- Research article
- Open Access
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.
- Small Molecule Inhibitor
- Asymmetric Unit
- Tyrosine Kinase Domain
- Polymer Interface
- Native Interface
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.
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.
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.
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