Doublesex and the Regulation of Sexual Dimorphism in Drosophila melanogaster

The DSX (Doublesex) transcription factor regulates somatic sexual differentiation in Drosophila. Female and male isoforms (DSXF and DSXM) are formed due to sex-specific RNA splicing. DNA recognition, mediated by a shared N-terminal zinc module (the DM domain), is enhanced by a C-terminal dimerization element. Sex-specific extension of this element in DSXF and DSXM leads to assembly of distinct transcriptional preinitiation complexes. Here, we describe the structure of the extended C-terminal dimerization domain of DSXF as determined by multidimensional NMR spectroscopy. The core dimerization element is well ordered, giving rise to a dense network of interresidue nuclear Overhauser enhancements. The structure contains dimer-related UBA folds similar to those defined by x-ray crystallographic studies of a truncated domain. Whereas the proximal portion of the female tail extends helix 3 of the UBA fold, the distal tail is disordered. Ala substitutions in the proximal tail disrupt the sex-specific binding of IX (Intersex), an obligatory partner protein and putative transcriptional coactivator; IX-DSXF interaction is, by contrast, not disrupted by truncation of the distal tail. Mutagenesis of the UBA-like dimer of DSXF highlights the importance of steric and electrostatic complementarity across the interface. Two temperature-sensitive mutations at this interface have been characterized in yeast model systems. One weakens a network of solvated salt bridges, whereas the other perturbs the underlying nonpolar interface. These mutations confer graded gene-regulatory activity in yeast within a physiological temperature range and so may provide novel probes for genetic analysis of a sex-specific transcriptional program in Drosophila development. The DSX (Doublesex) transcription factor regulates somatic sexual differentiation in Drosophila. Female and male isoforms (DSXF and DSXM) are formed due to sex-specific RNA splicing. DNA recognition, mediated by a shared N-terminal zinc module (the DM domain), is enhanced by a C-terminal dimerization element. Sex-specific extension of this element in DSXF and DSXM leads to assembly of distinct transcriptional preinitiation complexes. Here, we describe the structure of the extended C-terminal dimerization domain of DSXF as determined by multidimensional NMR spectroscopy. The core dimerization element is well ordered, giving rise to a dense network of interresidue nuclear Overhauser enhancements. The structure contains dimer-related UBA folds similar to those defined by x-ray crystallographic studies of a truncated domain. Whereas the proximal portion of the female tail extends helix 3 of the UBA fold, the distal tail is disordered. Ala substitutions in the proximal tail disrupt the sex-specific binding of IX (Intersex), an obligatory partner protein and putative transcriptional coactivator; IX-DSXF interaction is, by contrast, not disrupted by truncation of the distal tail. Mutagenesis of the UBA-like dimer of DSXF highlights the importance of steric and electrostatic complementarity across the interface. Two temperature-sensitive mutations at this interface have been characterized in yeast model systems. One weakens a network of solvated salt bridges, whereas the other perturbs the underlying nonpolar interface. These mutations confer graded gene-regulatory activity in yeast within a physiological temperature range and so may provide novel probes for genetic analysis of a sex-specific transcriptional program in Drosophila development. Sexual differentiation in Drosophila melanogaster is regulated by the X:autosome ratio and a sex-specific RNA-splicing pathway (Fig. 1A) (1Cline T.W. Meyer B.J. Annu. Rev. Genet. 1996; 30: 637-702Crossref PubMed Scopus (484) Google Scholar). A principal target is doublesex (dsx); expression of male- and female-specific transcription factors (DSXM and DSXF) in turn directs most aspects of somatic sexual differentiation (2Burtis K.C. Baker B.S. Cell. 1989; 56: 997-1010Abstract Full Text PDF PubMed Scopus (515) Google Scholar). The DSX isoforms are encoded by mRNAs sharing the first three exons; the C-terminal segment of DSXF is encoded by exon 4, whereas that of DSXM is encoded by exons 5 and 6. Male and female isoforms are thus identical for the first 397 residues but differ thereafter (Fig. 1B) (2Burtis K.C. Baker B.S. Cell. 1989; 56: 997-1010Abstract Full Text PDF PubMed Scopus (515) Google Scholar). DSXM and DSXF share two recognized domains, an N-terminal DNA-binding domain (3Erdman S.E. Burtis K.C. EMBO J. 1993; 12: 527-535Crossref PubMed Scopus (206) Google Scholar) and a C-terminal dimerization domain (4Erdman S.E. Chen H.J. Burtis K.C. Genetics. 1996; 144: 1639-1652Crossref PubMed Google Scholar, 5An W. Cho S. Ishii H. Wensink P.C. Mol. Cell. Biol. 1996; 16: 3106-3111Crossref PubMed Google Scholar). The DNA-binding domain (the DM motif) contains a nonclassical zinc module (6Zhu L. Wilken J. Phillips N.B. Narendra U. Chan G. Stratton S.M. Kent S.B. Weiss M.A. Genes Dev. 2000; 14: 1750-1764Crossref PubMed Scopus (95) Google Scholar). C-terminal dimerization enhances DNA binding (7Cho S. Wensink P.C. Biochemistry. 1998; 37: 11301-11308Crossref PubMed Scopus (28) Google Scholar) and is mediated by a novel α-helical dimer containing ubiquitin-associated (UBA 5The abbreviations used are:UBAubiquitin-associated domainX-gal5-bromo-4-chloro-3-indolyl-β-d-galactosideCTDC-terminal domainIPimmunoprecipitationdsxA and dsxBspecific DNA binding sites for Doublesex in the fat body enhancerHAhemagglutininNOEnuclear Overhauser enhancementNOESYNOE spectroscopyr.m.s.root mean squareHSQCheteronuclear single-quantum coherenceTOCSYtotal correlation spectroscopyY1Hyeast one-hybridY2Hyeast two-hybridADactivation domainDBDDNA binding domain.-like) folds (8Bayrer J.R. Zhang W. Weiss M.A. J. Biol. Chem. 2005; 280: 32989-32996Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). 6It is not known whether the structural analogy between the CTDs of the DSX isoforms implies a functional role of the ubiquitination machinery in sex-specific gene regulation or whether this resemblance is incidental (8Bayrer J.R. Zhang W. Weiss M.A. J. Biol. Chem. 2005; 280: 32989-32996Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Mutations in either the DM domain or dimerization domain have been isolated in association with intersexual phenotypes (3Erdman S.E. Burtis K.C. EMBO J. 1993; 12: 527-535Crossref PubMed Scopus (206) Google Scholar, 4Erdman S.E. Chen H.J. Burtis K.C. Genetics. 1996; 144: 1639-1652Crossref PubMed Google Scholar, 9Burtis K.C. Curr. Opin. Cell Biol. 1993; 5: 1006-1014Crossref PubMed Scopus (54) Google Scholar, 10Narendra U. Zhu L. Li B. Wilken J. Weiss M.A. J. Biol. Chem. 2002; 277: 43463-43473Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). 7Mutations in the DM motif (encoding residues 35-110) affect both DSXM and DSXF. Because the sex-specific region begins at residue 398 (Gly in DSXF and Ala in DSXM), mutations in exons 4-6 affect either DSXF (exon 4, encoding residues 398-427) or DSXM (exons 5 and 6, encoding residues 398-546). XX females homozygous for G398D, for example, are intersexual, whereas males are unaffected, since exon 5 is unchanged (3Erdman S.E. Burtis K.C. EMBO J. 1993; 12: 527-535Crossref PubMed Scopus (206) Google Scholar, 72Nothiger R. Leuthold M. Andersen N. Gerschwiler P. Grutter A. Keller W. Leist C. Roost M. Schmid H. Genet. Res. 1987; 50: 113-124Crossref Scopus (28) Google Scholar). Broad conservation of the DM motif in metazoan proteins related to sexual differentiation suggests that mechanisms of sexual dimorphism are in part universal (11Raymond C.S. Shamu C.E. Shen M.M. Seifert K.J. Hirsch B. Hodgkin J. Zarkower D. Nature. 1998; 391: 691-695Crossref PubMed Scopus (643) Google Scholar). ubiquitin-associated domain 5-bromo-4-chloro-3-indolyl-β-d-galactoside C-terminal domain immunoprecipitation specific DNA binding sites for Doublesex in the fat body enhancer hemagglutinin nuclear Overhauser enhancement NOE spectroscopy root mean square heteronuclear single-quantum coherence total correlation spectroscopy yeast one-hybrid yeast two-hybrid activation domain DNA binding domain. In this paper, we describe the solution structure of the C-terminal domain (CTD) of DSXF and its functional implications. This domain (residues 350-427) (4Erdman S.E. Chen H.J. Burtis K.C. Genetics. 1996; 144: 1639-1652Crossref PubMed Google Scholar) mediates both strong dimerization (Kd < 1 nm) and the sex-specific recruitment of an obligatory partner protein, Intersex (IX) (12Garrett-Engele C.M. Siegal M.L. Manoli D.S. Williams B.C. Li H. Baker B.S. Development. 2002; 129: 4661-4675PubMed Google Scholar). Such recruitment has provided a biochemical basis for observations that (i) the intersex gene (ix) interacts genetically with dsxF but not dsxM (13Baker B.S. Ridge K.A. Genetics. 1980; 94: 383-423PubMed Google Scholar, 14Waterbury J.A. Jackson L.L. Schedl P. Genetics. 1999; 152: 1653-1667PubMed Google Scholar) and (ii) mutations in ix cause an intersexual phenotype in XX:AA ix-null flies (15Morgan T.H. Redfield H. Morgan L.V. Yearbook Carnegie Institution, Washington. 1943; 42: 171-174Google Scholar) that is identical to that of XX:AA dsx-null flies. Such corresponding phenotypes make it unlikely that IX interacts with targets other than DSXF. The ix gene encodes a protein of 188 amino acids (12Garrett-Engele C.M. Siegal M.L. Manoli D.S. Williams B.C. Li H. Baker B.S. Development. 2002; 129: 4661-4675PubMed Google Scholar) that is homologous to a component of the mammalian Mediator transcriptional co-activation complex (16Sato S. Tomomori-Sato C. Banks C.A.S. Parmely T.J. Sorokina I. Brower C.S. Conaway R.C. Conaway J.W. J. Biol. Chem. 2003; 278: 49671-49674Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Whereas IX lacks recognizable motifs of DNA binding, its N-terminal region is also homologous to Caenorhabditis elegans SUR-2 (encoded by suppressor of ras) and to the SYT class of human synovial sarcoma translocation proteins; the latter can function as transcriptional co-activators in in vitro assays. Heterologous expression of insect and mammalian homologs of ix in transgenic flies provides evidence that its functional interactions with DSXF are (at least in part) conserved (17Siegal M.L. Baker B.S. Dev. Genes Evol. 2005; 215: 1-12Crossref PubMed Scopus (30) Google Scholar). Although IX is expressed in both female and male flies, it has no known function in males. The molecular basis of the sex-specific recruitment of IX by DSXF is not well understood but is presumably mediated by the female-specific C-terminal domain (CTDF). To investigate the structural basis of DSXF dimerization and IX recruitment, we have determined the structure of CTDF and explored its function by mutagenesis. Because this domain appears to be refractory to crystallization (18Bayrer J. Wan Z. Li B. Weiss M.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1328-1330Crossref PubMed Scopus (2) Google Scholar), heteronuclear multidimensional NMR methods were employed to define its ordered substructure and delineate disordered segments. A dense network of interresidue nuclear Overhauser effects (NOEs) was defined by three- and four-dimensional edited NMR spectroscopy; intra- and intermolecular contacts were distinguished by asymmetric isotopic labeling (19Weiss M.A. J. Magn. Reson. 1990; 86: 626-632Google Scholar, 20Lee W. Revington M.J. Arrowsmith C. Kay L.E. FEBS Lett. 1994; 350: 87-90Crossref PubMed Scopus (168) Google Scholar, 21Arrowsmith C. Pachter R. Altman R. Jardetzky O. Eur. J. Biochem. 1991; 202: 53-66Crossref PubMed Scopus (60) Google Scholar). The structure of the constituent UBA-like folds and their mode of dimerization in solution are similar to features observed in the crystal structure of a dimeric fragment comprising residues 350-412 (CTDF-pΔ) (8Bayrer J.R. Zhang W. Weiss M.A. J. Biol. Chem. 2005; 280: 32989-32996Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The female-specific portion of CTDF-p (residues 398-427) in part extends the C-terminal α-helix of the UBA fold (residues 350-408) but is otherwise disordered (residues 409-427). Deletion analysis and Ala-scanning mutagenesis collectively demonstrate that IX binding is mediated by the proximal helical portion of the female tail. The dimer interface of CTDF is remarkable for an electrostatic network of salt bridges and charge-stabilized hydrogen bonds braced by underlying nonpolar side chains. Two temperature-sensitive (ts) mutations at this interface have been characterized in yeast model systems. These mutations may provide in vivo probes to enable studies of sex-specific transcriptional regulation in D. melanogaster with application to developmental neurogenetics. Bacterial Expression and Isotopic Labeling—Uniformly 13C- and 15N-double-labeled domains CTDF-p (residues 350-427) and CTDF-pΔ (residues 350-412) were expressed as thrombin-cleavable fusion proteins (8Bayrer J.R. Zhang W. Weiss M.A. J. Biol. Chem. 2005; 280: 32989-32996Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) in modified M9 minimal medium containing 3 g/liter 13C-glucose, and 1 g/liter 15NH4Cl as sole carbon and nitrogen sources, respectively. The polypeptides each contained an additional N-terminal dipeptide (GS) derived from the vector. A 50-ml overnight culture was used to inoculate 1 liter of minimal medium; the starting cell density exhibited an absorbance value of 0.15 at 600 nm (A600). When aerobic bacterial growth reached an A600 value of 0.5 following shaking at 37 °C at 250 rpm, protein overexpression was induced for 6 h by the addition of isopropyl-β-d-thiogalactopy-ranoside to a final concentration of 1 mm. Cells were harvested by centrifugation at 6370 × g for 15 min at 4 °C. The cell pellet (from 4 liters of culture) was resuspended in 100 ml of 50 mm Tris-HCl buffer (pH 8.0) containing 250 mm NaCl and 60 μg/ml lysozyme. Cell lysis was affected by French press at least twice. The lysate was centrifuged at 20,100 × g for 45 min, and the supernatant was loaded onto a cobalt resin column equilibrated with 50 mm Tris-HCl buffer (pH 8.0) containing 250 mm NaCl. The DSX fusion protein was eluted with 150 mm imidazole and cleaved by thrombin. Final purification of the DSX domains was accomplished by gel filtration chromatography using an Amersham Biosciences Superdex-75 column. The protein was >98% pure as assessed by SDS-PAGE; molecular masses were verified by mass spectrometry. NMR Methods—Protein samples were prepared in nitrogen-purged H2O solution (7% D2O) containing 10 mm 2H-Tris-HCl (pH 6.5) and 250 mm NaCl in a 300-μl Shigemi NMR tube; the protein concentration was in each case ∼1.5 mm. One-, two-, and three-dimensional NMR spectra were acquired at 30 °C at 700 MHz using a triple resonance probe and a shielded (x, y, z)-gradient unit; four-dimensional 13C/13C NOESY spectra were acquired at 600 MHz. Spectra were in each case processed with the program nmrPipe (22Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11570) Google Scholar) and analyzed with pipp (23Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (802) Google Scholar). Three-dimensional NMR triple-resonance spectra (HNCACB, CBCA(CO)NH, C(CO)NH, H(CCO)NH, HCCH-TOCSY, and HNCO experiments) were acquired to enable complete main-chain and side-chain resonance assignments (24Bax A. Curr. Opin. Struct. Biol. 1994; 4: 738-744Crossref Scopus (193) Google Scholar, 25Grzesiek S. Bax A. J. Magn. Reson. 1992; 99: 201-207Google Scholar, 26Grzesiek S. Bax A. J. Am. Chem. Soc. 1992; 114: 6291-6293Crossref Scopus (928) Google Scholar). Stereospecific assignments were obtained by analysis of approximate intraresidue and sequential interresidue distances involving NH, CαH, and CβH protons, as derived from three-dimensional 15N-edited and 13C-edited NOESY spectra with short mixing times (35 ms) to avoid spin diffusion (27Powers R. Garrett D. March C.J. Frieden E.A. Gronenborn A.M. Clore G.M. Biochem. Biophys. Res. Commun. 1993; 32: 6744-6762Google Scholar). Three NOESY spectra (three-dimensional 13C-separated NOESY-HSQC, three-dimensional 15N-separated NOESY-HSQC, and four-dimensional 13C/13C-edited NOESY) were used to derive distance restraints. Three-dimensional 13C-edited (F3)/15N, 13C-filtered (F1) experiments were used to extract intermolecular NOEs between labeled and unlabeled protomers in a solution containing a 1:1.5 ratio of labeled and unlabeled domains (28Ikura M. Bax A. J. Am. Chem. Soc. 1992; 114: 2433-2440Crossref Scopus (284) Google Scholar, 29Zwahlen C. Legault P. Vincent S.J.F. Greenblatt J. Konrat R. Kay L.E. J. Am. Chem. Soc. 1997; 119: 711-721Crossref Scopus (537) Google Scholar). Four-dimensional 13C/13C-edited NOESY was employed to identify additional dimer-related NOEs. Structure Calculations and Molecular Modeling—An ensemble of structures was calculated using a hybrid distance-geometry dynamic simulated annealing method (30Nilges M. Clore G.M. Gronenborn A.M. FEBS Lett. 1988; 229: 317-324Crossref PubMed Scopus (772) Google Scholar, 31Omichinski J.G. Pedone P.V. Felsenfeld G. Gronenborn A.M. Clore G.M. Nat. Struct. Biol. 1997; 4: 122-132Crossref PubMed Scopus (174) Google Scholar) using the National Institutes of Health version of XPLOR (32Brünger A.T. XPLOR Version 3.1 Manual. Yale University Press, New Haven, CT1993Google Scholar, 33Schwieters C.D. Kuszewski J. Tjandra N. Clore G.M. J. Magn. Reson. 2003; 160: 65-73Crossref PubMed Scopus (1870) Google Scholar). Initial structures were calculated by employing only unambiguous NOEs; calculations were iteratively performed on further NOE analysis. Assigned intra- and intermolecular NOEs were classified as strong, medium, weak, and very weak, corresponding to respective interproton distance restraints of 1.8-2.7 Å (1.8-2.9 Å for NOEs involving NH protons), 1.8-3.3 Å (1.8-3.5 Å for NOEs involving NH protons), and 1.8-5.0 Å and 1.8-6.0 Å (34Williamson M.P. Havel T.F. Wüthrich K. J. Mol. Biol. 1985; 182: 295-315Crossref PubMed Scopus (531) Google Scholar, 35Clore G.M. Nigles M. Sukumaran D.K. Brünger A.T. Karplus M. Gronenborn A.M. EMBO J. 1986; 5: 2729-2735Crossref PubMed Google Scholar). Restraints on main-chain Φ and ψ torsion angles were generated using TALOS (36Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar), in which minimum error ranges were set to ±20 (Φ) and ±30 (ψ) from the average predicted value (37Caffrey M. Cai M.L. Kaufman J. Stahl S.J. Wingfield P.T. Covell D.G. Gronenborn A.M. Clore G.M. EMBO J. 1998; 17: 4572-4584Crossref PubMed Scopus (371) Google Scholar, 38Cai M. Huang Y. Ghirlando R. Wilson K.L. Craigie R. Clore G.M. EMBO J. 2001; 20: 4399-4407Crossref PubMed Scopus (172) Google Scholar). The structure of CTDF-pΔ was calculated based on 3057 NOE distance restraints, including 418 long range restraints and 275 contacts between protomers across the dimer interface. The structure of CTDF-p was obtained using 2847 NOE restraints. Ramachandran plots were calculated to examine the quality of the final structures using PROCHECK (39Laskowski R.A. MacArthur M. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Models were visualized using InsightII and molmol software (40Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 14 (29-32): 51-55Crossref PubMed Scopus (6490) Google Scholar). Mutagenesis—Site-directed substitutions were introduced by PCR-based two-stage overlap extension mutagenesis as described (8Bayrer J.R. Zhang W. Weiss M.A. J. Biol. Chem. 2005; 280: 32989-32996Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Random mutations were introduced by error-prone PCR, generating a DNA fragment spanning the CTDF coding region. Protocols for PCR amplification and subcloning have been described (41Zhang W. Li B. Singh R. Narendra U. Zhu L. Weiss M.A. Mol. Cell. Biol. 2006; 26: 535-547Crossref PubMed Scopus (20) Google Scholar). Yeast Assays—Dimerization of CTDF was probed using the Matchmaker GAL4 Y2H system (BD Clontech, Palo Alto, CA). For Y2H library screening, pGBKT7-CTDF wild-type and pGADT7-CTDF variants were co-transformed into yeast strain Y187 by the lithium acetate/polyethylene glycol method. Interactions were monitored on SD/-Leu/-Trp-selective plates supplemented with 80 μg/ml X-gal. Plates were incubated at 30 °C for 7 days. White colonies were picked and spotted on new selective plates, incubated at 20 °C until new colonies appeared. Plasmid DNA from blue colonies was recovered by PCR and sequencing. Interactions were otherwise characterized using a quantitative β-galatosidase enzymatic assay (see below). Specific DSXF-DNA binding was probed using a MATCHMAKER Y1H system (BD Clontech) in which lacZ is regulated by a 48-bp fragment of the DSX-responsive fbe (fat body enhancer in Drosophila yolk protein genes yp1 and yp2) containing binding sites dsxA and dsxB (42Burtis K.C. Coschigano K.T. Baker B.S. Wensink P.C. EMBO J. 1991; 10: 2577-2582Crossref PubMed Scopus (166) Google Scholar). The design and validation of this system have previously been described (41Zhang W. Li B. Singh R. Narendra U. Zhu L. Weiss M.A. Mol. Cell. Biol. 2006; 26: 535-547Crossref PubMed Scopus (20) Google Scholar). Expression levels of DSX fusion proteins were verified in each case by Western blot using anti-GAL4 antiserum (Upstate Group, Charlottesville, VA). DSXF-IX Interactions—The sex-specific interaction of DSXF and IX was probed by two assays developed by Baker and co-workers (12Garrett-Engele C.M. Siegal M.L. Manoli D.S. Williams B.C. Li H. Baker B.S. Development. 2002; 129: 4661-4675PubMed Google Scholar). (i) Initial studies employed a Y2H system in which the bait plasmid expressed a fusion protein containing the specific DNA-binding domain (DBD) of GAL4 linked to DSXF, DSXM, or respective fragments containing C-terminal deletions; the prey plasmid expressed the GAL4 activation domain (AD) linked to IX. In each construct, specific independent DNA binding by the DM domain of the DSX fusion proteins was blocked by the mutation R91Q to avoid toxicity (3Erdman S.E. Burtis K.C. EMBO J. 1993; 12: 527-535Crossref PubMed Scopus (206) Google Scholar). Yeast transformants (strain AH109) were grown at 30 °C on selective plates containing the His3 competitive inhibitor 3-AT. Under these conditions, little or no growth is observed in control studies of the DSXM fusion proteins. To identify determinants of IX binding, Ala-scanning mutagenesis of CTDF was conducted in this context. Ala substitutions were tested for effects on interaction with IX by Y2H as described above. (ii) Structure-activity relationships inferred from Y2H studies were verified by co-immunoprecipitation (co-IP) of recombinant tagged IX and DSXF proteins from Drosophila S2 nuclear extracts (12Garrett-Engele C.M. Siegal M.L. Manoli D.S. Williams B.C. Li H. Baker B.S. Development. 2002; 129: 4661-4675PubMed Google Scholar). Transfected S2 cells were grown at 25 °C, and nuclear extracts were obtained and analyzed at 4 °C. Wild-type or variant DSX isoforms were expressed as V5 epitope-tagged constructs, whereas IX was co-expressed as hemagglutinin (HA)-tagged constructs. Full-length IX, DSXM, DSXF, and DSXF deletion variant coding sequences were subcloned in frame into the pAc5.1/V5-HisA vector (Invitrogen). To generate HA-tagged pAc5.1-IX expression vector, an HA epitope was added to the N terminus of IX sequence by PCR. Constructs were verified in each case by DNA sequencing. Cells were transfected using Effectene transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's protocol. Nuclear extracts were prepared using a nuclear extract kit from Active Motif (Carlsbad, CA). 100-μl nuclear extracts were incubated with 2-μg polyclonal anti-HA antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) with gentle rocking overnight at 4 °C, followed by incubation with Protein A-agarose beads (Santa Cruz Biotechnology) for an additional 3 h. The beads were collected and gently washed threes times with cell lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerolphosphate, 1 mm Na3VO4, protease inhibitor mixture (Roche Applied Science) and 1 mm phenylmethanesulfonyl fluoride). Proteins were resolved on a 12% SDS-polyacrylamide gel and detected by immunoblotting with a mouse monoclonal anti-V5 antibody (Invitrogen). Enzyme Assays—Liquid state o-nitrophenyl-β-d-galactose assays were performed according to the vendor's protocol (BD Clontech). Results (given in Miller's units) represent the mean ± S.D. of triplicate experiments (43Oh D.B. Kim Y.G. Rich A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16666-16671Crossref PubMed Scopus (110) Google Scholar). Our study has three parts. We first undertook the heteronuclear NMR analysis of CTDF-p and its dimeric fragment CTDF-pΔ. This analysis permits comparison of the solution structure of the extended domain with the crystal structure of the fragment, in turn enabling characterization of the female-specific tail. We next investigated the role of the tail in IX binding by deletion analysis and Ala-scanning mutagenesis. Finally, we employed random- and site-directed mutagenesis to probe the dimer interface. Yeast models were exploited to identify ts mutations at this interface, which may be of future value as genetic probes. Solution Structure of DSX CTDF—NMR spectra of CTDF-p exhibit residue-specific spin systems corresponding to the number of residues in a protomer, indicating that the dimer is symmetric; within the dimer, any exchange between asymmetric conformations must be fast on the time scale of NMR chemical shifts. Despite the presence of two prolines (Pro370 and Pro375), minor conformations characteristic of cis-trans isomerization were not observed. 1H-15N HSQC "fingerprint" spectra of the 15N-labeled domains are superimposed in Fig. 2. The spectrum of CTDF-p contains additional cross-peaks that are poorly resolved near random coil chemical shifts. Patterns of chemical shifts are otherwise similar in the two spectra, indicating a correspondence of structures within respective UBA folds. Complete sequential assignment was in each case obtained. Analysis of Secondary Structure—Trends in the secondary chemical shifts of Hα, Cα, and Cβ resonances, together with diagnostic NOE patterns, indicate that CTDF-p contains three long α-helices (76% of the sequence): Gln353-Lys366 (α1), Trp371-Asn383 (α2), and Ile388-Gln408 (α3). These helices each exhibit large positive secondary Cα shifts, negative secondary Cβ shifts, and strong or medium strength NOEs between amide protons within the helix (HN(i, i + 1) and HN(i, i + 3) contacts in Wüthrich format (44Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley and Sons, Inc., New York1986: 117-174Google Scholar). Although such NOE patterns can sometimes be observed in nascent (but not stably folded) helical segments, observation of the accompanying canonical helix-associated 13Cα and 13Cβ chemical shifts indicates that these elements of secondary structure are well ordered. NMR-defined helical end points in CTDF-p are in accord with the crystal structure of CTDF-pΔ (8Bayrer J.R. Zhang W. Weiss M.A. J. Biol. Chem. 2005; 280: 32989-32996Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Long Range and Dimer-related NOEs—Analysis of tertiary structure requires assignment of long range contacts, which are predominantly between α-helices. Unambiguous assignment requires dimer-related NOEs to be distinguished from intramolecular NOEs. The latter are critical for determining the folding of individual protomers, whereas intermolecular NOEs aid in characterizing the dimer interface. The pattern of long range and dimer-related NOEs is in overall accord with the crystal structure; representative three- and four-dimensional NOESY spectra are provided as supplemental material. Of particular interest are two anomalous NOE cross-peaks involving in one dimension the side-chain HNϵ resonance of Arg394 at 8.55 ppm and in the other dimension respective resonances at 6.45 and 6.84 ppm (Fig. 3B). By elimination, the latter resonances cannot be due to aromatic, main-chain amide, or side-chain carboxyamide protons. These anomalous NOE cross-peaks are asymmetric, indicating that the unassigned resonances at 6.45 and 6.84 ppm are broader than that of Arg394 HNϵ. Four corresponding NOE cross-peaks are likewise observed in the 13C-edited NOESY spectrum involving the Arg394 CδH2 methylene group (Fig. 3A). We hypothesize that these resonances belong to the Arg394 NH2 side-chain guanidinium moieties, which are seldom observable in proteins due to rapid solvent exchange. The two-dimensional 1H-15N HSQC spectrum of CTDF-pΔ contains five cross-peaks in the chemical shift range characteristic of exchangeable Arg side-chain resonances (Fig. 3C). Four are assigned to Arg HNϵ resonances, whereas the additional cross-peak at 6.84/72.00 ppm is likely to be an Arg NH2 guanidinium resonance. We speculate that an electrostatic network, including charge-stabilized hydrogen bonds, protects this resonance from solvent exchange, enabling its observation (see "Discussion"). Mobility of C-terminal Tail—Whereas the initial residues of the female-specific tail of CTDF-p (residues 350-427) extend α-helix α3 of the UBA fold (see above), the remainder of the tail is disordered. Evidence of disorder is provided by four sets of observations. First, resonances in the C-terminal tail exhibit motional narrowing relative to resonances in the UBA folds. Such differences in line widths le