Native interface of the SAM domain polymer of TEL
© Tran et al; licensee BioMed Central Ltd. 2002
Received: 22 April 2002
Accepted: 22 August 2002
Published: 22 August 2002
TEL is a transcriptional repressor containing a SAM domain that forms a helical polymer. In a number of hematologic malignancies, chromosomal translocations lead to aberrant fusions of TEL-SAM to a variety of other proteins, including many tyrosine kinases. TEL-SAM polymerization results in constitutive activation of the tyrosine kinase domains to which it becomes fused, leading to cell transformation. Thus, inhibitors of TEL-SAM self-association could abrogate transformation in these cells. In previous work, we determined the structure of a mutant TEL-SAM polymer bearing a Val to Glu substitution in center of the subunit interface. It remained unclear how much the mutation affected the architecture of the polymer, however.
Here we determine the structure of the native polymer interface. To accomplish this goal, we introduced mutations that block polymer extension, producing a heterodimer with a wild-type interface. We find that the structure of the wild-type polymer interface is quite similar to the mutant structure determined previously. With the structure of the native interface, it is possible to evaluate the potential for developing therapeutic inhibitors of the interaction. We find that the interacting surfaces of the protein are relatively flat, containing no obvious pockets for the design of small molecule inhibitors.
Our results confirm the architecture of the TEL-SAM polymer proposed previously based on a mutant structure. The fact that the interface contains no obvious potential binding pockets suggests that it may be difficult to find small molecule inhibitors to treat malignancies in this way.
The proto-oncogene TEL (Translocation, Ets, Leukemia) is a transcriptional repressor that contains a C-terminal Ets family DNA binding domain; a central domain that together with co-repressors recruit histone deacetylases [1–3]; and an N-terminal SAM (sterile, alpha, motif) domain [4–6], which we have recently shown forms a polymer . Chromosomal translocations in a variety of leukemias result in fusion of the SAM domain of TEL to tyrosine kinase domains such as ABL, PDGFβ and JAK2 [8–14] or to the transcriptional activators AML1 and ARNT [15–17]. In the tyrosine kinase fusions, SAM domain polymerization leads to constitutive activation of the tyrosine kinase domains, which leads in turn to cell transformation [10, 12, 18, 19]. Thus, compounds that block TEL-SAM polymerization could be effective in treating these leukemias. To assess the feasibility of this approach it would be useful to have a structure of the polymer.
The wild-type TEL-SAM polymer forms large insoluble aggregates, which precludes structure determination. We were, however, able to obtain a structure of a mutant TEL-SAM polymer, V80E . The V80E mutation is in the center of the polymer interface and reduces the affinity of subunit association enough that the protein is relatively soluble above pH 7.0, where the Glu side chain is deprotonated. Sufficient affinity remains, however, that upon crystallization, the polymer reforms in the crystal. The structure of the V80E mutant TEL-SAM revealed a helical head-to-tail polymer in which the interface is made from two different surfaces on the protein. One binding surface, the mid-loop (ML) surface, consists of residues near the middle of the protein and the second surface, the end-helix (EH) surface, is centered around the C-terminal helix. Although the V80E mutant self-associates weakly under the high pH conditions used for crystallization, we were able to show that the native interface is quite strong. In particular, a protein with a mutation in the EH surface (V80E) could bind with high affinity (Kd = 2 nM) to a protein with a mutation in the ML surface (A61D) to form a heterodimer with a native interface. In addition, the wild-type protein forms fibers, visible by electron microscopy, that have a similar width to the V80E mutant polymer we observed in the crystal.
While the wild-type and V80E mutant SAM domains form fibers that are grossly similar, we cannot be certain that the mutation does not significantly alter the interface. Even a small change in subunit orientation could result in substantial alteration of the structure of the polymer, when propagated over many subunits. We have therefore determined the structure of a heterodimer with a native interface.
Results and Discussion
Crystal structure of the TEL-SAM dimer
Resolution Limits (Å)
a = 52.8; b = 60.3; c = 62.3; α = 116.2; β = 98.9; γ = 98.7
Non- protein molecules
As in the V80E structure, a network of salt-bridges surrounds the apolar core. The specific interactions in the salt-bridge network are somewhat variable and differ slightly from the V80E structure. In the structure of the native interface dimer reported here, all three dimers in the asymmetric unit contain salt-bridges from Glu44 to Lys60, Arg73 to Asp79, Asp69 to Arg71, Glu68 to Lys67, and Asp69 to Lys67. Salt-bridges from Glu56 to Arg48 and Glu68 to Arg71 were found in two of the three native interface dimers. All these salt-bridges were also observed in at least two out of the three molecules in the asymmetric unit of the V80E structure, with the exception of Glu68 to Lys67, which was surprisingly completely absent in the V80E structure . Thus, while the interfaces are grossly similar in all the structures, the salt-bridging interactions are malleable, shifting to accommodate slight changes in the geometry of the subunits.
Construction of native polymer model
The wild-type polymer is similar to its mutant counterpart
The wild-type and V80E mutant polymers are also very similar as shown in Figure 2B. Both polymers contain SAM subunits arranged as a left-handed helix with a 65 screw symmetry. The repeat distance of the polymers is essentially identical, differing by only one angstrom (53 Å for mutant and 52 Å for wild-type). These results confirm the architecture of the wild-type polymer proposed previously based on the V80E mutant structure.
SAM interface as possible drug target
In this report we have extracted a TEL-SAM dimer from the wild-type polymer and present its crystal structure. The native interface was found to be similar to the previously solved mutant interface. We also constructed a model of the native polymer and found it to be similar to the previously proposed mutant polymer. Thus, the polymer architecture is sufficiently robust to withstand a mutation from a hydrophobic to a charged residue in the center of the subunit interface. We have recently determined the polymer structure of the SAM domain from another protein involved in transcriptional repression, the polycomb group protein polyhomeotic (Ph) [Kim et al., in press]. The Ph-SAM polymer is quite similar to the TEL-SAM polymer even though the proteins have an unrelated domain structure and show less than 20% sequence identity between the SAM domains. Moreover, different residues are involved in the inter-subunit interactions. We therefore speculate that the polymer architecture is conserved for an important role in transcriptional repression, possibly involving in the generation of a repressed chromatin structure . A rigid, well-defined polymer structure may be important for organizing chromatin in this manner.
From the structure, we found that the interacting surfaces of the SAM domains are devoid of the deep pockets that are ideal for small molecule binding. Although it may still be possible to find small molecule inhibitors, these results are not encouraging. Perhaps a more effective strategy would be to develop protein inhibitors, such as the mutant SAM domains described here, that can bind with high affinity and block polymerization. This strategy is currently being tested.
Materials and Methods
Protein expression, mutagenesis and purification
We used the Quickchange kit (Stratagene) to generate site directed mutants, V80R and A61D from wild-type TEL-SAM cloned into a modified pET3c vector (Novagen) . The expressed protein sequence includes an MEKTR leader sequence, followed by residues 38–124 of the TEL protein and then a C-terminal His tag. Recombinant V80R and A61D mutants were expressed in E. Coli BL21 (DE3) pLysS cells (Novagen), and purified by Ni-NTA (Qiagen) and HiTrap SP (Pharmacia) affinity column chromatography followed by ammonium sulfate precipitation as described by Kim et al .
Crystallization, data collection and refinement
To generate the native dimer, equal amounts of each mutant dimer (both at 15 mg/ml) were mixed together prior to crystallization. Crystals were grown by the hanging drop method in which 2 μl of the 7.5 mg/ml dimer solution in 10 mM bis tris propane (pH 8.5) and 200 mM NaCl, were mixed with 2 μl of reservoir solution containing 5% PEG 4000 and 2.0 M ammonium sulfate. Hexagonal rod-like crystals grew at room temperature over a six-week period. Crystals were cryo-protected with the reservoir solution enriched with 30% (w/v) glycerol before data collection under a liquid nitrogen stream. The data was processed with DENZO/SCALEPACK . The molecular replacement solution was found using AMORE  with a previously solved TEL-SAM mutant (V80E) dimer structure as the search model. The program O  was used for model building and CNS  was used for refinement. Water molecules and sulfates were added to the model near the end of refinement using difference electron density maps. The final model has a crystallographic R-factor (Rcryst) of 23.0% and R-free of 27.2% on 10% of the data (Table 1). The program O was also used for subsequent construction of the native polymer.
Coordinates have been deposited in the Protein Data Bank (Accession Code 1LKY).
The authors would like to thank members of the lab for helpful comments on the manuscript. This work was supported by NIH grant RO1 CA81000-03. J.U.B. is a Leukemia and Lymphoma Society scholar.
- Chakrabarti SR, Nucifora G: The leukemia-associated gene TEL encodes a transcription repressor which associates with SMRT and mSin3A. Biochem Biophys Res Commun 1999, 264: 871–7. 10.1006/bbrc.1999.1605View ArticlePubMedGoogle Scholar
- Guidez F, Petrie K, Ford AM, Lu H, Bennett CA, MacGregor A, Hannemann J, Ito Y, Ghysdael J, Greaves M, Wiedemann LM, Zelent A: Recruitment of the nuclear receptor corepressor N-CoR by the TEL moiety of the childhood leukemia-associated TEL-AML1 oncoprotein [In Process Citation]. Blood 2000, 96: 2557–61.PubMedGoogle Scholar
- Fenrick R, Amann JM, Lutterbach B, Wang L, Westendorf JJ, Downing JR, Hiebert SW: Both TEL and AML-1 contribute repression domains to the t(12;21) fusion protein. Mol Cell Biol 1999, 19: 6566–74.PubMed CentralPubMedGoogle Scholar
- Kyba M, Brock HW: The SAM domain of polyhomeotic, RAE28, and scm mediates specific interactions through conserved residues. Dev Genet 1998, 22: 74–84. 10.1002/(SICI)1520-6408(1998)22:1<74::AID-DVG8>3.0.CO;2-4View ArticlePubMedGoogle Scholar
- Ponting CP: SAM: a novel motif in yeast sterile and Drosophila polyhomeotic proteins. Protein Sci 1995, 4: 1928–30.PubMed CentralView ArticlePubMedGoogle Scholar
- Schultz J, Ponting CP, Hofmann K, Bork P: SAM as a protein interaction domain involved in developmental regulation. Protein Sci 1997, 6: 249–53.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim CA, Phillips ML, Kim W, Gingery M, Tran HH, Robinson MA, Faham S, Bowie JU: Polymerization of the SAM domain of TEL in leukemogenesis and transcriptional repression. EMBO J 2001, 20: 4173–4182. 10.1093/emboj/20.15.4173PubMed CentralView ArticlePubMedGoogle Scholar
- Eguchi M, Eguchi-Ishimae M, Tojo A, Morishita K, Suzuki K, Sato Y, Kudoh S, Tanaka K, Setoyama M, Nagamura F, Asano S, Kamada N: Fusion of ETV6 to neurotrophin-3 receptor TRKC in acute myeloid leukemia with t(12;15)(p13;q25). Blood 1999, 93: 1355–63.PubMedGoogle Scholar
- Golub TR, Barker GF, Lovett M, Gilliland DG: Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell 1994, 77: 307–16.View ArticlePubMedGoogle Scholar
- Golub TR, Goga A, Barker GF, Afar DE, McLaughlin J, Bohlander SK, Rowley JD, Witte ON, Gilliland DG: Oligomerization of the ABL tyrosine kinase by the Ets protein TEL in human leukemia. Mol Cell Biol 1996, 16: 4107–16.PubMed CentralPubMedGoogle Scholar
- Knezevich SR, McFadden DE, Tao W, Lim JF, Sorensen PH: A novel ETV6-NTRK3 gene fusion in congenital fibrosarcoma. Nat Genet 1998, 18: 184–7.View ArticlePubMedGoogle Scholar
- Lacronique V, Boureux A, Valle VD, Poirel H, Quang CT, Mauchauffe M, Berthou C, Lessard M, Berger R, Ghysdael J, Bernard OA: A TEL-JAK2 fusion protein with constitutive kinase activity in human leukemia. Science 1997, 278: 1309–12. 10.1126/science.278.5341.1309View ArticlePubMedGoogle Scholar
- Papadopoulos P, Ridge SA, Boucher CA, Stocking C, Wiedemann LM: The novel activation of ABL by fusion to an ets-related gene, TEL. Cancer Res 1995, 55: 34–8.PubMedGoogle Scholar
- Peeters P, Raynaud SD, Cools J, Wlodarska I, Grosgeorge J, Philip P, Monpoux F, Van Rompaey L, Baens M, Van den Berghe H, Marynen P: Fusion of TEL, the ETS-variant gene 6 (ETV6), to the receptor-associated kinase JAK2 as a result of t(9;12) in a lymphoid and t(9;15;12) in a myeloid leukemia. Blood 1997, 90: 2535–40.PubMedGoogle Scholar
- Golub TR, Barker GF, Bohlander SK, Hiebert SW, Ward DC, Bray-Ward P, Morgan E, Raimondi SC, Rowley JD, Gilliland DG: Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in acute lymphoblastic leukemia. Proc Natl Acad Sci USA 1995, 92: 4917–21.PubMed CentralView ArticlePubMedGoogle Scholar
- Romana SP, Mauchauffe M, Le Coniat M, Chumakov I, Le Paslier D, Berger R, Bernard OA: The t(12;21) of acute lymphoblastic leukemia results in a tel-AML1 gene fusion. Blood 1995, 85: 3662–70.PubMedGoogle Scholar
- Salomon-Nguyen F, Della-Valle V, Mauchauffe M, Busson-Le Coniat M, Ghysdael J, Berger R, Bernard OA: The t(1;12)(q21;p13) translocation of human acute myeloblastic leukemia results in a TEL-ARNT fusion. Proc Natl Acad Sci USA 2000, 97: 6757–62. 10.1073/pnas.120162297PubMed CentralView ArticlePubMedGoogle Scholar
- Carroll M, Tomasson MH, Barker GF, Golub TR, Gilliland DG: The TEL/platelet-derived growth factor beta receptor (PDGF beta R) fusion in chronic myelomonocytic leukemia is a transforming protein that self-associates and activates PDGF beta R kinase-dependent signaling pathways. Proc Natl Acad Sci USA 1996, 93: 14845–50. 10.1073/pnas.93.25.14845PubMed CentralView ArticlePubMedGoogle Scholar
- Jousset C, Carron C, Boureux A, Quang CT, Oury C, Dusanter-Fourt I, Charon M, Levin J, Bernard O, Ghysdael J: A domain of TEL conserved in a subset of ETS proteins defines a specific oligomerization interface essential to the mitogenic properties of the TEL-PDGFR beta oncoprotein. EMBO J 1997, 16: 69–82. 10.1093/emboj/16.1.69PubMed CentralView ArticlePubMedGoogle Scholar
- Navaza J: Implementation of molecular replacement in AMoRe. Acta Crystallographica Section D Biological Crystallography 2001, 57: 1367–1372. 10.1107/S0907444901012422View ArticleGoogle Scholar
- Rubnitz JE, Pui CH, Downing JR: The role of TEL fusion genes in pediatric leukemias. Leukemia 1999, 13: 6–13. 10.1038/sj/leu/2401258View ArticlePubMedGoogle Scholar
- Kuntz ID: Structure-based strategies for drug design and discovery. Science (Washington D C) 1992, 257: 1078–1082.View ArticleGoogle Scholar
- Otwinowski Z, Minor W: Methods Enzymol 1997, 276: 307–326.View ArticleGoogle Scholar
- Perrakis A, Morris R, Lamzin VS: Automated protein model building combined with iterative structure refinement. Nat Struct Biol 1999, 6: 458–463. 10.1038/8263View ArticlePubMedGoogle Scholar
- Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL: Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 1998, 54: 905–21. 10.1107/S0907444998003254View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.