explanation for this is the increase of binding affinity due to interface, and appreciate the requirement of a Gly-selection gate the bivalent binding. This may be important for capturing Axin for Axin turnover. In addition, our structure made one solid step molecules in the cytosol that have a low concentration (10). towards the understanding of the overall structure of TNKS, and Alternatively, but nonexclusively, Axin binding promotes the allowed us to predict how a conserved TNKS binding motif dimerization of the ankyrin-repeat domain of TNKS, which may (TBM), which is shared by many TNKS partners, may dock on be important for Axin turnover. TNKS contains five ARCs and the TNKS surface. All these conclusions will contribute to not Axin can interact with ARC2, ARC4, and ARC5 independently only mechanistic understanding of Wnt signaling and other bio- (Fig. S1). Axin promotes the dimerization of ARC2-3, but logical processes that Axin and TNKS are involved, but also fu- probably not for ARC4-5, although we cannot rule out the possi- ture drug development targeting the Axin-TNKS interaction. bility that ARC4-5 may be also involved in TNKS dimerization in the context of full-length TNKS. Our modeling indicates that the Methods two Axin1 segments in the same subunit may also bind to ARC4 Expression, Purification, and Crystallization of TNKS/Axin Complex GST-tagged and ARC5 in the same TNKS molecule simultaneously. Previous mouse tankyrase-1 (TNKS) with a TEV protease-cleavage site following the work shows that deletion of TNKS ARC1-2 does not affect Axin1 GST tag or His-tagged mouse Axin1 with a TEV protease-cleavage site follow- binding, likely because of ARC4-5, but leads to a loss of Wnt sig- ing the His-tag were purified separately by affinity column, cleaved over- naling activity (11). This may be explained by the loss of Axin- night with TEV, and further purified by ion-exchange and gel-filtration. mediated TNKS dimerization that requires the binding of Axin to The two proteins were then incubated together on ice for 1 h with excess Axin1 and the complex separated from uncomplexed Axin1 by a final gel- ARC2. TNKS is known to form large polymers in the cell (21). filtration step. The fractions containing the complex were concentrated to It has a known oligomerization SAM domain, and the additional 15 mg∕mL. Optimized growth conditions produced single crystals at 22 °C induced dimerization in the ankyrin-repeat domain may impact in 4–5 d by mixing 1 μL of protein solution to 1 μL of reservoir solution con- on the overall networking and functioning of the TNKS polymers. sisting of 0.06 M sodium citrate tribasic dihydrate pH 5.6, 0.12 M ammonium In this regard, it is interesting that while TNKS catalyzes the acetate, 18% MPD, and 20 mM DTT.

1504 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1116618109 Morrone et al. Downloaded by guest on September 30, 2021 X-ray Data Collection and Structure Determination For data collection, crystals in a modified pGEX-4T-1 vector. All TNKS constructs contained an N-terminal of the mouse TNKS1(308-655)/Axin1(1-80) complex were dehydrated by soak- GST-tag with a TEV-cleavage site following the tag and were expressed in ing overnight in the crystallization buffer with 25% MPD, and flash frozen in pGEX-4T-1. Growth, induction, and harvesting of cells was identical to liquid nitrogen. Crystals belong to the space group C2 with cell parameters wild-type. a ¼ 131.78 Å, b ¼ 106.59 Å, c ¼ 73.48Å, β ¼ 105.76°. X-ray data were col- lected at ALS, BL8.2.2, and processed with Mosflm in the CCP4 package Isothermal Titration Calorimetric Studies All titrations were performed at (27). The structure of the complex was determined by molecular replacement 30 °C using a VP-isothermal calorimeter (MicroCal). Protein samples were with program PHASER (28) using a partial mouse Notch-1 ankyrin domain dialyzed for 18 h against 10mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT (accession code 1YMP) (29) as a search model. After several cycles of refine- and degassed. TNKS was placed into the 1.42 mL cell of the calorimeter at ment by REFMAC5 (30) and building by Coot (31), the electron density maps 15 μM and titrated with 300 μM of Axin1. Each titration started with a (2fo-fc and fo-fc) at the 2.0 Å resolution were clear enough to build two 2 μL injection followed by 32 subsequent injections (5 μL) at 4 min intervals. Axin1 regions from residues 18-30 and residues 60-76. Final crystallographic The data were analyzed and thermodynamic parameters were determined refinement was done with REFMAC5 with TLS parameters (27). The refinement statistics are summarized in Table S1. using the Origin software package (MicroCal) with one-site mode.

In Vitro Binding Assays GST-tagged Axin1 or GST-tagged TNKS1 was first in- Cellular Axin1 Stabilization Assay GFP-Axin1 was cloned into a lentiviral con- cubated for 20 min on ice with 40 μL glutathione beads; washed twice in struct under control of the metallotheonein promoter. SW480 cells were in- μ 200 μL PBS, 0.1% Triton X-100, 1 mM DTT (pulldown buffer); and incubated fected with GFP-Axin1 lentivirus, and treated with DMSO or 5 M XAV939 for 20 min with 40 μg of either untagged TNKS1 or Axin1. The mixture was overnight. Cells were lysed using RIPA buffer supplemented with protease then washed four times in 200 μL pulldown buffer, before boiled for SDS- inhibitors and phosphatase inhibitors. Cell lysates were resolved by SDS- PAGE. For competition assays, 5–10 μM GST-tagged Axin1, excessive ARC2 PAGE, and blotted with anti-GFP antibody (Clontech) or anti-Tubulin anti- and variable amounts of the competitor protein were mixed, before glu- body (Sigma). tathione beads were added. ACKNOWLEDGMENTS. We are grateful to the staff at ALS beamlines BL 8.2.1 Mutagenesis and Mutant Protein Purification Point mutations were introduced and 8.2.2 for assistance with synchrotron data collection. This work was sup- into TNKS or Axin by the QuikChange procedure (Stratagene). N-terminal ported by the National Institutes of Health Grant CA90351 to W.X. R.T.M. is GST-tagged Axin1 with a TEV-cleavage site following the tag were expressed an investigator of the HHMI.

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Morrone et al. PNAS ∣ January 31, 2012 ∣ vol. 109 ∣ no. 5 ∣ 1505 Downloaded by guest on September 30, 2021