The NF-YB/NF-YC Structure Gives Insight into DNA Binding and Transcription Regulation by CCAAT Factor NF-Y

Artigo Acesso aberto Revisado por pares

The NF-YB/NF-YC Structure Gives Insight into DNA Binding and Transcription Regulation by CCAAT Factor NF-Y

2003; Elsevier BV; Volume: 278; Issue: 2 Linguagem: Inglês

10.1074/jbc.m209635200

ISSN

1083-351X

Autores

Christophe Romier, Fabienne Cocchiarella, Roberto Mantovani, Dino Moras,

Tópico(s)

RNA Research and Splicing

Resumo

The heterotrimeric transcription factor NF-Y recognizes with high specificity and affinity the CCAAT regulatory element that is widely represented in promoters and enhancer regions. The CCAAT box acts in concert with neighboring elements, and its bending by NF-Y is thought to be a major mechanism required for transcription activation. We have solved the structure of the NF-YC/NF-YB subcomplex of NF-Y, which shows that the core domains of both proteins interact through histone fold motifs. This histone-like pair is closely related to the H2A/H2B and NC2α/NC2β families, with features that are both common to this class of proteins and unique to NF-Y. The structure together with the modeling of the nonspecific interaction of NF-YC/NF-YB with DNA and the full NF-Y/CCAAT box complex highlight important structural features that account for different and possibly similar biological functions of the transcriptional regulators NF-Y and NC2. In particular, it emphasizes the role of the newly described αC helix of NF-YC, which is both important for NF-Y trimerization and a target for regulatory proteins, such as MYC and p53. The heterotrimeric transcription factor NF-Y recognizes with high specificity and affinity the CCAAT regulatory element that is widely represented in promoters and enhancer regions. The CCAAT box acts in concert with neighboring elements, and its bending by NF-Y is thought to be a major mechanism required for transcription activation. We have solved the structure of the NF-YC/NF-YB subcomplex of NF-Y, which shows that the core domains of both proteins interact through histone fold motifs. This histone-like pair is closely related to the H2A/H2B and NC2α/NC2β families, with features that are both common to this class of proteins and unique to NF-Y. The structure together with the modeling of the nonspecific interaction of NF-YC/NF-YB with DNA and the full NF-Y/CCAAT box complex highlight important structural features that account for different and possibly similar biological functions of the transcriptional regulators NF-Y and NC2. In particular, it emphasizes the role of the newly described αC helix of NF-YC, which is both important for NF-Y trimerization and a target for regulatory proteins, such as MYC and p53. general transcription factors electrophoretic mobility shift assay Transcription initiation by RNA polymerase II at class II gene promoters is a finely regulated process requiring the interplay of many different transcription factors (1Hampsey M. Microbiol. Mol. Biol. Rev. 1998; 62: 465-503Crossref PubMed Google Scholar). General transcription factors (GTFs),1 namely TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, recognize specifically the core promoters, recruit the RNA polymerase, and help melt the DNA, thus enabling the initiation of transcription at the correct start site (2Roeder R.G Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar). Assembly of this preinitiation complex is controlled by a large set of transcriptional activators and repressors that recognize, in a sequence-specific way, DNA sequences located on proximal or distal enhancer regions of the promoters and function by contacting either directly or indirectly, through co-activators and co-repressors, the GTFs (1Hampsey M. Microbiol. Mol. Biol. Rev. 1998; 62: 465-503Crossref PubMed Google Scholar). The eukaryotic transcription factor NF-Y (also termed CBF) specifically recognizes the regulatory CCAAT element found in either orientation in the proximal and distal enhancer regions of many genes (3Maity S.N. de Crombrugghe B. Trends Biochem. Sci. 1998; 23: 174-178Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 4Mantovani R. Gene (Amst.). 1999; 239: 15-27Crossref PubMed Scopus (690) Google Scholar). In higher eukaryotes, this element is found in about 30% of the promoters, preferentially in the −60/−100 region, and analysis of various CCAAT boxes showed that specific flanking sequences are required for efficient binding (5Bucher P. J. Mol. Biol. 1990; 212: 563-578Crossref PubMed Scopus (975) Google Scholar, 6Bi W., Wu, L. Coustry F. de Crombrugghe B. Maity S.N. J. Biol. Chem. 1997; 272: 26562-26572Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 7Mantovani R. Nucleic Acids Res. 1998; 26: 1135-1143Crossref PubMed Scopus (447) Google Scholar). NF-Y is a heterotrimeric complex composed of NF-YA, NF-YB, and NF-YC which are all required for CCAAT binding (8Sinha S. Maity S.N., Lu, J. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1624-1628Crossref PubMed Scopus (251) Google Scholar). Each subunit contains a core region that has been highly conserved throughout evolution and that is sufficient for subunit interactions and CCAAT binding, whereas the flanking regions, which include the activation domains, are much less conserved (8Sinha S. Maity S.N., Lu, J. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1624-1628Crossref PubMed Scopus (251) Google Scholar, 9Hooft van Huijsduijnen R., Li, X.Y. Black D. Matthes H. Benoist C. Mathis D. EMBO J. 1990; 9: 3119-3127Crossref PubMed Scopus (210) Google Scholar, 10McNabb D.S. Xing Y. Guarente L. Genes Dev. 1995; 9: 47-58Crossref PubMed Scopus (233) Google Scholar, 11Coustry F. Maity S.N. Sinha S. de Crombrugghe B. J. Biol. Chem. 1996; 271: 14485-14491Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 12de Silvio A. Imbriano C. Mantovani R. Nucleic Acids Res. 1999; 27: 2578-2584Crossref PubMed Scopus (53) Google Scholar, 13Gusmaroli G. Tonelli C. Mantovani R. Gene (Amst.). 2001; 264: 173-185Crossref PubMed Scopus (112) Google Scholar). In yeast, the activation function is encoded by a fourth subunit with no apparent homologues in other species (14Forsburg S.L. Guarente L. Genes Dev. 1989; 3: 1166-1178Crossref PubMed Scopus (308) Google Scholar). NF-YC and NF-YB core regions are homologous in sequence to histones H2A and H2B, respectively, and are required for heterodimerization, a prerequisite for NF-YA association and CCAAT binding (8Sinha S. Maity S.N., Lu, J. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1624-1628Crossref PubMed Scopus (251) Google Scholar, 15Kim I.S. Sinha S. de Crombrugghe B. Maity S.N. Mol. Cell. Biol. 1996; 16: 4003-4013Crossref PubMed Scopus (128) Google Scholar, 16Sinha S. Kim I.S. Sohn K.Y. de Crombrugghe B. Maity S.N. Mol. Cell. Biol. 1996; 16: 328-337Crossref PubMed Scopus (144) Google Scholar). NF-YC and NF-YB show an even higher sequence similarity with subunits α and β of NC2 (16Sinha S. Kim I.S. Sohn K.Y. de Crombrugghe B. Maity S.N. Mol. Cell. Biol. 1996; 16: 328-337Crossref PubMed Scopus (144) Google Scholar), a protein that represses TATA box-dependent transcription, while increasing the activity of the distal promoter element (17Maldonado E. Hampsey M. Reinberg D. Cell. 1999; 99: 455-458Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 18Willy P.J. Kobayashi R. Kadonaga J.T. Science. 2000; 290: 982-985Crossref PubMed Scopus (134) Google Scholar). The recent structure of a NC2/TBP/TATA element complex confirmed that NC2α and -β subunits interact through histone fold motifs and that NC2 recognizes the preformed TBP/TATA complex (19Kamada K. Shu F. Chen H. Malik S. Stelzer G. Roeder R.G. Meisterernst M. Burley S.K. Cell. 2001; 106: 71-81Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The NF-YA core domain is less than 60 amino acids long and is sufficient for DNA binding when complexed with NF-YC/NF-YB (9Hooft van Huijsduijnen R., Li, X.Y. Black D. Matthes H. Benoist C. Mathis D. EMBO J. 1990; 9: 3119-3127Crossref PubMed Scopus (210) Google Scholar,20Olesen J.T. Guarente L. Genes Dev. 1990; 4: 1714-1729Crossref PubMed Scopus (144) Google Scholar, 21Maity S.N. de Crombrugghe B. J. Biol. Chem. 1992; 267: 8286-8292Abstract Full Text PDF PubMed Google Scholar). Contrary to NF-YC and NF-YB, careful examination of available data bases failed to reveal homologues of NF-YA. Several studies have divided the NF-YA core domain into two segments: an N-terminal domain responsible for NF-YC/NF-YB binding, and a C-terminal domain implicated in specific recognition of the CCAAT element (20Olesen J.T. Guarente L. Genes Dev. 1990; 4: 1714-1729Crossref PubMed Scopus (144) Google Scholar, 21Maity S.N. de Crombrugghe B. J. Biol. Chem. 1992; 267: 8286-8292Abstract Full Text PDF PubMed Google Scholar, 22Xing Y. Fikes J.D. Guarente L. EMBO J. 1993; 12: 4647-4655Crossref PubMed Scopus (139) Google Scholar, 23Xing Y. Zhang S. Olesen J.T. Rich A. Guarente L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3009-3013Crossref PubMed Scopus (65) Google Scholar, 24Mantovani R., Li, X.Y. Pessara U. Hooft V.H. Benoist C. Mathis D. J. Biol. Chem. 1994; 269: 20340-20346Abstract Full Text PDF PubMed Google Scholar). Once the trimeric complex is formed, it binds DNA with very high specificity and affinity (6Bi W., Wu, L. Coustry F. de Crombrugghe B. Maity S.N. J. Biol. Chem. 1997; 272: 26562-26572Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 25Kim C.G. Sheffery M. J. Biol. Chem. 1990; 265: 13362-13369Abstract Full Text PDF PubMed Google Scholar). Specific recognition of the bases seems to involve both minor and major groove interactions, and circular permutation assays indicated that, upon binding, the DNA is bent by about 60–80° (26Ronchi A. Bellorini M. Mongelli N. Mantovani R. Nucleic Acids Res. 1995; 23: 4565-4572Crossref PubMed Scopus (86) Google Scholar, 27Liberati C. Ronchi A. Lievens P. Ottolenghi S. Mantovani R. J. Biol. Chem. 1998; 273: 16880-16889Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Footprinting and photocross-linking experiments have shown that the DNA is contacted by a subset of the three subunits at three different locations, spanning about 24–26 nucleotides on each strand (6Bi W., Wu, L. Coustry F. de Crombrugghe B. Maity S.N. J. Biol. Chem. 1997; 272: 26562-26572Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 28Liang S.G. Maity S.N. J. Biol. Chem. 1998; 273: 31590-31598Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In agreement with these results, it was shown that two CCAAT boxes cannot be occupied simultaneously, unless they are separated by at least 22–24 bp (27Liberati C. Ronchi A. Lievens P. Ottolenghi S. Mantovani R. J. Biol. Chem. 1998; 273: 16880-16889Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 29Liberati C. di Silvio A. Ottolenghi S. Mantovani R. J. Mol. Biol. 1999; 285: 1441-1455Crossref PubMed Scopus (53) Google Scholar). A major role of NF-Y is to act synergistically with other transcription factors for activation. The CCAAT box is generally found in the close vicinity of other promoter elements, and in many cases a precise distance is required for proper transcription. Evidence that this process requires CCAAT box bending and/or direct protein/protein interactions has been reported repeatedly (4Mantovani R. Gene (Amst.). 1999; 239: 15-27Crossref PubMed Scopus (690) Google Scholar). Several lines of evidence also indicate that NF-Y interacts directly with GTFs, especially TFIID (30Bellorini M. Lee D.K. Dantonel J.C. Zemzoumi K. Roeder R.G. Tora L. Mantovani R. Nucleic Acids Res. 1997; 25: 2174-2181Crossref PubMed Scopus (104) Google Scholar, 31Coustry F. Sinha S. Maity S.N. Crombrugghe B. Biochem. J. 1998; 331: 291-297Crossref PubMed Scopus (38) Google Scholar, 32Frontini M. Imbriano C. diSilvio A. Bell B. Bogni A. Romier C. Moras D. Tora L. Davidson I. Mantovani R. J. Biol. Chem. 2002; 277: 5841-5848Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Additionally, NF-Y has been shown to be the target of regulatory proteins such as c-Myc (33Izumi H. Molander C. Penn L.Z. Ishisaki A. Kohno K. Funa K. J. Cell Sci. 2001; 114: 1533-1544Crossref PubMed Google Scholar) and p53. 2C. Imbriano, A. Gurtner, F. Cocchiarella, M. Gostissa, G. del Sal, G. Piaggio, and R. Mantovani, manuscript in preparation.2C. Imbriano, A. Gurtner, F. Cocchiarella, M. Gostissa, G. del Sal, G. Piaggio, and R. Mantovani, manuscript in preparation. We have started the structural characterization of transcription factor NF-Y and have solved the structure of the complex between the conserved regions of human NF-YB and NF-YC by x-ray crystallography. The structure was refined at 1.6 Å resolution and shows that both proteins interact through histone fold motifs in a head-to-tail fashion. The structure is very close to that of H2A/H2B and especially of NC2α/NC2β, but changes at the sequence and secondary structure level provide explanations for various functional roles played by these different complexes. Based on this overall structural homology, which extends up to the electrostatic properties, the interaction between the NF-YC/NF-YB dimer and DNA was modeled and further extended to the full NF-Y/CCAAT element complex, in agreement with several biochemical studies performed on NF-Y, including footprinting experiments and mutational analyses. EMSA experiments were also carried out which emphasized the importance of the NF-YC/NF-YB histone-like pair in DNA binding and bending. Finally, the structure reveals an important element of secondary structure, the αC helix of NF-YC, which is not only involved in NF-YA binding but plays also a role in the regulatory pathway of important growth regulators such as MYC and p53. The various constructs used for the co-expression study were amplified by standard PCR procedures. All NF-YB constructs were inserted in the pACYC184-11b vector (34Fribourg S. Romier C. Werten S. Gangloff Y.G. Poterszman A. Moras D. J. Mol. Biol. 2001; 306: 363-373Crossref PubMed Scopus (58) Google Scholar), whereas all NF-YC constructs were inserted in the pET15b (Novagen) and pGEX4T-2 (Amersham Biosciences) vectors, usingNdeI and BamHI restriction sites. Co-expression tests were carried out using a standard procedure described previously (34Fribourg S. Romier C. Werten S. Gangloff Y.G. Poterszman A. Moras D. J. Mol. Biol. 2001; 306: 363-373Crossref PubMed Scopus (58) Google Scholar). For large scale expression, 6× 1-liter cultures were grown, either in 2× LB medium for native complexes or in M9 medium supplemented with seleno-methionine (Sigma) for seleno-methionylated complexes. Cells were grown at 37 °C to an absorbance of 0.3 at 600 nm, and the temperature was then switched to 25 °C. Growth was then carried on until cells reached an absorbance of 0.8–1.0 at 600 nm. At this point, co-expression of the complex was induced by adding a final concentration of 1 mmisopropyl-β-d-thiogalactopyranoside (Euromedex), and cells were further grown overnight at 25 °C. Cells were then collected by low speed centrifugation, resuspended in buffer A (10 mm Tris, pH 8.0; 400 mm NaCl), and lysed by sonication. The soluble fraction recovered by high speed centrifugation was mixed with 1 ml of Talon resin (Clontech) in the case of a His-tagged complex or 1 ml of glutathione-Sepharose 4B resin (Amersham Biosciences) in the case of a GST-tagged complex. After 1 h of incubation, the supernatant was removed and the resin washed extensively with buffer A. The resin was then resuspended in 2 ml of buffer A, and 5 units of bovine thrombin (Sigma) were added overnight at 4 °C for cleaving off the tag. The supernatant containing the soluble dimer was recovered and applied onto a gel filtration column Hiload 16/60 Superdex 75 (Amersham Biosciences) equilibrated with buffer B (buffer A + 2 mm1.4-dithiothreitol, Roche Molecular Biochemicals). The purified complexes were concentrated on Microsep 10K Omega (Pall Filtron) to a final concentration of ∼10–14 mg/ml as assayed with Bio-Rad protein assay (Bio-Rad). For crystallization of the NF-YC3/NF-YB3 complex, 2 μl of protein complex solution were mixed with an equal volume of the reservoir solution containing 0.1m NaHepes (Sigma), pH 7.5, 0.2 mMg(OAc)2 (Merck), and 10–14% PEG 4000 (Fluka). Crystals appeared after a few hours and continued to grow for a few days or weeks to reach a size of approximately 0.5 × 0.1 × 0.1 mm (3Maity S.N. de Crombrugghe B. Trends Biochem. Sci. 1998; 23: 174-178Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). For the NF-YC2/NF-YB3 complex, the percentage of PEG 4000 had to be raised to 20–24% to obtain crystals that were smaller and more clustered than for the NF-YC3/NF-YB3 complex. Only the latter dimer was used for solving the phase problem with seleno-methionylated proteins. For data collection, crystals were briefly transferred in a cryoprotectant solution of 0.05 m NaHepes, 0.1m Mg(OAc)2, 0.2 m NaCl, 13 or 22% PEG 4000, 20% glycerol, and then quickly frozen in liquid ethane. Data collection on native and seleno-methionylated crystals was carried out on beamline BM30A at the European Synchrotron Radiation Facility. A three-wavelength multiwavelength anomalous diffraction experiment with collection of data up to 1.8 Å resolution was first carried out, and native data sets for the NF-YC3/NF-YB3 and NF-YC2/NF-YB3 complexes were then collected at 1.57 and 1.67 Å resolution, respectively. All data were processed and scaled using Denzo/Scalepack (35Otwinowski Z. Proceedings of the CCP4 Study Weekend 1993. SERC Daresbury Laboratory, UK1993: 55-62Google Scholar). Location of 5 of the 6 selenium atoms was done using Shake and Bake (see Ref. 36Miller R. Gallo S.M. Khalak H.G. Weeks C. J. Appl. Crystallogr. 1994; 27: 613-621Crossref Scopus (241) Google Scholar). Their positions were refined within the phasing program SHARP (37La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar) and the phases further improved with the solvent flattening program SOLOMON (38Abrahams J.P. Leslie A.G.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 30-42Crossref PubMed Scopus (1142) Google Scholar). Model building was carried out using program TURBO-FRODO (39Roussel A. Cambillau C. Silicon Graphics Geometry Partners Directory. Silicon Graphics, Mountain View, CA1992: 86Google Scholar). The model built in the initial 1.9-Å resolution multiwavelength anomalous diffraction electron density map was further refined independently against both native data sets by several cycles of manual building and refinement using standard protocols within the CNS (40Brünger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kussweski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar). B-factor restraints for bonded main chain and side chain atoms were 1.5 and 2.0, respectively. B-factor restraints for angle main chain and side chain were 2.0 and 2.5, respectively. The coordinates of the NF-YC2/NF-YB3 complex have been deposited in the Protein Data Bank with code1N1J. Superimposition of NF-YC/NF-YB, H2A/H2B, H3/H4, and NC2α/NC2β complexes was carried out using polyglycine models with our in-house program Superpose, 3B. Rees, unpublished data. and the transformations were applied onto the full models of the nucleosome core particle (Protein Data Bank code 1AOI) and of the NC2/TBP/TATA element complex (Protein Data Bank code 1JFI). The root mean square differences were obtained from the superimposition of the polyglycine model, removing additional residues but also helix α1-loop L1 in the case of superimpositions with H2A and H3, because these elements clearly have a different trajectory with respect to those of NF-YC or NC2α. Modeling of the NF-YC/NF-YB/DNA complex was made by extracting a DNA fragment from the structure of the nucleosome core particle once superimposed as described above. Replacement of the bases and the modeling of the interaction between NF-YA α-helices and the NF-YC/NF-YB/DNA complex was carried out manually in TURBO-FRODO. The coordinates of the model are available upon request. NF-YC mutants were produced by PCR mutagenesis with the appropriate oligonucleotides in the backbone of the YC5 mutant (41Bellorini M. Zemzoumi K. Farina A. Berthelsen J. Piaggio G. Mantovani R. Gene (Amst.). 1997; 193: 119-125Crossref PubMed Scopus (0) Google Scholar). The recombinant His-tagged YC5 mutants were obtained in inclusion bodies from BL21 bacteria, renatured with equimolar amounts of NF-YB, and purified over nitrilotriacetic acid columns (29Liberati C. di Silvio A. Ottolenghi S. Mantovani R. J. Mol. Biol. 1999; 285: 1441-1455Crossref PubMed Scopus (53) Google Scholar, 42Zemzoumi K. Frontini M. Bellorini M. Mantovani R. J. Mol. Biol. 1999; 286: 327-337Crossref PubMed Scopus (50) Google Scholar). The resulting dimers were assayed in immunoprecipitations and EMSA experiments with recombinant NF-YA and the monoclonal antibody 7 monoclonal antibody (42Zemzoumi K. Frontini M. Bellorini M. Mantovani R. J. Mol. Biol. 1999; 286: 327-337Crossref PubMed Scopus (50) Google Scholar). Production and purifications of NF-Y and off-rate EMSA experiments were done under conditions described previously (29Liberati C. di Silvio A. Ottolenghi S. Mantovani R. J. Mol. Biol. 1999; 285: 1441-1455Crossref PubMed Scopus (53) Google Scholar,43Caretti G. Motta M.C. Mantovani R. Mol. Cell. Biol. 1999; 19: 8591-8603Crossref PubMed Scopus (59) Google Scholar). All three subunits of NF-Y contain a core region that has been highly conserved throughout evolution and, in the case of NF-YC and NF-YB, that displays sequence homology to the histone fold motifs of H2A/NC2α and H2B/NC2β, respectively (Fig. 1). The core domains of NF-YB and NF-YC have been shown to be necessary and sufficient for DNA binding in the context of the trimeric complex (15Kim I.S. Sinha S. de Crombrugghe B. Maity S.N. Mol. Cell. Biol. 1996; 16: 4003-4013Crossref PubMed Scopus (128) Google Scholar, 16Sinha S. Kim I.S. Sohn K.Y. de Crombrugghe B. Maity S.N. Mol. Cell. Biol. 1996; 16: 328-337Crossref PubMed Scopus (144) Google Scholar, 20Olesen J.T. Guarente L. Genes Dev. 1990; 4: 1714-1729Crossref PubMed Scopus (144) Google Scholar, 41Bellorini M. Zemzoumi K. Farina A. Berthelsen J. Piaggio G. Mantovani R. Gene (Amst.). 1997; 193: 119-125Crossref PubMed Scopus (0) Google Scholar). However, less conserved stretches at their N and C termini seem to influence this process (29Liberati C. di Silvio A. Ottolenghi S. Mantovani R. J. Mol. Biol. 1999; 285: 1441-1455Crossref PubMed Scopus (53) Google Scholar). The majority of histone fold proteins are produced in bacteria as insoluble material in inclusion bodies. We have studied the formation of the NF-YC/NF-YB pair with protein constructs of different lengths, by testing protein solubilization using the technique of co-expression in Escherichia coli (34Fribourg S. Romier C. Werten S. Gangloff Y.G. Poterszman A. Moras D. J. Mol. Biol. 2001; 306: 363-373Crossref PubMed Scopus (58) Google Scholar). The results summarized in Table I indicate that only the evolutionary conserved domains of NF-YC and NF-YB, but not the less conserved regions, are necessary for complex formation.Table ISummary of co-expression experimentsNF-YC1 (21–120)1-aHuman numbering.NF-YC2 (27–120)NF-YC3 (44–120)1-bEvolutionary conserved domains.NF-YB1 (49–122)−−−NF-YB2 (49–131)−−−NF-YB3 (49–141)1-bEvolutionary conserved domains.+++NF-YB4 (49–149)+++1-a Human numbering.1-b Evolutionary conserved domains. Open table in a new tab For the subsequent crystallization trials, four of the six soluble complexes obtained were used: NF-YC2/NF-YB3, NF-YC2/NF-YB4, NF-YC3/NF-YB3, and NF-YC3/NF-YB4 (see Table I). Small crystals were initially obtained with the NF-YC3/NF-YB3 pair. Further refinement of the crystallization conditions showed that crystals of NF-YC2/NF-YB3 could also be obtained. Both crystals belong to the same space group with the same cell parameters (TableII). Crystals of the seleno-methionylated NF-YC3/NF-YB3 complex were also grown and used for solving the phase problem by multiwavelength anomalous diffraction (44Hendrickson W.A. Science. 1991; 254: 51-58Crossref PubMed Scopus (1015) Google Scholar). An initial model was built manually into the experimental electron density map at 1.9 Å resolution and was further refined independently against native NF-YC3/NF-YB3 and NF-YC2/NF-YB3 data sets at 1.57 and 1.67 Å resolution, respectively. The final models include 87 residues of NF-YB, 78 residues of NF-YC, about 300 water molecules, and have R factors around 18% and R-free factors around 20%, with very good deviations from ideal geometry (Table II).Table IIData collection and refinement statisticsNF-YC/NF-YB pairsC2/B3C3/B3C3/B3C3/B3C3/B3Data sets wavelength (Å)Native 0.920023Native 0.920023Se-Met λ 1 0.979650Se-Met λ 2 0.979407Se-Met λ 3 0.977775Space groupP212121P212121P212121P212121P212121Cell constants a(Å)51.651.651.651.651.6 b (Å)60.360.660.660.660.6 c (Å)61.661.862.062.062.0Resolution (Å)24–1.6724–1.5724–1.7824–1.7824–1.78Reflections measured/unique110,854/22,726151,344/27,411131,407/35,755130,502/35,753132,289/36,007Redundancy overall/last shell4.9/2.55.5/2.93.7/1.93.7/1.93.7/1.9Completeness (%) overall/last shell97.9/99.097.9/93.598.7/99.598.8/99.398.9/99.0R sym (%) overall/last shell2.6/5.34.5/8.34.8/6.55.0/6.24.6/10.6I/ς(I) overall/last shell43.8/17.328.9/13.620.2/10.819.8/11.219.3/8.8Refinement statisticsResolution (Å)24–1.6724–1.57No. protein atoms13371337No. water molecules298307No. reflections (working/test sets)21,368/109525,803/1325R factor (%)18.118.3R·free (%)20.620.5Deviations from ideal geometry Bonds (Å)0.0060.006 Angles (°)1.11.1Mean temperature factors (Å2)16.615.1The pairs used are NFY-C2/NF-YB3 and NF-YC3/NF-YB3. The wavelengths (Å) for the data sets are as follows: native, 0.920023; native, 0.920023; Seleko-Met λ1, 0.979650; Seleko-Met λ2, 0.979407; and Seleko-Met λ3, 0.977775. Open table in a new tab The pairs used are NFY-C2/NF-YB3 and NF-YC3/NF-YB3. The wavelengths (Å) for the data sets are as follows: native, 0.920023; native, 0.920023; Seleko-Met λ1, 0.979650; Seleko-Met λ2, 0.979407; and Seleko-Met λ3, 0.977775. In NF-YB3, no density was observed for the first seven residues. Mass spectrometry revealed that all these residues except for the initial methionine were present in the protein used for crystallization and also in the crystals (data not shown). Thus, the N-terminal residues of NF-YB3, which point toward a solvent channel, are probably disordered. In NF-YC3, only the residual thrombin site residues Gly-Ser at the N terminus were not unambiguously found in density. In the case of the NF-YC2 construct, which is 16 residues longer than NF-YC3, no additional residues could be built at the N terminus either. Once again, mass spectrometry revealed that all the unobserved residues are present in the crystals (data not shown). Because the initial experimental phases were obtained for the NF-YC3/NF-YB3 complex, it could be assumed that the initial model was not good enough to provide phases for these residues. However, several loops in other parts of the structure, which could not be seen in the initial electron density map, appeared during refinement, whereas density at the N terminus of NF-YC2 never improved. Because a large solvent channel was found where the residues should be located, it seems reasonable to assume that these residues are disordered. As expected, the core domains of NF-YB and NF-YC adopt a histone-like fold and interact in a head-to-tail fashion, forming a histone-like pair (Fig. 2 A). Interestingly, comparison of the NF-YC/NF-YB, NC2α/NC2β, H2A/H2B, but also H3/H4 histone pairs, reveals relatively little differences between their core histone motifs (helix α1-loop L1-helix α2-loop L2-helix α3; see Fig. 2 B) both in terms of sequence identity (ranging from 10 to 20%) or pairwise main chain root mean square differences (ranging from 1.5 to 1.1). Actually, that NF-YC/NF-YB belongs to the H2A/H2B family is confirmed by the presence of additional elements of secondary structure, at the C termini of both proteins, characteristic of H2A and H2B, although H3/H4 features are also observed (see below). The interactions between the various elements of secondary structure of NC2α/NC2β and the comparison of this pair with the H2A/H2B dimer have already been described at length (19Kamada K. Shu F. Chen H. Malik S. Stelzer G. Roeder R.G. Meisterernst M. Burley S.K. Cell. 2001; 106: 71-81Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The conclusions mostly apply to NF-YC/NF-YB and will not be discussed further. Rather, we will focus on the differences and the specificities we observe. One feature concerns the presence in both NF-YB and NF-YC of an intra-chain arginine-aspartate bidentate pair which is found in histones H3 and H4 but not in H2A and H2B (45Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6885) Google Scholar) (Fig. 2 A). In NF-YC this pair is formed by residues Arg-93 (loop L2) and Asp-100 (helix α3), and both are absolutely conserved in the NF-YC but neither in H2A nor in NC2α families (Fig. 1 C). In NF-YB, residues Arg-108 (loop L2) and Asp-115 (helix α3) form an identical pair and are also absolutely conserved throughout evolution. Once again, this pair is not conserved in NC2 and is replaced by an arginine/lysine-glutamate pair in H2B (Fig. 1 B). In this latter case, however, the pair is not formed, and the arginine contacts the DNA; interestingly, this is not seen in H3 and H4, where the pairs are formed even if the arginines are in the vicinity of the DNA backbone (45Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6885) Google Scholar). Another specific feature is the presence in NF-YC of an

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The NF-YB/NF-YC Structure Gives Insight into DNA Binding and Transcription Regulation by CCAAT Factor NF-Y