Mutagenesis of the Runt Domain Defines Two Energetic Hot Spots for Heterodimerization with the Core Binding Factor β Subunit
2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês
10.1074/jbc.m303972200
ISSN1083-351X
AutoresLina Zhang, Zhe Li, Jiangli Yan, Padmanava Pradhan, Takeshi Corpora, Matthew D. Cheney, Jerónimo Bravo, Alan J. Warren, John H. Bushweller, Nancy A. Speck,
Tópico(s)RNA Research and Splicing
ResumoCore-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in several developmental pathways and in human disease. Mutations in CBF genes are found in leukemias, bone disorders, and gastric cancers. CBFs consist of a DNA-binding CBFα subunit (Runx1, Runx2, or Runx3) and a non-DNA-binding CBFβ subunit. CBFα binds DNA in a sequence-specific manner, whereas CBFβ enhances DNA binding by CBFα. Both DNA binding and heterodimerization with CBFβ are mediated by a single domain in the CBFα subunits known as the “Runt domain.” We analyzed the energetic contribution of amino acids in the Runx1 Runt domain to heterodimerization with CBFβ. We identified two energetic “hot spots” that were also found in a similar analysis of CBFβ (Tang, Y.-Y., Shi, J., Zhang, L., Davis, A., Bravo, J., Warren, A. J., Speck, N. A., and Bushweller, J. H. (2000) J. Biol. Chem. 275, 39579–39588). The importance of the hot spot residues for Runx1 function was demonstrated in in vivo transient transfection assays. These data refine the structural analyses and further our understanding of the Runx1-CBFβ interface. Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in several developmental pathways and in human disease. Mutations in CBF genes are found in leukemias, bone disorders, and gastric cancers. CBFs consist of a DNA-binding CBFα subunit (Runx1, Runx2, or Runx3) and a non-DNA-binding CBFβ subunit. CBFα binds DNA in a sequence-specific manner, whereas CBFβ enhances DNA binding by CBFα. Both DNA binding and heterodimerization with CBFβ are mediated by a single domain in the CBFα subunits known as the “Runt domain.” We analyzed the energetic contribution of amino acids in the Runx1 Runt domain to heterodimerization with CBFβ. We identified two energetic “hot spots” that were also found in a similar analysis of CBFβ (Tang, Y.-Y., Shi, J., Zhang, L., Davis, A., Bravo, J., Warren, A. J., Speck, N. A., and Bushweller, J. H. (2000) J. Biol. Chem. 275, 39579–39588). The importance of the hot spot residues for Runx1 function was demonstrated in in vivo transient transfection assays. These data refine the structural analyses and further our understanding of the Runx1-CBFβ interface. Core-binding factors (CBFs) 1The abbreviations used are: CBFs, core-binding factors; RD, Runt domain; EMSAs, electrophoretic mobility shift assays; TCRβ, T cell receptor β; SMMHC, smooth muscle myosin heavy chain; HSQC, heteronuclear single quantum correlation.1The abbreviations used are: CBFs, core-binding factors; RD, Runt domain; EMSAs, electrophoretic mobility shift assays; TCRβ, T cell receptor β; SMMHC, smooth muscle myosin heavy chain; HSQC, heteronuclear single quantum correlation. are a small family of heterodimeric transcription factors that play critical roles in development and in human disease. CBFs contain a DNA-binding CBFα subunit and a CBFβ subunit that does not contact DNA directly (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (437) Google Scholar, 2Ogawa E. Maruyama M. Kagoshima H. Inuzuka M. Lu J. Satake M. Shigesada K. Ito Y. Proc. Natl. Acad. Sci. U. S. 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Kumasaka T. Yamamoto M. Ishii S. Ogata K. Cell. 2001; 104: 755-767Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 26Bravo J. Li Z. Speck N.A. Warren A.J. Nat. Struct. Biol. 2001; 8: 371-377Crossref PubMed Scopus (135) Google Scholar, 29Nagata T. Werner M.H. J. Mol. Biol. 2001; 308: 191-203Crossref PubMed Scopus (34) Google Scholar, 30Bartfeld D. Shimon L. Couture G.C. Rabinovich D. Frolow F. Levanon D. Groner Y. Shakked Z. Structure. 2002; 10: 1395-1407Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar).Comparison of the Runt domain-DNA and Runt domain-CBFβ-DNA complexes (25Tahirov T.H. Inoue-Bungo T. Morii H. Fujikawa A. Sasaki M. Kimura K. Shiina M. Sato K. Kumasaka T. Yamamoto M. Ishii S. Ogata K. Cell. 2001; 104: 755-767Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 26Bravo J. Li Z. Speck N.A. Warren A.J. Nat. Struct. Biol. 2001; 8: 371-377Crossref PubMed Scopus (135) Google Scholar, 31Warren A.J. Bravo J. Williams R.L. 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Wolf-Watz M. Grundström C. Härd T. Grundström T. Sauer U.H. J. Mol. Biol. 2002; 322: 259-272Crossref PubMed Scopus (51) Google Scholar). The dramatic change in the βG-G′ loop and the changes in the βC-D loop are in agreement with an earlier study by NMR chemical shift perturbation that showed dramatic changes in the Cα chemical shift for residues in the βG-G′ loop indicative of a significant conformational change (28Tang Y.-Y. Crute B.E. Kelley III, J.J. Huang X. Yan J. Shi J. Hartman K.L. Laue T.M. Speck N.A. Bushweller J.H. FEBS Lett. 2000; 470: 167-172Crossref PubMed Scopus (44) Google Scholar).Previous studies in our laboratory and others identified amino acid side chains in CBFβ that contribute functionally to heterodimerization with the Runt domain (25Tahirov T.H. Inoue-Bungo T. Morii H. Fujikawa A. Sasaki M. Kimura K. Shiina M. Sato K. Kumasaka T. Yamamoto M. Ishii S. Ogata K. 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Here we determine the energetic contribution of the amino acids in the Runt domain that contact CBFβ to heterodimer formation. In doing so we identify two energetic hot spots at the heterodimerization interface involving Runt domain residues Asn-109 and Thr-161, and we discuss the importance of these contacts in terms of the overall ternary complex structure.EXPERIMENTAL PROCEDURESProtein Purification—We introduced alanine substitutions into a cDNA encoding the murine Runx1 Runt domain and subcloned the cDNA sequences into the bacterial pET-3c (Novagen) expression vector as described by Li et al. (35Li Z. Yan J. Matheny C.J. Corpora T. Bravo J. Warren A.J. Bushweller J.H. Speck N.A. J. Biol. Chem. 2003; 278: 33088-33096Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). We labeled the wild type and mutated Runt domains (residues 41–214, numbered according to Bae et al. (36Bae S.C. Yamaguchi-Iwai Y. Ogawa E. Maruyama M. Inuzuka M. Kagoshima H. Shigesada K. Satake M. Ito Y. Oncogene. 1993; 8: 809-814PubMed Google Scholar)) with 15N and purified them as described previously (23Berardi M. Sun C. Zehr M. Abildgaard F. Peng J. Speck N.A. Bushweller J.H. Struct. Fold Des. 1999; 7: 1247-1256Abstract Full Text Full Text PDF Scopus (69) Google Scholar, 37Crute B.E. Lewis A.F. Wu Z. Bushweller J.H. Speck N.A. J. Biol. Chem. 1996; 271: 26251-26260Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar).We subcloned the CBFβ141 cDNA, which encodes the CBFβ heterodimerization domain (amino acids 1–141), into the bacterial expression vector pET3c (Novagen) using NdeI and BamHI sites. We transformed the resulting plasmid pET3c-CBFβ141 into Rosetta (DE3) cells (Novagen) and overexpressed the CBFβ (141) protein at 37 °C. We harvested cells from a 1-liter culture and resuspended them in 10 ml of 50 mm Tris-Cl (pH 7.5), 10 mm EDTA, and 25% sucrose. We then sonicated the cells, collected the insoluble inclusion body by centrifugation, and denatured it with 7 m urea. We passed the denatured protein through a 50-ml DEAE-Sephacel (Amersham Biosciences) column in the presence of 7 m urea, collected the protein fractions, and refolded the CBFβ (141) protein by serial dialysis in 25 mm Tris-Cl (pH 7.5), 200 mm NaCl, 1 mm EDTA, 1 mm EGTA, 0.05% Triton X-100, 10% ethylene glycol, 0.5 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride with 1, 0.5, and 0 m urea. We concentrated the proteins and loaded them onto a 550-ml S-100 (Amersham Biosciences) gel filtration column. We pooled and concentrated the fractions of interest, which contained a single protein band on SDS-PAGE gels as visualized by Coomassie Brilliant Blue staining (not shown).NMR Spectroscopy—We performed all measurements on a Varian Inova 500-MHz NMR spectrometer equipped with an actively shielded triple resonance probe from Nalorac Corp. We prepared samples of Runt domain-DNA complexes and recorded 15N-1H HSQC spectra at 40 °C as described previously (23Berardi M. Sun C. Zehr M. Abildgaard F. Peng J. Speck N.A. Bushweller J.H. Struct. Fold Des. 1999; 7: 1247-1256Abstract Full Text Full Text PDF Scopus (69) Google Scholar).Urea Denaturation Monitored by Fluorescence Spectroscopy—We detected the urea denaturation of the Runt domain by tryptophan fluorescence as described previously (37Crute B.E. Lewis A.F. Wu Z. Bushweller J.H. Speck N.A. J. Biol. Chem. 1996; 271: 26251-26260Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We performed all fluorescence experiments using a protein concentration of 20–40 μm in a total volume of 0.8 ml of 25 mm Tris-HCl (pH 7.5), 50 μm EDTA, and 1 mm dithiothreitol on a SPEX Fluoromax spectrofluorometer (Spex Industries, Edison, NJ). We incubated individual 0.8-ml solutions of the Runt domain with increasing concentrations of urea (0–6.0 m) and equilibrated the samples for 2 h at 4 °C prior to fluorescence measurement to follow the Runt domain denaturation. We excited the samples at a wavelength of 280 nm and detected the emission at 340 nm. We took background spectra of the buffer alone for later subtraction. We then recorded spectra in the range of 300–370 nm with an increment of 4 nm and an integration time of 1 s, and averaged two scans to minimize error. We obtained the parameters for the unfolding curves by nonlinear, least squares fitting using two-state equations (Equation 1 and Equation 2) as described elsewhere (38Santoro M.M. Bolen D.W. Biochemistry. 1988; 27: 8063-8068Crossref PubMed Scopus (1593) Google Scholar, 39Bullock A.N. Henckel J. DeDecker B.S. Johnson C.M. Nikolova P.V. Proctor M.R. Lane D.P. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14338-14442Crossref PubMed Scopus (341) Google Scholar, 40Panda M. Horowitz P.M. J. Protein Chem. 2000; 19: 399-409Crossref PubMed Scopus (9) Google Scholar). Fobs=(αN+βN[D])+(αD+βD[D])exp{m([D]-[D]50&x0025;)/RT}1+exp{m([D]-[D]50&x0025;)/RT}(Eq. 1) We fit the data using Equation 1, where F obs is the fluorescence observed; αN and αD are the intercepts; βN and βD are the slopes of the base lines at low (N) and high (D) denaturant concentrations, respectively; R is the gas constant; T is the absolute temperature in K, [D] is the denaturant concentration; [D]50% is the concentration of the denaturant when half of the protein is denatured; and m is the slope of the unfolding transition. We used Equation 2 to determine ΔG N-D. Fobs=(αN+βN[D])+(αD+βD[D])(exp-{ΔGN-D/RT-m[D]/RT})1+exp-{ΔGN-D/RT-m[D]/RT}(Eq. 2) ΔG N-D is the free energy of unfolding in the absence of denaturant. We used Origin 5.0 software for nonlinear least square fitting.Equilibrium Binding Constant Measurements—We determined equilibrium binding constants for the wild type and mutated Runt domains by electrophoretic mobility shift assays (EMSA) using conditions described previously (6Wang Q. Stacy T. Miller J.D. Lewis A.F. Huang X. Bories J.-C. Bushweller J.H. Alt F.W. Binder M. Marín-Padilla M. Sharpe A. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar, 28Tang Y.-Y. Crute B.E. Kelley III, J.J. Huang X. Yan J. Shi J. Hartman K.L. Laue T.M. Speck N.A. Bushweller J.H. FEBS Lett. 2000; 470: 167-172Crossref PubMed Scopus (44) Google Scholar, 33Tang Y.-Y. Shi J. Zhang L. Davis A. Bravo J. Warren A.J. Speck N.A. Bushweller J.H. J. Biol. Chem. 2000; 275: 39579-39588Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 41Huang X. Crute B.E. Sun C. Tang Y.-Y. Kelley III, J.J. Lewis A.F. Hartman K.L. Laue T.M. Speck N.A. Bushweller J.H. J. Biol. Chem. 1998; 273: 2480-2487Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 42Lewis A.F. Stacy T. Green W. Taddesse-Heath L. Hartley J.W. Speck N.A. J. Virol. 1999; 73: 5535-5547Crossref PubMed Google Scholar, 43Gu T.-L. Goetz T.L. Graves B.J. Speck N.A. Mol. Cell. Biol. 2000; 20: 91-103Crossref PubMed Scopus (126) Google Scholar). We fit all data points to a rearranged mass action equation, [PD]/[D t ] = 1/(1 + Kd /[P]), using nonlinear least squares analyses (Kaleidagraph, Synergy Software).Transient Transfection of P19 Cells and Luciferase Activity Measurements—We performed site-directed mutagenesis of a cDNA encoding the full-length (451 amino acid) murine Runx1 protein in pBluescript SK+ as described above. We prepared pcDNA/Runx1 expression vectors by subcloning 1714-bp Runx1 cDNA fragments from pBluescript SK+ between the EcoRI and XhoI sites of pcDNA3.1(+) (Invitrogen). We used the TCRβ-LUC plasmid, which contains the firefly luciferase gene driven by a nucleotide 617–735 fragment from the T cell receptor β chain enhancer (44Krimpenfort P. de Jong R. Uematsu Y. Dembic Z. Ryser S. von Boehmer H. Steinmetz M. Berns A. EMBO J. 1988; 7: 745-750Crossref PubMed Scopus (111) Google Scholar), and the TCRβ(core)-LUC plasmid which contains the same fragment with mutations in the three CBF-binding sites in the enhancer (45Sun W. Graves B.J. Speck N.A. J. Virol. 1995; 69: 4941-4949Crossref PubMed Google Scholar) to detect Runx1 activity. We constructed both reporter plasmids by isolating a BamHI fragment containing the TCRβ enhancer fragments from the previously described pTCRβCAT plasmids (45Sun W. Graves B.J. Speck N.A. J. Virol. 1995; 69: 4941-4949Crossref PubMed Google Scholar), filling in the overhangs with Klenow polymerase and ligating the fragment into the SmaI site of pT81-LUC (46Nordeen S.K. BioTechniques. 1988; 6: 454-457PubMed Google Scholar).We seeded P19 cells at 2 × 106 cells per well in 6-well plates the day before transfection, and we performed transient transfections with LipofectAMINE 2000 (Invitrogen) following the manufacturer's instructions. Typically, we mixed 1 μg of the TCRβ-LUC reporter plasmids, 2 μg of the pcDNA/Runx1 expression vectors, and 2 ng of transfection efficiency control plasmid pRL-SV40 (Promega) with 250 μl of Opti-MEM (Invitrogen) and then added LipofectAMINE 2000 (7.5 μl) with 250 μl of Opti-MEM and incubated for 5 min at room temperature. We then added the plasmid DNA mixture to the LipofectAMINE 2000 mixture and incubated for 30–45 min at room temperature. We rinsed P19 cells with 2 ml of serum-free Dulbecco's modified Eagle's media and added another 2 ml of the same media to each well. The DNA/LipofectAMINE 2000 mixture was then added, and the cells were incubated for 5 h at 37 °C, and then 350 μl of Dulbecco's modified Eagle's medium with 70% fetal calf serum was added to each well. We prepared cell lysates 48 h post-transfection, and we measured their luciferase activity following instructions provided in the manual accompanying the Dual-Luciferase Reporter Assay System (Promega). We used pRL-SV40, which carries the Renilla luciferase gene, to monitor the transfection efficiency for each sample, and we used P19 cells that were transfected in the absence of DNA as blanks. We calculated the reporter activity as follows: reporter activity = (firefly luciferase activity – Blank)/(Renilla luciferase activity – Blank).RESULTSPreparation of Alanine-substituted Runt Domain Proteins and Assessment of Their Structural Integrity—Crystal structures of the Runt domain-CBFβ complex (31Warren A.J. Bravo J. Williams R.L. Rabbitts T.H. EMBO J. 2000; 19: 3004-3115Crossref PubMed Google Scholar) and of the ternary Runt domain-CBFβ-DNA complex (25Tahirov T.H. Inoue-Bungo T. Morii H. Fujikawa A. Sasaki M. Kimura K. Shiina M. Sato K. Kumasaka T. Yamamoto M. Ishii S. Ogata K. Cell. 2001; 104: 755-767Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 26Bravo J. Li Z. Speck N.A. Warren A.J. Nat. Struct. Biol. 2001; 8: 371-377Crossref PubMed Scopus (135) Google Scholar) identified 16 amino acids in the Runt domain within 3.5 Å of 12 amino acids in CBFβ (Table I). Eight of these amino acids in the Runt domain (Asn-69, Met-106, Tyr-113, Ser-14, Thr-149, Pro-156, Pro-157, and Thr-161) make side chain contacts to CBFβ that involve atoms beyond the CB carbon. To determine which of these Runt domain side chains are energetically important for the interaction with CBFβ, we substituted all but the two prolines with alanine (Fig. 1) (35Li Z. Yan J. Matheny C.J. Corpora T. Bravo J. Warren A.J. Bushweller J.H. Speck N.A. J. Biol. Chem. 2003; 278: 33088-33096Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Substitution with alanine should remove interactions involving atoms beyond the CB of an amino acid side chain (47Wells J.A. Methods Enzymol. 1991; 202: 390-411Crossref PubMed Scopus (309) Google Scholar). Alanine substitution, on the other hand, will not allow us to examine the energetic contribution of main chain atoms or the CB moieties to heterodimerization. We also substituted Asp-66 and Val-159 with alanines as controls. Asp-66 and Val-159 make backbone contacts to CBFβ, and Asp-66 also makes a CB contact. Alanine substitution of these residues should not affect heterodimerization unless the Runt domain structure is perturbed. We substituted Asn-109, which makes backbone O, C, and CA contacts with CBFβ with alanine because Asn-109 maps to a previously defined energetic hot spot at the heterodimerization interface identified in a similar analysis of CBFβ (33Tang Y.-Y. Shi J. Zhang L. Davis A. Bravo J. Warren A.J. Speck N.A. Bushweller J.H. J. Biol. Chem. 2000; 275: 39579-39588Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar).Table IContacts of amino acids in the RD to CBFβAmino acid in RDLocation in RDAtom in RDContacts to atoms in CBFβaListed are atoms within the Runt domain and CBFβ within 3.5 Å from one another. Molecules A (Runt domain) and B (CBFβ) from Protein Data Bank entry 1H9D were used for the analysis. Contacts were determined using the CNS module of Endscript 1.0.Asp-66βO-AOAsn-104 CB, Asn-104 CG, Asn-104 OD1CAsn-104 OD1CBAsn-104 CG, Asn-104 OD1Pro-68βO-AOPro-2 N, Pro-2 CD, Pro-2 OCPro-2 OAsn-69βO-ANPro-2 OCAPro-2 OCBPro-2 OCGPro-2 C, Pro-2 OOD1Pro-2 N, Pro-2 CA, Pro-2 C, Pro-2 OMet-106βCCELeu-64 C, Ser-65 N, Ser-65 OGAla-107βCOAsn-63 ND2Gly-108βCCGly-61 OCAGly-61 OAsn-109βCOGly-61 CACThr-60 OCAThr-60 OTyr-113βC-DOAsn-63 ND2CE2Arg-33 NH2CZLys-28 CDOHLys-28 CD, Arg-33 NE, Arg-33 CZSer-114βDCBAsn-63 OD1OGThr-30 CB, Thr-30 CG2, Asn-63 OD1T
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