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Structural basis for dimerization and DNA binding of transcription factor FLI1 Caixia Hou, and Oleg V Tsodikov Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01121 • Publication Date (Web): 30 Nov 2015 Downloaded from http://pubs.acs.org on December 6, 2015

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Structural basis for dimerization and DNA binding of transcription factor FLI1 Caixia Hou and Oleg V. Tsodikov*

Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536-0596, USA

*Correspondence: Oleg V. Tsodikov, College of Pharmacy, University of Kentucky, 789 South Limestone St., Lexington, KY 40536-0596, USA Tel: +1-859-218-1687 Fax: +1-859-257-7585 E-mail: [email protected]

Running title: Crystal structures of a FLI1 dimer on and off DNA

ACS Paragon Plus Environment

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Abstract FLI1 (Friend leukemia integration 1) is a metazoan transcription factor that is upregulated in a number of cancers. In addition, rearrangements of the fli1 gene cause sarcomas, leukemias and lymphomas. These rearrangements encode oncogenic transcription factors, in which the DNA binding domain (DBD or ETS domain) of FLI1 on the C-terminal side is fused to a part of an another protein on the N-terminal side. Such abnormal cancer cell-specific fusions retain the DNA binding properties of FLI1 and acquire non-native protein-protein or protein-nucleic acid interactions of the substituted region. As a result, these fusions trigger oncogenic transcriptional reprogramming of the host cell. Interactions of FLI1 fusions with other proteins and with itself play a critical role in the oncogenic regulatory functions, and they are currently under intense scrutiny, both mechanistically and as potential novel anticancer drug targets. We report elusive crystal structures of the FLI1 DBD, alone and in complex with cognate DNA containing a GGAA recognition sequence. Both structures reveal a previously unrecognized dimer of this domain, consistent with its dimerization in solution. The homodimerization interface is helix-swapped and dominated by hydrophobic interactions, including those between two interlocking Phe362. A mutation of Phe362 to an alanine disrupted the propensity of this domain to dimerize without perturbing its structure or the DNA binding function, consistent with the structural observations. We propose that the FLI1 DBD dimerization plays a role in transcriptional activation and repression by FLI1 and its fusions at promoters containing multiple FLI1 binding sites.

Keywords: Ets family; Ewing sarcoma; Friend leukemia; transcriptional regulator; oncogenic protein; cancer target


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Introduction Transcriptional regulators that belong to the ETS (E twenty six; named by avian leukemia E26 virus) family are unique to metazoa, where they are involved in a variety of processes among which are cell proliferation, differentiation and apoptosis.1 Disregulation of these functions, either through changes in expression or through genetic alterations, results in syndromes and malignancies. The members of the ETS family share a wing-turn-helix DNA binding domain (DBD) (the ETS domain) with a highly conserved sequence (Fig. 1), structure and affinity to a GAA(A/T) sequence of DNA.2 Despite this conservation, even minor differences among ETS family members result in distinct DNA sequence preferences outside of this core sequence as well as unique protein-protein interactions. These differences as well as the functions of the additional, divergent, domains of the ETS family proteins define whether an ETS-domain protein is a transcriptional activator, repressor, or both, and what biological function it performs. FLI1, ERG and FEV are ETS family transcription factors that constitute a FLI1 subfamily. FLI1 and ERG were shown to regulate megakaryocyte differentiation and angiogenesis,3-6 whereas FEV (also known as Pet-1) was demonstrated to regulate genes involved in the serotonergic pathway in early brain development.7-9 Disregulation or mutations in this family of transcription factors have been associated with blood- or bone-related disorders and cancers. Expression of FLI1 is upregulated in hematological tumors induced by retroviruses in mice10 and is essential in human erythroleukemia.11 Conversely, downregulation of FLI1 expression was found to be correlated with invasiveness of breast cancer in mice.12 Genetic translocations resulting in fusions of the DBD of ERG with parts of TMPRSS2 protein (TMPRSS2-ERG) are present in ~50% of prostate cancers13, 14

and are associated with invasiveneness, aggressiveness and recurrence of these cancers.14, 15 Most

recently, it was found that a point mutation in the region coding for the FLI DBD containing the


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Arg324Trp mutation caused an inherited platelet-related bleeding disorder that mimicked ParisTrousseau thrombocytopenia.16 In addition, fusions of the DBDs of FLI1, ERG and occasionally other ETS-domain proteins with an N-terminal region of EWS protein (EWS-FLI1 and EWS-ERG) are abnormal oncogenic transcription factors that drive tumorigenesis of Ewing sarcoma, a devastating cancer of bone and soft tissue that is most commonly diagnosed in children and young adults.17-19 Approximately 85% of Ewing sarcomas are driven by EWS-FLI1 and ~10% by EWSERG.20 These abnormal fusions are present only in cancer cells, but not in normal cells; therefore, they are important not only for understanding the mechanism of oncogenic transcriptional reprogramming, but also as potential targets for novel anti-Ewing sarcoma agents. Indeed, YK-4279, a small molecule that was discovered to inhibit interactions of EWS-FLI1 with RNA helicase A is being developed as a selective anti-Ewing sarcoma therapeutic.21, 22 Another Ewing sarcoma drug candidate, a DNA binding natural product mithramycin, is a potent antagonist of EWS-FLI1mediated activation of oncogenes23 that has recently undergone Phase I and II clinical trials as a drug against Ewing sarcoma (clinical trial ID: NCT01610570). Activation of some genes and repression of others by EWS-FLI1 are required for its oncogenic function as a Ewing sarcoma driver. These functions arise, in part, because EWS-FLI1 shares the DNA sequence recognition properties of FLI1 and the RNA polymerase II binding function of EWS.24, 25 Furthermore, other functions that are not present in germline EWS and FLI1 appear to be unmasked in EWS-FLI1 fusion. For example, EWS-FLI1, but not FLI1, is able to form homooligomers in the nucleus.26 The C-terminal region of FLI1 containing its DBD, was shown to be critical for the dimerization of EWS-FLI1 with itself.26 In addition, ERG was demonstrated to homodimerize and even form heterodimers with FLI1.27 Both the N-terminal domain and the DBD of ERG and were shown to be required for its homodimerization. Therefore, FLI1 DBD likely plays


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a key role in EWS-FLI1 homodimerization; possibly because the interface involved in the homodimer formation is exposed only in the fusion, but not in the germline FLI1. Dimerization of other ETS-domain proteins, Elk-1 and Ets-1, was reported to play a role in the DNA binding function and cellular stability of these factors.28-30 The DBDs of FEV and ETV1/4/5 were recently shown to form dimers via an intermolecular disulfide bond involving a Cys residue located close to the C-terminus of the DBDs of these proteins (Cys135 in FEV).31 The dimerization of ETV1/4/5 and, presumably, FEV, is critically dependent on the formation of the disulfide bridge and observed in solution only upon protein oxidation, making this effect a potential redox signaling mechanism.31 The Cys residue critical for FEV and ETV/1/4/5 dimerization is absent in FLI1; therefore, dimerization of FLI1 DBD may occur through a different interface. Thus, the structural logic of dimerization of FLI1 DBD remains unclear. FL11 DBD was the first among ETS domains to be elucidated structurally (in the DNA-bound state), by solution NMR.32 Since this elegant pioneering study by Fesik and co-workers, crystal structures of DBDs of many ETS family members were determined, but a crystal structure of FLI1 remained elusive. In this study, we report crystal structures of the FLI1 DBD alone and in complex with DNA and demonstrate structurally and biochemically the previously unrecognized dimerization of this domain.

Materials and methods Protein purification and preparation of protein-DNA complexes for crystallization A previously reported pET28a plasmid containing a gene encoding a type I EWS-FLI1 fusion protein 33 containing an intact DNA binding domain (DBD) of FLI1 was a kind gift from Dr. Jeffrey Toretsky. This construct was used as template to amplify by PCR two regions encoding the DBD of


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FLI1: a minimal region (residues 276-399) used for crystallization and a longer region (residues 259-399) used for dimerization and DNA binding studies in solution. These inserts were cloned into a modified pET19b vector 34 between NdeI and XhoI restriction sites. The Phe362Ala mutant of this construct was generated by using a QuikChange mutagenesis kit (Agilent Technologies) with two primers, 5'-AAAGATATGCTTACAAATTTGACGCCCACGGCATTGCCCAGG-3' and 5'CCTGGGCAATGCCGTGGGCGTCAAATTTGTAAGCATATCTTT-3' (the mutated codon is underlined), as described in the kit protocol. As a result, the recombinant proteins contain an Nterminal decahistidine tag cleavable by a Prescission protease (GE Healthcare). BL21(DE3)/RIL (Agilent Technologies) cells transformed with these plasmids were grown with shaking in Luria Bertani medium supplemented with ampicillin and chloramphenicol at 100 µg/mL and 25 µg/mL, respectively, at 12 ºC. Upon reaching attenuance of 0.4, the cells were induced with IPTG at 1 mM and incubated for 18 hours at 12 ºC. All subsequent purification steps and dialysis were carried out at 4 ºC. The cells were pelleted and the pellet was resuspended in lysis buffer (40 mM Tris pH 8.0 adjusted at room temperature with HCl, 400 mM NaCl, 10% glycerol and 2 mM βmercaptoethanol). The cells were disrupted by sonication and the lysate was clarified by centrifugation at 40,000×g for 45 min. The clarified lysate was loaded on a 5 mL Ni2+-IMAC HiTrap HP column (GE Healthcare) equilibrated in the lysis buffer and washed first with 50 mL of wash buffer A (40 mM Tris-HCl pH 8.0, 1 M NaCl, 10% glycerol, 50 mM imidazole, 2 mM βmercaptoethanol) to ensure removal of DNA, and then with 50 mL of wash buffer B (40 mM TrisHCl pH 8.0, 400 mM NaCl, 10% glycerol, 50 mM imidazole, 2 mM β-mercaptoethanol). The proteins were eluted in 10 mL of elution buffer (40 mM Tris-HCl pH 8.0, 400 mM NaCl, 10% glycerol, 500 mM imidazole, 2 mM β-mercaptoethanol). The tag was removed by overnight cleavage with PreScission Protease simultaneously with the dialysis into dialysis buffer (20 mM


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Tris-HCl pH 8.0, 400 mM NaCl, 1 mM dithiotheitol). Upon tag cleavage, the proteins were purified on a size-exclusion Sephacryl S-200 column (GE Healthcare) equilibrated in gel filtration buffer (40 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM β-mercaptoethanol). The fractions containing the monomeric FLI1 DBD (see the main text) were pooled and the proteins were concentrated using an Amicon Ultra-15 centrifugal filter device (Millipore) to 12 mg/ml, rapidly frozen in liquid nitrogen and stored at -80 ºC. Dimerization of the FLI1 DBD was observed by size-exclusion chromatography on the same S-200 column run in the gel filtration buffer. To prepare the double-stranded DNA oligomer used for co-crystallization with the FLI1 DBD, we dissolved two complementary single-stranded 10-mers: 5´-ACCGGAAGTG-3´ and 5´CACTTCCGGT-3´ (purchased from IDT) in annealing buffer (10 mM Na cacodylate, pH 6.0, 50 mM NaCl) at the concentrations of 1 mM of each strand. The DNA was then annealed by heating the mixture to 95 C ºC for 5 min followed by slowly cooling it down to 4 ºC. This annealed substrate (1 mM) was added to purified FLI1 DBD (4 mg/mL) at the molar ratio of 1.2:1 of DNA:FLI1. This protein-DNA mixture was then diluted 5-fold with a buffer not containing NaCl (40 mM Tris pH 8.0, 2 mM β-mercaptoethanol), incubated on ice for 15 min and then purified on a size-exclusion Sephacryl S-200 column equilibrated in 25 mM Tris pH 8.0. An elution peak at a new position represented the protein-DNA complex, judged by the absorbances at 260 and 280 nm. The peak fractions were pooled, concentrated to ~16 mg/mL, rapidly frozen in liquid nitrogen and stored at -80 ºC for crystallization and DNA binding experiments.

Crystallization, data collection and crystal structure determination Initial crystallization conditions for FLI1 DBD were found by 1536-condition high-throughput screening carried out at the Hauptman-Woodward Medical Research Institute by the microbatch


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method.35 The crystallization was then optimized by the hanging drop method. At the optimized conditions, the drops contained 1 µL of FLI1 DBD and 1 µL of the reservoir solution (0.1 M sodium acetate pH 5.5, 0.1 M Co2+ sulfate heptahydrate, 24% PEG 4000), and the drops were incubated against 1 mL of the reservoir solution. The crystals of FLI1 DBD were grown in 2-4 days at 21 ºC and were pink due to bound Co2+. The crystals were gradually transferred into the cryoprotectant solution (0.1 M sodium acetate pH 5.5, 0.1 M Co2+ sulfate heptahydrate, 24% w/v PEG 4000, 20% v/v glycerol) and then frozen in liquid nitrogen by quick immersion. Both FLI1 constructs yielded crystal in the same crystal form, but better quality data were obtained with the crystals of the smaller construct (residues 276-399), and these data were used for crystal structure determination. The initial crystallization condition for FLI1 DBD-DNA complex was found by sparse incomplete factorial screening (Hampton Research Crystal Screen). After growth optimization, the crystals were grown at 21 ºC by vapor diffusion in hanging drops containing a mixture of 1 µL of the concentrated protein-DNA complex and 1 µL of reservoir solution (0.1 M Na cacodylate pH 6.5, 0.2 M CaCl2, 14% w/v PEG 8000). The crystals were gradually transferred into the cryoprotectant solution (0.1 M Na cacodylate pH 6.5, 0.2 M CaCl2, 14% w/v PEG 8000, 20% v/v glycerol) and then frozen in liquid nitrogen by quick immersion. X-ray diffraction data for the crystals of FLI DBD and FLI1 DBD-DNA complex were collected at 100 K at the Advanced Photon Source of the Argonne National Laboratory (Argonne, IL), at beamlines 21ID-G and 22ID-D, respectively. The data were processed with HKL200036. The crystal structure of FLI1 DBD were determined by molecular replacement with MOLREP37, by using the crystal structure of the DNA binding domain of ERG38 (PDB accession code 4IRG) as a search model. The crystal structure of FLI1 DBD-DNA complex was determined by molecular replacement with PHASER,39 by using the crystal structure of ERG DBD-DNA complex38 (PDB


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accession code 4IRI). The structures was then iteratively adjusted and refined with COOT40 and REFMAC41, respectively. The data collection and refinement statistics are given in Table 1. Solvent-accessible surface area calculations were performed with Surface Racer software.42 The crystal structure coordinates and structure factor amplitudes for FLI1 DBD and FLI1 DBD-DNA were deposited in the Protein Data Bank under accession codes 5E8G and 5E8I, respectively.

DNA binding assays A 12-mer single-stranded DNA oligomer 5′-GACCGGAAGTGG-3′ and its complementary oligomer 5′-CCACTTCCGGTC-3′ labeled on the 5′-end with 6-carboxyfluorescein (6-FAM) were purchased from IDT. To anneal the two strands, these oligomers were dissolved at 2 mM in 10 mM Tris-HCl, pH 8.0, and the mixture was heated to 90 ºC for 1 min and then slowly cooled down to 4 °C. FLI1 DBD-DNA binding was monitored by measuring fluorescence anisotropy upon titrating into the DNA three different FLI1 DBD (residues 259-399) samples: 1) the wild-type protein that was eluted from the S-200 column as a monomeric species, 2) the dimeric species, and 3) the monomeric Phe362Ala mutant. All titrations were performed in triplicate. The assays were carried out in the binding buffer containing 20 mM Tris-HCl, pH 8.0, 50 mM NaCl and 5 mM MgCl2 at 22 °C. Each 100 µL binding mixture contained 20 nM DNA and FLI1 DBD at concentrations specified in Results. The fluorescence was measured after the mixture was incubated for 10 min at 22 °C. Fluorescence anisotropy measurements were carried out in triplicate on a SpectraMax M5 microplate reader (Molecular Devices) with excitation and emission wavelengths set at 494 nm and 525 nm, respectively. Nonlinear regression fit of the 1:1 isotherm to the titration data was performed by using SigmaPlot (SysStat) as described previously,43 to obtain the best-fit values of the equilibrium binding constants (Kd).


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Results Crystal structure of the FLI1 DBD 1,536-condition high-throughput screening yielded crystallization conditions for FLI1 DBD, with Co2+ as a critical crystallization component. The crystal structure of FLI1 DBD (Fig. 2A) was determined by molecular replacement. The winged helix-turn-helix structure of the FLI DBD very closely resembles that of the DBD of ERG and other ETS family transcription factors.1 This structural conservation is expected from the nearly identical sequences of FLI1 and ERG and very high sequence conservation of the DBDs among ETS family members in general. Ala295 and Ala297 are the only two residues that differ between the FLI1 and ERG DBDs and are both Ser in ERG (Fig. 1). These two residues are solvent exposed and are located on the same face of a 310 helix. The structure contains Co2+ ions unambiguously identified by very strong electron density that mediating protein-protein crystal packing interactions (Fig. 2B). The Co2+ is coordinated to the two carboxyl oxygen atoms of Asp361, an imidazole group nitrogen of His363 and three water molecules. The coordination geometry appears to be distorted octahedral, as the bidentate coordination by Asp361 does not appear to be consistent with ideal octahedral coordination. As a result, one of the oxygen atoms of Asp361 is too far ~2.5 Å from the Co2+ and it does not lie on the line drawn between the Co2+ and the opposing coordinating N atom of His363. Asp313 of the crystal packing neighbor forms a hydrogen bond with one of the Co2+-coordinating water molecules and interacts electrostatically with His363, explaining the critical role of Co2+ in crystallization of FLI1 DBD.


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The crystals contained two nearly identical dimers of FLI1 DBD per asymmetric unit. The monomers in each dimer are related by a dyad symmetry axis (Fig. 3A). The dimeric interface is characterized by swapping of the C-terminal helices (helix α4 in Fig. 1) of the respective DBDs. Some of the residues in the dimer interface (Trp302, Phe360) are highly conserved among many ETS family members, whereas other residues are conserved only in the FLI1 sub-family, FLI1, ERG and FEV (Fig. 1). The interface is dominated by hydrophobic contacts, including those between the residues of these two helices (Fig. 3B). A few interactions between polar residues occur on the periphery of the interface and include hydrogen bonds of side chains of Gln367 and Gln370 with the backbone amides of the other monomer. The total of 1,745 Å2 of solvent accessible surface is buried in this interface by both monomers, most of which, 1,237 Å2 is non-polar and the rest, 508 Å2, is polar, reflecting the nature of this interaction. In the middle of the dimer interface, two buried Phe362, one from each swapped helix, are stacked against each other and contacted by other hydrophobic residues of the other monomer (Fig. 3B). In order to test whether the dimer observed in the crystal structure is formed in solution, we generated the Phe362Ala mutant of FLI1 DBD and investigated the oligomeric states of the wildtype FLI1 DBD and this point mutant by size-exclusion chromatography. The wild-type FLI1 DBD eluted as a mixture of monomers and dimers (Fig. 4A). The separation of the chromatographic peaks corresponding to the monomer and the dimer suggests that the monomer-dimer equilibration is much slower than the elution time (~1 hour). To test this more directly, we incubated the protein from the chromatographic peak corresponding to the dimer for 48 hours at 4 °C and then performed a similar size-exclusion chromatographic experiment. The chromatogram (Fig. 4B) showed that the majority of the protein remained dimeric, consistent with a slow monomer-dimer equilibration process. In contrast to the wild-type, the Phe362Ala mutant eluted exclusively as monomers (Fig.


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4C). These observations indicate that, indeed, FLI1 DBD dimerizes in solution and that it does so through the interface consistent with that observed in our crystal structure. Crystal structure of FLI1 DBD-DNA complex We crystallized and determined the structure of the complex of FLI1 DBD with a doublestranded 10-base pair oligomer ACCGGAAGTG harboring a central GGAA ETS family binding motif. The crystals that diffracted X-rays were obtained only when FLI1-DBD was purified in complex with DNA (Materials and Methods). The crystals contained four protein-DNA complexes per asymmetric unit (Fig. 5A), where the four monomers of FLI1 DBD are assembled into the same dimers as those observed in the crystals of the protein alone (Fig. 3A). The fact that the crystal packing of protein alone and those of protein-DNA complex are different in the two crystal forms, together with the size-exclusion chromatography data described in the previous section, one can conclude that this dimeric organization of FLI1 DBD on and off DNA is not a crystallization artifact. The two DNA oligomers bound to the dimer of FLI1 DBD are oriented unidirectionally, with the two helical axes forming a ~40° angle. The binding of FLI1 DBD to DNA is very similar to that of ERG DBD38 and other ETS family proteins,1 as expected based on nearly absolute conservation of all DNA interacting residues in this family (Fig. 1). The FLI1 DBD-DNA interactions are shown schematically in Fig. 5C. Analogous interactions have been previously described for other ETS family members; for example, in the seminal structural studies of Elk-1-DNA and SAP-1-DNA complexes by Marmorstein and coworkers;44, 45 therefore, we will summarize only briefly the main features of the FLI1-DNA interface and focus on the unique aspects of FLI1. Similarly to all known ETS domain-DNA complexes, all interactions of FLI1 DBD with DNA bases are made by residues located in helix α3 bound in the major groove (Figs. 1, 5B and 5C). All but one of these interactions are made with the


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core sequence GGAA (Fig. 5C). The DNA is nearly straight, with only a ~3° global bend, according to 3DNA software.46 The minor groove at the GG of the GGAA motif is ~14 Å, consistent with the widening of this groove at repeating G·C base pairs. The residues that change conformation the most between the DNA-bound and unbound states are Arg337, Tyr341, Tyr343, Lys345 and His352 (Fig. 5B). These residues were also observed in different conformations in DNA-bound and unbound ERG DBD. Notably, the conformations of Tyr341 in the unbound form of FLI1 DBD is analogous to that of Tyr66 of Elk-1 in Elk-1-DNA complex.44 In contrast, the conformations of Lys345 in the unbound and DNA-bound FLI1 DBD structures are relatively similar and both consistent with a salt bridge with a phosphate backbone, whereas in the Elk-1-DNA complex, the analogous residue, Lys70 does not interact with DNA and instead forms a salt bridge with Asp67 (Asp344 in FLI1), which adopts a suitable rotamer conformation.44 Asp344 is in the same conformation in the two FLI1 DBD structures. The conformation of Tyr66 was proposed to be coupled to that of Asp67 through Lys70 in Elk-1.44 In contrast, in FLI1 this coupling appears to be absent, as Tyr341 appears to change conformations whereas the Lys345 and Asp344 do not. These observations, however, do not rule out control of Lys345 by Asp344 in FLI1 DBD. Dimerization of FLI1 DBD and its binding to a short DNA oligomer do not affect each other To probe whether the dimerization of FLI1 DBD interferes with or promotes its binding to DNA or, conversely, whether DNA binding affects the dimerization of FLI1 DBD, we carried out equilibrium DNA binding assays with the wild-type FLI1 DBD purified as a monomer, dimer (Fig. 4), and with the monomeric Phe362Ala mutant. These proteins were titrated into 6carboxyfluorescein (6FAM) end-labeled 12-base pair base-pair double-stranded DNA oligomer containing the same high-affinity FLI1 recognition sequence CCGGAA as that used for crystallization. The anisotropy of 6FAM fluorescence was monitored as a function of protein


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concentration (Fig. 6). These data demonstrate that wild-type FLI1 DBD monomers and the Phe89Ala mutant bind DNA with the same affinities within the experimental uncertainty, indicating that the ability of FLI1 DBD to form dimers does not affect DNA binding. A larger fluorescence anisotropy increase was observed for the wild-type dimers, consistent with the formation of larger FLI1-DNA complexes (e.g. as in Fig. 5A). The affinity of dimeric FLI1 DBD for DNA is only 2.5fold weaker than that of the monomeric species. As the titration data demonstrate, dimers and monomers of FLI1 DBD bind DNA in the same concentration range. Consistent with the previous observations, these data also demonstrate that the monomer-dimer conversion is very slow (since it does not occur during the titration) and that it is not affected by DNA binding. We can, therefore, conclude that FLI1 DBD dimerization does not interfere with the binding of this protein to short oligomeric DNA and, conversely, binding to oligomeric DNA does not interfere with the dimerization of FLI1 DBD.

Discussion FLI1 DBD has been a challenging crystallographic target. Co2+ was crucial to obtaining its crystals, and the crystal structure indeed revealed Co2+-mediated crystal packing interactions. Not surprisingly, FLI1 DBD does not form crystals at crystallization conditions for ERG DBD (not containing Co2+). The side chain hydroxyl groups of Ser308 and Ser310 of ERG DBD in its crystals (PDB accession code: 4IRG)38 form direct or water-mediated intermolecular hydrogen bonds with the crystal packing neighbors, whereas these crystal packing interactions would be impossible for FLI1 DBD, as these two residues in FLI1 are alanines (Ala295 and Ala297; Fig. 1). In FLI1 DBD, a His and a nearby Asp form a suitable intramolecular chelation interface for a Co2+ ion, in turn


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creating a new surface for crystal packing. This transition metal ion coordination enabling new crystal packing interactions is reminiscent of the elegant metal-mediated synthetic symmetrization methodology introduced by Yeates and coworkers.47 In this method, pairs of His or Cys residues adjacent to each other in space are introduced at solvent exposed ends of helices to chelate divalent metal ions, thereby creating potential intra- and inter-molecular chelation sites for divalent metal ions, in order to expand a range of possible crystal packing arrangements. The Co2+-chelating His in FLI1 is the second residue in a helix and the Asp is immediately preceding the helix. We propose that introducing a similar solvent-exposed Asp-X-His motif at ends of helices for Co2+-mediated protein crystallization can be a useful addition to the Yeates’ methodology. The region immediately N-terminal to the DBD in FLI1 and ERG is highly similar for the two proteins and has been observed to be inhibitory to DNA binding of ERG, causing a ~4-fold increase in Kd for DNA binding of a construct of ERG DBD containing this additional region.38 Examination of our FLI1 DBD and FLI1 DBD-DNA crystal structures together with the recently reported crystal structures of ERG DBD with and without this N-terminal auto-inhibitory region and that of ERGDBD-DNA complex containing the auto-inhibited ERG38 has prompted us to reevaluate the role of the N-terminal auto-inhibitory region. Our FLI1 DBD construct used for crystallization with and without DNA lacks the N-terminal auto-inhibitory region. Our crystal structure of FLI1 DBD-DNA closely resembles the structure of ERG DBD-DNA complex, where the N-terminal auto-inhibitory region is present, but is disordered. Likewise, the structure of unbound FLI1 DBD is similar to the structures of unbound ERG DBD,38 either with or without the N-terminal auto-inhibitory region (Fig. 5B and Supplementary Fig. S1). In particular, FLI1-DNA and ERG-DNA interactions and the conformations of the DNA-interacting residues in the DNA-bound FLI1 and ERG are very similar to each other, as are the conformations of these residues in unbound FLI1 DBD and the respective


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so-called uninhibited ERG DBD (Fig. 5B and Supplementary Fig. S1). For example, residue Tyr341 is in the same conformation in FLI1 as the analogous Tyr354 of ERG with or without the Nterminal auto-inhibitory region,38 when crystallized in the absence of DNA (Fig. 5B and Supplementary Fig. S1). Therefore, the conformational differences of several DNA interacting residues between the DNA-bound and unbound states of both FLI1 and ERG (Fig. 5B and Supplementary Fig. S1) appear to be a result of DNA binding and not of the presence of the Nterminal auto-inhibitory region. For the ERG DBD construct containing the N-terminal autoinhibitory region in the DNA-unbound state, the authors argued that the conformation of Tyr354 was additionally stabilized by a hydrogen bond with Ser283 in this auto-inhibitory region. 38 This argument served as a structural basis for the allosteric effect of the auto-inhibition, consistent with the NMR studies of different ERG constructs.38 When we superimposed the structure of unbound auto-inhibited ERG DBD onto the structure of this ERG construct in complex with DNA, we found that the N-terminal Ser283 of the auto-inhibitory region sterically clashes with the DNA backbone (Supplementary Fig. S1). This additional, direct, auto-inhibition effect of the N-terminal autoinhibitory region was not described previously. Indeed, mutating Ser283 to an Ala significantly (causing a ~2-fold reduction in Kd), but not completely, relieved the effect of auto-inhibition,38 further supporting the contribution of steric interference, in addition to the allosteric effects of this N-terminal region. Based on the structural and sequence similarity of ERG and FLI1 DBDs, we expect similar auto-inhibitory effects to be observed for FLI1. Alterations of fli1 and erg genes involving the DBD coding regions are strongly linked to or causative of human disease. Point mutations in FLI1 DBD are associated with blood disorders, whereas translocations of fli1 and erg genes fusing the DBDs of FLI1 and ERG to other proteins result in abnormal transcription factors driving oncogenesis in Ewing sarcoma and prostate cancers.


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The Arg324Trp mutant of FLI1 was recently shown to have a defect in transcriptional regulation and to cause a bleeding phenotype in mice similar to that observed in the afflicted patients bearing this mutation.16 Arg324 is the last residue of helix α2 preceding a loop containing DNA interacting residues (Fig. 1). Even though Arg324 itself does not interact with DNA, it forms a partially buried salt bridge with Asp289, which would be disrupted by the Arg324Trp substitution. This electrostatic interaction likely provides a structural stability to the C-terminal end of helix α2 and aids in positioning the loop between α2 and α3 for DNA binding. The degenerate 4-base pair DNA recognition sequence GGA(A/T) of the ETS family proteins is short enough to be present at many promoters. In some FLI1-regulated promoters such as NR0B1, this sequence is repeated consecutively >20 times, and these repeats are essential for the oncogenic regulation by EWS-FLI1.48 Frequent occurrence of this sequence at many promoters explains pleiotropic transcriptional programs of FLI1 and other ETS family members. Furthermore, the transcriptional effects of FLI1 are different at different promoters49, 50 and these effects are, in turn, different from those of the EWS-FLI1 fusion. The diverse regulatory functions among different promoters must arise due to different interactions of FLI1 with the transcriptional machinery. Formation of heterodimers of between the DBDs of different ETS family members, including FLI1/EWS-FLI1 and other transcription factors has been amply demonstrated.51, 52 In addition to the heterodimer formation, FLI1 DBD has also been implicated in homodimerization, but this self-association has remained unclear at the structural level.26 This study demonstrated FLI1 DBD dimerization in solution and revealed its structural logic. We surveyed homodimers of the DBDs of other structurally elucidated ETS family transcription factors and, to our surprise, found that, with the sole exception of ERG, their dimeric organizations differed from that of FLI1 (Fig. 7). In the recently reported crystal structures of ERG DBD unbound and bound to DNA,38 this DBD


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forms the same dimer as FLI1. This interface has been overlooked, possibly because the two ERG DBD monomers of a dimer are related by a crystal symmetry operation in the structures of ERG DBD alone and in complex with DNA. In all proteins shown in Fig. 7, except Elk-1, the same face of the different DBDs as in FLI1 is involved in dimerization. The most similar to FLI1 organization is displayed by ETV1, whose domains are related to those in FLI1 by a ~25° rotation and a minor translational shift of one DBD monomer relative to another. Intriguingly, in FEV, a close genetic relative of FLI1, this rotation is much larger, ~90°. Elk-1 represents an extreme case, where the dimer interface is entirely different. The structure of Ets-1 is somewhat divergent from the rest and it contains an additional N-terminal region, which is involved in dimerization. Indeed, not all residues of FLI1 DBD involved in dimerization (Fig. 1) are conserved and even less or no conservation is observed outside of the DBD, which apparently results in a different dimer organization. These divergent dimer arrangements result in different mutual orientation of the DNA binding helices in the monomers of the same dimer (Fig. 7). We propose that simultaneous binding of one ETS family dimer to two DNA sites must play a role in the diverse array of regulatory functions among ETS family members. The affinity of an ETS family dimer to DNA may be modulated by properly phased DNA binding sites, as it is observed for the paradigm of gene regulation, lac repressor.53, 54 A particular disposition of the monomers in an ETS family dimer would then uniquely regulate a specific pattern on repeating cognate sites at different promoters. Future studies will need to clarify these functional implications of dimerization among ETS family members.

Acknowledgements


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We thank Dr. Jeff Toretsky for the EWS-FLI1 encoding plasmid and the staff of sectors LS-CAT and SER-CAT of the Advanced Synchrotron Source at the Argonne National Laboratory (Argonne, IL) for assistance with X-ray diffraction data collection.

Supporting Information The Supporting Information is available on the ACS Publications website. Figure S1 (PDF)

Funding information This project was supported in part by a pilot award from the Markey Cancer Center, University of Kentucky. Support of sector SER-CAT at the Advanced Photon Source of the Argonne National Laboratory was provided, in part, by the Center of Structural Biology at the University of Kentucky.

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Table 1. X-ray diffraction data collection and refinement statistics for the crystal structures of the FLI1 DBD and the FLI1 DBD-DNA complex. FLI1 DBD FLI1 DBD -DNA PDB accession code 5E8G 5E8I Data collection Space group Number of monomers/asymmetric unit Unit cell dimensions a, b, c (Å) α, β, γ (°) Resolution (Å) I/σ Completeness (%) Redundancy Rmerge Number of unique reflections

R3 4

P3121 4 complexes

140.6, 140.6, 85.1 90, 90, 120 50.0-2.7 (2.75-2.70)a 27 (2.3) 100.0 (100.0) 4.6 (4.7) 0.09 (0.67) 17,449

86.6, 86.6, 230.3 90, 90, 120 50.00-3.45 (3.51-3.45) 19 (1.9) 99.9 (100.0) 8.2 (7.9) 0.1 (0.87) 13,800

Structure refinement statistics Resolution (Å) R (%) Rfree (%)

40.0-2.7 19.9 24.8

35.00-3.45 22.5 27.1

R.m.s.d.b from ideal bond lengths (Å) bond angles (°)

0.009 1.23

0.006 0.88

Ramachadran plot statisticsc % of residues in favored regions 95.9 98.6 allowed regions 4.1 1.4 outlier regions 0.0 (0 residues) 0.0 (0 residues) a Numbers in parentheses indicate the values in the highest-resolution shell. b R.m.s.d. stands for root-mean-square deviation. c Indicates Rampage statistics.55


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Biochemistry

Figure legends

Figure 1. A multiple sequence alignment of the structurally characterized DBDs of the ETS family transcription factors. The DNA interacting residues are denoted by orange circles and the residues in the dimerization interface of FLI1 DBD are denoted by grey circles.

Figure 2. The structure of a monomer of FLI1 DBD and a coordinated Co2+ ion. A. A monomer of FLI1 DBD. B. The crystal packing interface mediated by Co2+ coordination. The Co2+ ion (yellow sphere) is coordinated to Asp361 and His363 of one monomer (in pale yellow) and three water molecules (red spheres). Also shown is Asp313 of a crystal packing neighbor (in grey) in this interface.

Figure 3. The dimer of FLI1 DBD. A. The overall view of the dimer, where the residues in the monomer-monomer interface are shown as sticks. B. The zoomed-in view of the interface, in which the residues of one of the monomers (shown in pale yellow) labeled. The residues of the other monomer (in light blue) are related by a dyad symmetry, where the symmetry axis is perpendicular to the view.

Figure 4. The size-exclusion chromatogram of FLI1 DBD. A. The chromatogram of wild-type FLI1 DBD. B. The chromatogram of wild-type FLI1 DBD, initially purified as a dimer, followed by


Biochemistry

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incubation for 48 hours at 4 °C. The protein was reloaded onto the size-exclusion column without concentrating. C. The chromatogram of the Phe362Ala mutant of FLI1 DBD.

Figure 5. The crystal structure of the FLI1 DBD dimer bound to DNA. A. The overall view of the complex. The dimer (in pale yellow and light blue) is oriented as in Fig. 3. DNA is shown in orange and bound Ca2+ ions are shown as purple spheres. B. The view of the protein-DNA interface. A superimposed monomer from the structure of FLI1 DBD unbound to DNA is shown in grey. The DNA interacting residues whose conformations differ in the two structures are shown as sticks and labeled. C. A schematic of the interactions between FLI1 DBD and the DNA oligomer used in this study. Bases, sugar moieties and phosphate groups are shown as ovals, rectangles and black connector lines, respectively.

Figure 6. Titration of dimers of wild-type FLI1 DBD (filled circles), monomers of wild-type FLI1 DBD (open circles) and monomeric Phe362Ala FLI1 DBD (open squares) into 6FAM-labeled DNA oligomer bearing one FLI1 binding site (at 20 nM). The solid, long-dashed and short-dashed curves correspond to the best fit to the 1:1 isotherm to these data with Kd = 41 ± 5 nM (wild-type dimer), 16 ± 3 nM (wild-type monomer) and 17 ± 1 nM (Phe362Ala mutant).

Figure 7. Diverse dimeric arrangements of FLI1 DBD and the DBDs of other ETS family transcription factors. One of the monomers (in light blue) is oriented similarly in all structures. The structure of Ets-1 is shown on a smaller scale than the rest due to its larger size. The PDB accession


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

numbers for the previously reported structures are: FEV- 2YPR31, Ets-1- 1K7956, ETV1- 4BNC31, Elk-1- 1DUX44.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 1

α1

η1

β1

FLI1 FLI1 ERG FEV Ets-1 ETV1 ETV4 ETV5 Elk-1

β2

α2

α3

β3

β4

α4

TT 276

290

300

310

320

330

340

350

360

370

PGSGQIQLWQFLLELLSDSANASCITWEGT.NGEFKMTDPDEVARRWGERKSKPNMNYDKLSRALRYYYDKNIMTKVHGKRYAYKFDFHGIAQALQP.... PGSGQIQLWQFLLELLSDSSNSSCITWEGT.NGEFKMTDPDEVARRWGERKSKPNMNYDKLSRALRYYYDKNIMTKVHGKRYAYKFDFHGIAQALQPHPP E KGSGQIQLWQFLLELLADRANAGCIAWEGG.HGEFKLTDPDEVARRWGERKSKPNMNYDKLSRALRYYYDKNIMSKVHGKRYAYRFDFQGLAQACQPPPAH TGSGPIQLWQFLLELLTDKSCQSFISWTGD.GWEFKLSDPDEVARRWGKRKNKPKMNYEKLSRGLRYYYDKNIIHKTAGKRYVYRF.VCDLQSLLGYTPEE QRRGSLQLWQFLVALLDDPSNSHFIAWTGR.GMEFKLIEPEEVARRWGIQKNRPAMNYDKLSRSLRYYYEKGIMQKVAGERYVYKFVCDPEALFSMAFPDN ..RGALQLWQFLVALLDDPTNAHFIAWTGR.GMEFKLIEPEEVARLWGIQKNRPAMNYDKLSRSLRYYYEKGIMQKVAGERYVYKFVCEPEALFSLAFPDN .....LQLWQFLVTLLDDPANAHFIAWTGR.GMEFKLIEPEEVARRWGIQKNRPAMNYDKLSRSLRYYYEKGIMQKVAGERYVYKFVCDPDALFSMAFPDN .MDPSVTLWQFLLQLLREQGNGHIISWTSRDGGEFKLVDAEEVARLWGLRKNKTNMNYDKLSRALRYYYDKNIIRKVSGQKFVYKFVSYPEVAGC......


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 2

A

B

D313

D361 H363


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 3

A C-ter

C-ter Q367 Q370 D361 A366 L369 K359 F360

F362

L288 N306 W302

E289


B

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Figure 4

Absorbance at 280 nm

A 0.30 wild-type monomer

0.25 0.20

dimer

0.15 0.10 0.05 0.00 100 Time, min


B 0.030 wild-type 0.025

dimer

0.020 0.015 0.010 0.050 0.000 100 Time, min

C 0.30 Absorbance at 280 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

F362A

monomer

0.25 0.20 0.15 0.10 0.05 0.00 100 Time, min


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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Figure 6

0.30 Fluorescence anisotropy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

wt dimer 0.25 F362A 0.20 wt monomer 0.15 0.10 0.05 0

50

100

150

200

[FLI1 DBD], nM


250

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 7

FLI1 A DN

gh din bin

elix

FEV

Ets-1

Elk-1

ETV1


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Table of Contents graphic


Biochemistry

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Figure 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

FLI1 FLI1 ERG FEV Ets-1 ETV1 ETV4 ETV5 Elk-1

α1 276

η1 290

β1 300

TT

β2 310

α2 320

α3 330

β3 340

350

β4

α4 360

370

PGSGQIQLWQFLLELLSDSANASCITWEGT.NGEFKMTDPDEVARRWGERKSKPNMNYDKLSRALRYYYDKNIMTKVHGKRYAYKFDFHGIAQALQP.... P G S G Q I Q L W Q F L L E L L S D S S N S S C I T W E G T . N G E F K M T D P D E V A R R W G E R K S K P N M N Y D K L S R A L R Y Y Y D K N I M T K V H G K R Y A Y K F D F H G I A Q A L Q P H P PE KGSGQIQLWQFLLELLADRANAGCIAWEGG.HGEFKLTDPDEVARRWGERKSKPNMNYDKLSRALRYYYDKNIMSKVHGKRYAYRFDFQGLAQACQPPPAH TGSGPIQLWQFLLELLTDKSCQSFISWTGD.GWEFKLSDPDEVARRWGKRKNKPKMNYEKLSRGLRYYYDKNIIHKTAGKRYVYRF.VCDLQSLLGYTPEE QRRGSLQLWQFLVALLDDPSNSHFIAWTGR.GMEFKLIEPEEVARRWGIQKNRPAMNYDKLSRSLRYYYEKGIMQKVAGERYVYKFVCDPEALFSMAFPDN ..RGALQLWQFLVALLDDPTNAHFIAWTGR.GMEFKLIEPEEVARLWGIQKNRPAMNYDKLSRSLRYYYEKGIMQKVAGERYVYKFVCEPEALFSLAFPDN .....LQLWQFLVTLLDDPANAHFIAWTGR.GMEFKLIEPEEVARRWGIQKNRPAMNYDKLSRSLRYYYEKGIMQKVAGERYVYKFVCDPDALFSMAFPDN .MDPSVTLWQFLLQLLREQGNGHIISWTSRDGGEFKLVDAEEVARLWGLRKNKTNMNYDKLSRALRYYYDKNIIRKVSGQKFVYKFVSYPEVAGC......


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 2

A

B

D313

D361


H363

Biochemistry

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Figure 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

C-ter

C-ter Q367 Q370 D361 K359 F360 L288

A366 L369 F362

N306 W302

E289


B

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Figure 4


A 0.30 wild-type monomer

0.25 0.20

dimer

0.15 0.10 0.05 0.00 100 Time, min


B 0.030 wild-type 0.025

dimer

0.020 0.015 0.010 0.050 0.000 100 Time, min

C 0.30 Absorbance at 280 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

F362A

monomer

0.25 0.20 0.15 0.10 0.05 0.00 100 Time, min


Biochemistry

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Figure 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

B H352

R337

Y343

Y341

K345

N-ter

C

K327 M330 K325 A338Q282 Y342 K345 D333 N329 K334 W321 L283

hydrophobic H-bond ionic

T

G

G T

C G

C

T

T

C

A

C

A

C

C A

G

G

A

A

G

T

G

1

2

3

4

5

6

7

8

9

10

Y332 Y343 R340 Y356 R355 K350 H352 R337

Y341


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Biochemistry

Figure 6

0.30 Fluorescence anisotropy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

wt dimer

0.25

F362A

0.20

wt monomer

0.15 0.10 0.05

0

50

100

150

200

[FLI1 DBD], nM


250

Biochemistry

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Figure 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

FLI1 Ab

DN

elix

gh

in ind

FEV

Ets-1

Elk-1

ETV1


Structural Basis for Dimerization and DNA Binding ... - ACS Publications

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