Characterization of the N-terminal Domain of the Yeast Transcriptional Repressor Tup1
2000; Elsevier BV; Volume: 275; Issue: 12 Linguagem: Inglês
10.1074/jbc.275.12.9011
ISSN1083-351X
AutoresCarole Jabet, Elizabeth R. Sprague, Andrew P. VanDemark, Cynthia Wolberger,
Tópico(s)Genomics and Chromatin Dynamics
ResumoThe yeast Tup1 and Ssn6 proteins form a transcriptional repression complex that represses transcription of a broad array of genes. It has been shown that the N-terminal domain of the Tup1 protein interacts with a region of the Ssn6 protein that consists of 10 tandem copies of a tetratricopeptide motif. In this work, we use a surface plasmon resonance assay to measure the affinity of the N-terminal domain of Tup1 for a minimal 3-TPR domain ofSaccharomyces cerevisiae Ssn6 that is sufficient for binding to Tup1. This domain of Ssn6 binds with comparable affinity to S. cerevisiae and Candida albicans Tup1, but with 100-fold lower affinity to Tup1 protein containing a point mutation that gives rise to a defect in repressionin vivo. Results from studies using analytical ultracentrifugation, CD spectroscopy, limited proteolysis, and1H NMR show that this domain of Tup1 is primarily α-helical and forms a stable tetramer that is highly nonglobular in shape. X-ray diffraction recorded from poorly ordered crystals of the Tup1 tetramerization domain contains fiber diffraction typical of a coiled coil. Our results are used to propose a model for the structure of the N-terminal domain of Tup1 and its interaction with the Ssn6 protein. The yeast Tup1 and Ssn6 proteins form a transcriptional repression complex that represses transcription of a broad array of genes. It has been shown that the N-terminal domain of the Tup1 protein interacts with a region of the Ssn6 protein that consists of 10 tandem copies of a tetratricopeptide motif. In this work, we use a surface plasmon resonance assay to measure the affinity of the N-terminal domain of Tup1 for a minimal 3-TPR domain ofSaccharomyces cerevisiae Ssn6 that is sufficient for binding to Tup1. This domain of Ssn6 binds with comparable affinity to S. cerevisiae and Candida albicans Tup1, but with 100-fold lower affinity to Tup1 protein containing a point mutation that gives rise to a defect in repressionin vivo. Results from studies using analytical ultracentrifugation, CD spectroscopy, limited proteolysis, and1H NMR show that this domain of Tup1 is primarily α-helical and forms a stable tetramer that is highly nonglobular in shape. X-ray diffraction recorded from poorly ordered crystals of the Tup1 tetramerization domain contains fiber diffraction typical of a coiled coil. Our results are used to propose a model for the structure of the N-terminal domain of Tup1 and its interaction with the Ssn6 protein. tetratricopeptide repeats protein phosphatase 5 Transcriptional repression of a variety of yeast genes is mediated by two proteins that act in concert, Ssn6 and Tup1. These proteins are required for the repression of at least five independently regulated sets of genes: the mating type a- and haploid-specific genes (1.Mukai Y. Harashima S. Oshima Y. Mol. Cell. Biol. 1991; 11: 3773-3779Crossref PubMed Google Scholar, 2.Keleher C.A. Redd M.J. Schultz J. Carlson M. Johnson A.D. Cell. 1992; 68: 709-719Abstract Full Text PDF PubMed Scopus (544) Google Scholar), glucose-repressed genes (3.Schultz J. Carlson M. Mol. Cell. Biol. 1987; 7: 3637-3645Crossref PubMed Scopus (135) Google Scholar, 4.Trumbly R.J. Mol. Microbiol. 1992; 6: 15-21Crossref PubMed Scopus (269) Google Scholar), hypoxic genes (5.Zitomer R.S. Lowry C.V. Microbiol. Rev. 1992; 56: 1-11Crossref PubMed Google Scholar), and DNA damage-inducible genes (6.Elledge S.J. Zhou Z. Allen J.B. Navas T.A. Bioessays. 1993; 15: 333-339Crossref PubMed Scopus (209) Google Scholar). Tup1 and Ssn6 have been shown to form a corepressor complex that is recruited to the DNA by interaction with sequence-specific DNA-binding proteins such as Matα2, Rox1, Mig1, and the Crt repressor (2.Keleher C.A. Redd M.J. Schultz J. Carlson M. Johnson A.D. Cell. 1992; 68: 709-719Abstract Full Text PDF PubMed Scopus (544) Google Scholar, 7.Nehlin J.O. Carlberg M. Ronne H. EMBO J. 1991; 10: 3373-3377Crossref PubMed Scopus (304) Google Scholar, 8.Zhou Z. Elledge S.J. Genetics. 1992; 131: 851-866Crossref PubMed Google Scholar, 9.Balasubramanian B. Lowry C.V. Zitomer R.S. Mol. Cell. Biol. 1993; 13: 6071-6078Crossref PubMed Scopus (123) Google Scholar, 10.Treitel M.A. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3132-3136Crossref PubMed Scopus (342) Google Scholar, 11.Tzamarias D. Struhl K. Genes Dev. 1995; 9: 821-831Crossref PubMed Scopus (242) Google Scholar). The Tup1·Ssn6 corepressor complex has been estimated by sucrose gradient sedimentation and by gel densitometry to contain three or four Tup1 subunits and one Ssn6 subunit (12.Varanasi U.S. Klis M. Mikesell P.B. Trumbly R.J. Mol. Cell. Biol. 1996; 16: 6707-6714Crossref PubMed Scopus (105) Google Scholar, 13.Redd M.J. Arnaud M.B. Johnson A.D. J. Biol. Chem. 1997; 272: 11193-11197Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Although mutations in either protein give rise to defects in transcriptional repression (1.Mukai Y. Harashima S. Oshima Y. Mol. Cell. Biol. 1991; 11: 3773-3779Crossref PubMed Google Scholar, 3.Schultz J. Carlson M. Mol. Cell. Biol. 1987; 7: 3637-3645Crossref PubMed Scopus (135) Google Scholar, 14.Schultz J. Marshall-Carlson L. Carlson M. Mol. Cell. Biol. 1990; 10: 4744-4756Crossref PubMed Scopus (93) Google Scholar, 15.Thrash-Bingham C. Fangman W.L. Mol. Cell. Biol. 1989; 9: 809-816Crossref PubMed Scopus (31) Google Scholar, 16.Trumbly R.J. J. Bacteriol. 1986; 166: 1123-1127Crossref PubMed Google Scholar, 17.Carlson M. Osmond B.C. Neigeborn L. Botstein D. Genetics. 1984; 107: 19-32Crossref PubMed Google Scholar, 18.Flick J.S. Johnston M. Mol. Cell. Biol. 1990; 10: 4757-4769Crossref PubMed Scopus (125) Google Scholar), Tup1 appears to play the predominant role, because the defect in repression that results from deletion of both the Ssn6 and Tup1 genes can be overcome by overexpression of Tup1, but not Ssn6 (19.Komachi K. Redd M.J. Johnson A.D. Genes Dev. 1994; 8: 2857-2867Crossref PubMed Scopus (189) Google Scholar). Moreover, Ssn6-LexA fusions repress transcription in a Tup1-dependent manner, whereas Tup1-LexA fusions can repress transcription in the absence of Ssn6 (20.Tzamarias D. Struhl K. Nature. 1994; 369: 758-761Crossref PubMed Scopus (284) Google Scholar). The mechanism of repression by Tup1·Ssn6 is not well understood, although there is evidence that it occurs by either direct interaction with the polymerase II holoenzyme (21.Wahi M. Komachi K. Johnson A.D. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 447-457Crossref PubMed Scopus (32) Google Scholar, 22.Kuchin S. Carlson M. Mol. Cell. Biol. 1998; 18: 1163-1171Crossref PubMed Scopus (111) Google Scholar) or by altering local chromatin structure (23.Cooper J.P. Roth S.Y. Simpson R.T. Genes Dev. 1994; 8: 1400-1410Crossref PubMed Scopus (166) Google Scholar, 24.Edmondson D.G. Smith M.M. Roth S.Y. Genes Dev. 1996; 10: 1247-1259Crossref PubMed Scopus (406) Google Scholar). The functional domains of the 713-amino acid Tup1 protein from the yeast Saccharomyces cerevisiae have been analyzed by genetic and biochemical approaches. The N-terminal residues 1–72 contain sequences that are involved in complex formation with Ssn6 (20.Tzamarias D. Struhl K. Nature. 1994; 369: 758-761Crossref PubMed Scopus (284) Google Scholar) as well as mediating multimerization of Tup1 (12.Varanasi U.S. Klis M. Mikesell P.B. Trumbly R.J. Mol. Cell. Biol. 1996; 16: 6707-6714Crossref PubMed Scopus (105) Google Scholar, 20.Tzamarias D. Struhl K. Nature. 1994; 369: 758-761Crossref PubMed Scopus (284) Google Scholar). Residues 120–334 contain sequences that mediate transcriptional repression, as determined by the inability of Tup1 fragments lacking this region to repress transcription (20.Tzamarias D. Struhl K. Nature. 1994; 369: 758-761Crossref PubMed Scopus (284) Google Scholar). The C-terminal half of Tup1 (residues 334–713) consists of a domain that contains seven WD40 repeats. These repeats, also known as β-transducin motifs (1.Mukai Y. Harashima S. Oshima Y. Mol. Cell. Biol. 1991; 11: 3773-3779Crossref PubMed Google Scholar, 25.Fong H.K. Hurley J.B. Hopkins R.S. Miake-Lye R. Johnson M.S. Doolittle R.F. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2162-2166Crossref PubMed Scopus (322) Google Scholar, 26.Williams F.E. Trumbly R.J. Mol. Cell. Biol. 1990; 10: 6500-6511Crossref PubMed Scopus (161) Google Scholar), are present in many proteins that are involved in diverse cellular processes and have been suggested to mediate protein-protein interactions. In the case of Tup1, the WD motif has been shown to interact with the mating-type regulator, Matα2 (19.Komachi K. Redd M.J. Johnson A.D. Genes Dev. 1994; 8: 2857-2867Crossref PubMed Scopus (189) Google Scholar, 27.Komachi K. Johnson A.D. Mol. Cell. Biol. 1997; 17: 6023-6028Crossref PubMed Scopus (85) Google Scholar). Both the sequence and the biological function of Tup1 are conserved in the yeast Candida albicans whose 515-residue Tup1 protein is 48% identical to theS. cerevisiae Tup1 protein. Moreover, expression of theC. albicans Tup1 gene fully complements a tup1deletion in S. cerevisiae (28.Braun B.R. Johnson A.D. Science. 1997; 277: 105-109Crossref PubMed Scopus (458) Google Scholar). Ssn6 is a 966-amino acid protein that contains at its N terminus a domain of 10 tetratricopeptide repeats (TPR)1 (residues 46–398) that is essential for its function (3.Schultz J. Carlson M. Mol. Cell. Biol. 1987; 7: 3637-3645Crossref PubMed Scopus (135) Google Scholar, 14.Schultz J. Marshall-Carlson L. Carlson M. Mol. Cell. Biol. 1990; 10: 4744-4756Crossref PubMed Scopus (93) Google Scholar). The TPR is a motif of 34 amino acids found in over 30 proteins from a variety of organisms that have diverse cellular functions (for review see Ref. 29.Goebl M. Yanagida M. Trends Biochem. Sci. 1991; 16: 173-177Abstract Full Text PDF PubMed Scopus (376) Google Scholar). Distinct combinations of TPR motifs are required for direct interaction with Tup1 and for repression of distinct classes of genes (11.Tzamarias D. Struhl K. Genes Dev. 1995; 9: 821-831Crossref PubMed Scopus (242) Google Scholar). For example, the first three TPR motifs are sufficient for binding to Tup1 (11.Tzamarias D. Struhl K. Genes Dev. 1995; 9: 821-831Crossref PubMed Scopus (242) Google Scholar) and to Matα2 (30.Smith R.L. Redd M.J. Johnson A.D. Genes Dev. 1995; 9: 2903-2910Crossref PubMed Scopus (81) Google Scholar) and for repression of mating-type regulated genes (11.Tzamarias D. Struhl K. Genes Dev. 1995; 9: 821-831Crossref PubMed Scopus (242) Google Scholar). Repeats 1–7 are necessary for the repression of oxygen-regulated genes, whereas all the TPR motifs are required for repression of DNA damage-regulated genes (11.Tzamarias D. Struhl K. Genes Dev. 1995; 9: 821-831Crossref PubMed Scopus (242) Google Scholar). As seen in the crystal structure of the 3-TPR domain of the protein phosphatase 5 (PP5) (31.Das A.K. Cohen P.W. Barford D. EMBO J. 1998; 17: 1192-1199Crossref PubMed Scopus (710) Google Scholar), a single TPR folds to form a pair of antiparallel α-helices of equal length. Successive TPRs pack against one another in tandem and are related by a small rotation. The uniform angular and spatial arrangement of neighboring α-helices generates a right-handed superhelix with a central groove (31.Das A.K. Cohen P.W. Barford D. EMBO J. 1998; 17: 1192-1199Crossref PubMed Scopus (710) Google Scholar). In the present study, we use a surface plasmon resonance-based assay to quantitate the interaction between the N-terminal tetramerization domain of Tup1 and the minimal 3-TPR domain of S. cerevisiaeSsn6 that is sufficient for mediating interactions with Tup1 (20.Tzamarias D. Struhl K. Nature. 1994; 369: 758-761Crossref PubMed Scopus (284) Google Scholar). We show that the affinity for Ssn6 of both the S. cerevisiaeand C. albicans Tup1 is comparable, whereas the S. cerevisiae Tup1 containing an L62R substitution binds 100-fold more weakly to Ssn6. Equilibrium ultracentrifugation of the wild type and mutant proteins shows that this mutation, which has a deleterious effect on Tup1-mediated repression in vivo, does not interfere with the tetramerization of Tup1 and is therefore likely to lie on the surface of the Tup1 tetramer. Using a combination of analytical ultracentrifugation, circular dichroism (CD) spectroscopy, proteolysis, 1H NMR, and x-ray fiber diffraction, we characterize structural features of the tetramerization domain. We find that this domain is highly nonglobular in shape and associates to form a type of α-helical coiled coil. These results are used to propose a model for the structure of the N-terminal domain of Tup1 and its interaction with the TPR domain of Ssn6. The modification of the cDNA encoding the N-terminal domain of Tup1 carrying the mutation Leu-62 → Arg (Sc mut62 Tup1) was obtained with the QuickChangeTM mutagenesis kit (Stratagene) following the instructions of the manufacturer. The cDNAs encoding the respective N-terminal domains of S. cerevisiae Tup1 (Sc N91 Tup1; residues 1–91) and of C. albicans Tup1 (Ca N92 Tup1; residues 1–92) as well as the N-terminal domain of Tup1 carrying the mutation Leu-62 → Arg (Sc mut62 Tup1) were cloned in the pET 3d vector (Novagen). Each fragment is preceded by an additional methionine and is expressed in Escherichia coli under control of the T7 promoter. BL21 (DE3) pLysS cells were grown at 37 °C in LB medium with 100 μg/ml ampicillin, induced at mid-log phase with 1 mm isopropyl-1-thio-β-d-galactopyranoside, and grown for 3 h at 25 °C. The bacterial cell pellet was sonicated in 1× phosphate-buffered saline, 0.8 m NaCl, 10% glycerol, 1% Igepal, and 1 mm EDTA. The lysate was centrifuged at 8500 rpm in a GSA rotor for 15 min. The protein was precipitated with 20% ammonium sulfate and resuspended in 50 mm Tris (pH 8), 50 mm NaCl, and 1 mm EDTA. Ion exchange chromatography was performed on the protein solution with a Q Fast Flow column (Amersham Pharmacia Biotech) followed by a MonoQ column (Amersham Pharmacia Biotech). In each case, protein solutions were loaded onto columns equilibrated in 50 mm Tris (pH 8), 50 mm NaCl, and 1 mm EDTA and eluted with a 0.05–1 m NaCl gradient. Peak fractions were pooled and purified by gel filtration using a Superdex 75 column (Amersham Pharmacia Biotech) in 50 mm Tris (pH 8), 150 mm NaCl, and 1 mm EDTA. Proteins were then dialyzed against 10 mm Tris (pH 8) and 25 mm NaCl, concentrated to 10 mg/ml, and stored at −80 °C. The cDNA encoding the first three TPR motifs of S. cerevisiae Ssn6 (residues 31–149) was cloned in the pET 16b vector (Novagen), which directs protein expression in E. coli under control of the T7 promoter. The Ssn6 fragment is preceded by a 10 histidine tag and the sequence SSGHIQGAH, which contains a factor Xa cleavage site. BL21 (DE3) pLysS cells were grown at 37 °C in LB medium with 100 μg/ml ampicillin, induced at mid-log phase with 1 mmisopropyl-1-thio-β-d-galactopyranoside, and grown for 3 h at 25 °C. The bacterial cell pellet was sonicated in 1× phosphate-buffered saline, 0.8 m NaCl, 10% glycerol, 1% Igepal, 1 mm EDTA, and 2 m urea. The lysate was centrifuged in a GSA rotor at 8500 rpm for 15 min, after which the protein was found in the insoluble fraction. This fraction was resuspended in 20 mm Tris (pH 8), 500 mm NaCl, and 6M urea, filtered, and loaded onto a HisTrap column (Amersham Pharmacia Biotech) equilibrated in 20 mm Tris (pH 8), 500 mm NaCl, and 6 m urea. The elution was performed with a 0–1 m imidazole gradient. Peak fractions were pooled, diluted to 10 μg/ml, and dialyzed against 50 mm Tris (pH 8) and 50 mm NaCl. Sedimentation equilibrium experiments were conducted using a Beckman Optima XL-A analytical ultracentrifuge equipped with an optical absorbance system. Runs were carried out at 9000, 10,000, 13,000, 15,000, 20,000, and 27,000 rpm at 20 °C. Six-channel, charcoal-filled epon centerpieces with quartz windows were used in an An-60 Ti rotor. Samples at concentrations of 1.7, 0.8, and 0.4 mg/ml, in 50 mm Tris (pH 8) and 150 mm NaCl were analyzed. Cells were loaded with 100 μl of protein sample and 110 μl of reference buffer. Radial scans at 280 nm were collected between 5.9 and 7.2 cm as the average of five measurements, with a step size of 0.001 cm. The samples were allowed to achieve sedimentation equilibrium over the course of 26 h and were judged to be at equilibrium when sequential scans 2 h apart were superimposable. The proteins' partial specific volumes (Sc N91 Tup1, 0.726 g/ml and Sc mut62 Tup1, 0.7241 g/ml), buffer density (1.0058 g/ml), buffer viscosity (1.0312 × 10−2 poise), and temperature corrections were determined using standard methods (for review of methods, see Ref. 32.Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.M. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, London1992: 90-125Google Scholar), as implemented in the SEDNTERP program. Sedimentation equilibrium data were analyzed using the appropriate functions by nonlinear least squares procedures (33.Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar) provided in the Beckman Optima XL-A software package. For data analysis according to discrete self-association models, the following general equation was used, C(r)=δ+C1.0exp(ς(r2−r02))+∑N>1C1.0NKNexp(Nς(r2−r02))Equation 1 where C(r) is the total concentration at radius r, δ is the base-line offset,C 1,0 is the monomer concentration at the reference radius r0 , N is the stoichiometry of the reaction, and KN is the equilibrium association constant. ς is defined as follows, ς=M1(1−ν¯ρ)ω2/2RTEquation 2 where M 1 is the monomer molecular weight,ν̄ is the partial specific volume, ρ is the solvent density, ω is the angular velocity of the rotor, R is the gas constant, and T is the absolute temperature of the sedimentation equilibrium experiment. Global molecular weights were obtained for several rotor speeds and protein concentrations by fitting the equilibrium sedimentation data to a single species using the equations above. Sedimentation velocity experiments were carried out at 60,000 rpm at 20 °C for sample concentrations of 0.18, 1.1, and 1.3 mg/ml. Protein and buffer samples were prepared as described above. Two-sector charcoal-filled epon 12-mm centerpieces with quartz windows were loaded with 420 μl of protein in the sample well and 426 μl of buffer in the reference well. Radial scans at 230 or 280 nm were collected with a step size of 0.003 cm in continuous mode at intervals of about 4 min for a period of 4 h. Sedimentation coefficients corrected for diffusion were calculated for each boundary by the method of van Holde and Weischet (34.van Holde K.E. Weischet W.O. Biopolymers. 1975; 17: 1387-1403Crossref Scopus (318) Google Scholar) using the program Ultrascan II (B. Demeler, University of Texas Health Sciences Center at San Antonio). Thes 20,w for each protein concentration was determined at the boundary midpoint from a plot of the boundary fraction versus S 20,w. Values ofs 20,w at three different protein concentrations did not show evidence of concentration dependence when extrapolated to infinite dilution. Thus, the s20,w0 of 2.24 ± 0.01 S was obtained by averaging the three sedimentation coefficients. The translational frictional coefficient, f, was calculated from the Svedberg equation, f=M(1−ν¯ρ)NAsEquation 3 where ν̄ is the partial specific volume, ρ is the solvent density, M is the molecular weight,N A is Avogadro's number, and s is the sedimentation coefficient. The frictional coefficient for a rigid spherical molecule of equal (anhydrous) volume,f o, was calculated from Stokes law,f o = 6πηR o where η is the buffer viscosity and Ro is the radius of the molecule. Based on the frictional coefficient and an estimated value for hydration from the amino acid composition (δ = 0.4244) (32.Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.M. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, London1992: 90-125Google Scholar), axial ratios for simple ellipsoidal and cylindrical models were estimated using SEDNTERP. Assays of the Tup1-Ssn6 interaction were performed on a Biacore system with certified nitrilotriacetic acid sensor chips. Flow cells were coated with nickel as described by the manufacturer. Immobilization of the His-3TPR Ssn6 and of the His-AraC (control) fragments was performed as follows. A continuous flow of running buffer (50 mm Tris (pH 8) and 150 mm NaCl) over the sensor surface at 10 μl/min was maintained and between 60 and 150 μl of HisTag fragments (200 nm in 50 mm Tris (pH 8) and 50 mmNaCl) were injected at 3 μl/min. The relative amount of protein immobilized ranged from 250 to 400 response units. Binding of the N-terminal Tup1 fragments to the immobilized Ssn6 3 TPR protein was monitored by injecting 20 μl of Tup1 at increasing concentrations (2–20 μm) over the chip surface at a flow rate of 10 μl/min at 25 °C. After 15 min of dissociation, the surface was regenerated. Interaction curves were obtained by subtracting the experimental curves from the control. Data were analyzed using the Langmuir model functions provided in the Biacore Biaevaluation 3.0 software package. According to this model, data were evaluated using the following rate equation, dR/dt=kaC(Rmax−Rt)−kdRtEquation 4 assuming a single site interaction between the Ssn6 3 TPR protein and each N-terminal Tup1 tetramer, wheredR/dt is the rate of formation of surface complexes, C is the concentration of N-terminal Tup1 tetramer, R max is the total amount of immobilized ligand expressed as surface plasmon resonance response,Rt is the response observed at timet. ka and kd are the association and dissociation rate constants, respectively. The equilibrium dissociation constant, KD , was calculated from the ratiokd /ka . The fit of the data to the model was assessed by examining the residuals and by minimizing the χ2 values. CD spectra of the N-terminal Tup1 fragments were measured at 20 °C in a AVIV DS 60 spectrometer with a quartz cell of 0.1-mm path length. The protein concentration was 1 mg/ml in 10 mm phosphate buffer (pH 7). Molar ellipticity was calculated as described by Delage and Geourjon (35.Delage G. Geourjon C. Comp. Appl. Biosci. 1993; 9: 197-199PubMed Google Scholar). Estimates of the secondary structure were made using the CDNN program (36.Andrade M.A. Chacon P. Merelo J.J. Moran F. Protein Eng. 1993; 4: 383-390Crossref Scopus (949) Google Scholar, 37.Bohm G. Muhr R. Jaenicke R. Protein Eng. 1992; 3: 191-195Crossref Scopus (1015) Google Scholar). The Sc N91 Tup1 fragment was concentrated to 1 mm in 20 mm phosphate buffer (pH 7). The 1H nuclear Overhauser effect spectroscopy spectrum was collected with Brucker DMX 600 MHz at 25 °C in 90% H2O − 10% D2O. The spectrum was processed with NMRPipe (38.Delaglio F. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11555) Google Scholar). Crystals of the N-terminal domain of Tup1 were grown by the method of hanging drop vapor diffusion from protein purified and concentrated as described above. Protein was mixed on a siliconized coverslip with an equal volume of well solution containing 12% (w/v) polyethylene glycol 4000, 100 mm Bis-Tris propane (pH 9), and 1 mmdithiothreitol and suspended over the well solution, after which crystals appeared in 1–2 weeks. Crystals of dimension 600 × 400 × 50 μm were mounted and sealed in a glass capillary. X-ray diffraction was recorded at room temperature with an RAXIS II image plate detector equipped with Osmic double-focusing mirrors using copper Kα radiation generated by a Rigaku RU-200 rotating anode x-ray generator. Unit cell and fiber diffraction parameters were measured directly from diffraction images using the program IPDISP (54.Collaborative Computational Project, November 4Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19762) Google Scholar). Comparison of several fungal Tup1 proteins reveals the presence of a conserved 10-kDa N-terminal domain containing residues that have been found to mediate tetramerization ofS. cerevisiae Tup1 (12.Varanasi U.S. Klis M. Mikesell P.B. Trumbly R.J. Mol. Cell. Biol. 1996; 16: 6707-6714Crossref PubMed Scopus (105) Google Scholar, 20.Tzamarias D. Struhl K. Nature. 1994; 369: 758-761Crossref PubMed Scopus (284) Google Scholar) and interaction with Ssn6 (20.Tzamarias D. Struhl K. Nature. 1994; 369: 758-761Crossref PubMed Scopus (284) Google Scholar). The corresponding domain of S. cerevisiae Tup1 containing residues 1–91 (Sc N91 Tup1) was expressed and purified, and its oligomeric state was assayed by sedimentation equilibrium analytical ultracentrifugation. Typical data collected at one initial loading concentration and at three different speeds are shown in Fig.1 A. These data, as well as additional data collected at different initial loading concentrations (data not shown), were well described by a model for a single homogeneous species (“Experimental Procedures,” Equation 1) as evaluated by randomness of the residuals and minimization of the variance (Fig. 1 A). The single species model yielded an average molecular mass of 43,000 Da ± 1500, which compared with the calculated monomer molecular mass of 11,088 Da, strongly suggests that Sc N91 Tup1 self-associates as a tetramer. To examine whether monomer, dimer, or octamer species were also present, the data were also fit to models describing monomer-dimer-tetramer, monomer-tetramer, and monomer-tetramer-octamer equilibria. Attempts to fit the data to each of these models were unsuccessful as reflected in increased variances as well as unrealistic values for equilibrium constants. We therefore conclude that the tetramer is the predominant species of Sc N91 Tup1 and that this tetramer is not in a detectable reversible equilibrium with other species. Carrico and Zitomer (39.Carrico P.M. Zitomer R.S. Genetics. 1998; 148: 637-644PubMed Google Scholar) identified a Tup1 mutant protein with leucine 62 replaced by an arginine, L62R, which is unable to form a complex with Ssn6 or to repress expression of hypoxic and glucose-repressed reporter genes. Repression of the a-mating type reporter gene, however, is unaffected by this substitution. To better understand the effects of this mutation, we used equilibrium analytical ultracentrifugation to examine whether the mutation Leu-62 → Arg impairs the ability of Sc N91 Tup1 to form a tetramer. Data collected for one loading concentration of Sc mut62 Tup1 at three different speeds are shown in Fig. 1 B. These data with additional data at two other loading concentrations (data not shown) fit best to a model with a single species of molecular mass 40,000 ± 1000 Da. Because the calculated monomer molecular mass of Sc mut62 Tup1 is 11,131 Da, the predominant oligomeric state of Sc mut62 Tup1 is a tetramer, as was observed for the wild-type fragment. In light of the 4500 Da difference between the observed molecular mass and the theoretical one, it is possible that some monomer or dimer states are also present in solution. However, attempts to fit the data to monomer-dimer-tetramer, monomer-tetramer, or dimer-tetramer models were unsuccessful, as shown again by increased variances and unrealistic values for equilibrium constants. These results show that the tetramerization of the N-terminal domain of Tup1 is very stable and is not in a reversible equilibrium with lower oligomeric states. They also demonstrate that the mutation L62R, which weakens the interaction with Ssn6, does not notably impair the tetramerization of Tup1. A surface plasmon resonance-based biosensor assay was used to carry out a kinetic analysis of the Tup1-Ssn6 interaction. The protein fragments assayed were those that had been previously shown to be the minimal domains required for the two proteins to bind to one another (11.Tzamarias D. Struhl K. Genes Dev. 1995; 9: 821-831Crossref PubMed Scopus (242) Google Scholar, 20.Tzamarias D. Struhl K. Nature. 1994; 369: 758-761Crossref PubMed Scopus (284) Google Scholar). An Ssn6 fragment consisting of the first three TPR motifs (residues 31–149) and preceded by an N-terminal His tag was immobilized on a Ni2+-coated biosensor chip surface (see “Experimental Procedures”). The binding of the Tup1 protein to the surface of the chip and its subsequent dissociation were monitored by surface plasmon resonance, which yields a signal proportional to the mass detected. The sensorgram in Fig. 2 A shows the binding of the Sc N91 Tup1 fragment to the immobilized Ssn6 3 TPR fragment. As a control for the specificity of the interaction, this was compared with the binding of Tup1 to a surface coupled with the N-terminal domain of the E. coli AraC protein fused to a His tag (Fig. 2 A) and to a Ni2+-coated surface lacking immobilized protein (Fig. 2 A). Binding of Tup1 to either surface was negligible. Furthermore, a Tup1 fragment containing the WD region of the protein but lacking the N-terminal residues that interact with Ssn6 (Sc WD Tup1), did not bind to the immobilized Ssn6 3TPR fragment, even at high concentrations (Fig. 2 B). Binding of Sc N91 Tup1 to the immobilized Ssn6 3 TPR fragment was studied over a Sc N91 Tup1 concentration range of 2–15 μm. Fig. 3 Ashows representative sensorgrams of association and dissociation at various Sc N91 Tup1 concentrations. These data were analyzed by simultaneous global fitting of both association and dissociation phases for all sets of concentrations, using the model AB 219 A+B and assuming a single site interaction between a Tup1 tetramer and Ssn6 (“Experimental Procedures,” Equation 1). The best fit was achieved by considering the first three minutes of the dissociation (χ2 = 17.5, Table I). According to this fit, the dissociation rate,kd , was equal to 8 × 10−4s−1, and
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