Investigating the structural stability of the Tup1-interaction domain of Ssn6: Evidence for a conformational change on the complex
Maria Palaiomylitou
Institute of Biology, National Centre for Scientific Research “Demokritos”, 15310 Ag. Paraskevi Attikis, Greece
Maria Palaiomylitou and Athanassios Tartas contributed equally to this work.
Search for more papers by this authorAthanassios Tartas
Institute of Biology, National Centre for Scientific Research “Demokritos”, 15310 Ag. Paraskevi Attikis, Greece
Maria Palaiomylitou and Athanassios Tartas contributed equally to this work.
Search for more papers by this authorDimitrios Vlachakis
Institute of Biology, National Centre for Scientific Research “Demokritos”, 15310 Ag. Paraskevi Attikis, Greece
Search for more papers by this authorDimitris Tzamarias
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, 71110 Heraklion, Greece
Search for more papers by this authorCorresponding Author
Metaxia Vlassi
Institute of Biology, National Centre for Scientific Research “Demokritos”, 15310 Ag. Paraskevi Attikis, Greece
Institute of Biology, National Centre for Scientific Research “Demokritos”, 15310 Ag. Paraskevi Attikis, Greece===Search for more papers by this authorMaria Palaiomylitou
Institute of Biology, National Centre for Scientific Research “Demokritos”, 15310 Ag. Paraskevi Attikis, Greece
Maria Palaiomylitou and Athanassios Tartas contributed equally to this work.
Search for more papers by this authorAthanassios Tartas
Institute of Biology, National Centre for Scientific Research “Demokritos”, 15310 Ag. Paraskevi Attikis, Greece
Maria Palaiomylitou and Athanassios Tartas contributed equally to this work.
Search for more papers by this authorDimitrios Vlachakis
Institute of Biology, National Centre for Scientific Research “Demokritos”, 15310 Ag. Paraskevi Attikis, Greece
Search for more papers by this authorDimitris Tzamarias
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, 71110 Heraklion, Greece
Search for more papers by this authorCorresponding Author
Metaxia Vlassi
Institute of Biology, National Centre for Scientific Research “Demokritos”, 15310 Ag. Paraskevi Attikis, Greece
Institute of Biology, National Centre for Scientific Research “Demokritos”, 15310 Ag. Paraskevi Attikis, Greece===Search for more papers by this authorAbstract
Ssn6, a tetratricopeptide repeat (TPR) containing protein, associates with the Tup1 repressor to form a global transcriptional co-repressor complex, which is conserved across species. The three N-terminal TPR repeats of Ssn6, out of a total of 10, are involved in this particular interaction. Our previously reported 3D-modeling and mutagenesis data suggested that the structural integrity of TPR1 and its correct positioning relatively to TPR2 are crucial for Tup1 binding. In this study, we first investigate the structural stability of the Tup1 binding domain of Ssn6, in pure form, through a combination of CD spectroscopy and limited proteolysis mapping. The obtained data were next combined with molecular dynamics simulations and disorder/order predictions. This combined study revealed that, although competent to fold, in the absence of Tup1, TPR1 is partially unfolded with its helix B being highly dynamic exposing an apolar surface to the solvent. Subsequent CD spectroscopy on this domain complexed with a Tup1 fragment comprising its Ssn6 binding region provided strong evidence for a conformational change consisting of acquisition of α-helical structure with simultaneous stabilization of a coiled-coil configuration upon complex formation. We propose that this conformational change occurs largely in the TPR1 of Ssn6 and is in accord with the concept of folding coupled to binding, proposed for other TPR domains. A possible implication of the structural flexibility of Ssn6 TPR1 in Tup1 recognition is discussed and a novel mode of interaction is proposed for this particular TPR-mediated complex. Proteins 2008. © 2007 Wiley-Liss, Inc.
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REFERENCES
- 1 Varanasi US,Klis M,Mikesell PB,Trumbly RJ. The Cyc8 (Ssn6)-Tup1 corepressor complex is composed of one Cyc8 and four Tup1 subunits. Mol Cell Biol 1996; 16: 6707–6714.
- 2 Smith RL,Redd MJ,Johnson AD. The tetratricopeptide repeats of Ssn6 interact with the homeo domain of α 2. Genes Dev 1995; 9: 2903–2910.
- 3 Williams FE,Varanasi U,Trumbly RJ. The CYC8 and TUP1 proteins involved in glucose repression in Saccharomyces cerevisiae are associated in a protein complex. Mol Cell Biol 1991; 11: 3307–3316.
- 4 Keleher CA,Redd MJ,Schultz J,Carlson M,Johnson AD. Ssn6-Tup1 is a general repressor of transcription in yeast. Cell 1992; 68: 709–719.
- 5 Tzamarias D,Struhl K. Functional dissection of the yeast Cyc8-Tup1 transcriptional co-repressor complex. Nature 1994; 369: 758–761.
- 6 Grbavec D,Lo R,Liu Y,Greenfield A,Stifani S. Groucho/transducin- like enhancer of split (TLE) family members interact with the yeast transcriptional co-repressor SSN6 and mammalian SSN6-related proteins: implications for evolutionary conservation of transcription repression mechanisms. Biochem J 1999; 337 (Part 1): 13–17.
- 7 Sikorski RS,Boguski MS,Goebl M,Hieter P. A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell 1990; 60: 307–317.
- 8 Main ER,Xiong Y,Cocco MJ,D'Andrea L,Regan L. Design of stable α-helical arrays from an idealized TPR motif. Structure 2003; 11: 497–508.
- 9 Gounalaki N,Tzamarias D,Vlassi M. Identification of residues in the TPR domain of Ssn6 responsible for interaction with the Tup1 protein. Febs Lett 2000; 473: 37–41.
- 10 Limbach MP,Zitomer RS. The isolation and characterization of missense mutants in the general repressor protein Ssn6 of Saccharomyces cerevisiae. Mol Gen Genet 2000; 263: 455–462.
- 11 Magliery TJ,Regan L. Beyond consensus: statistical free energies reveal hidden interactions in the design of a TPR motif. J Mol Biol 2004; 343: 731–745.
- 12 Goebl M,Yanagida M. The TPR snap helix: a novel protein repeat motif from mitosis to transcription. Trends Biochem Sci 1991; 16: 173–177.
- 13 Lamb JR,Tugendreich S,Hieter P. Tetratrico peptide repeat interactions: to TPR or not to TPR? Trends Biochem Sci 1995; 20: 257–259.
- 14
Blatch GL,Lassle M.
The tetratricopeptide repeat: a structural motif mediating protein-protein interactions.
Bioessays
1999;
21:
932–939.
10.1002/(SICI)1521-1878(199911)21:11<932::AID-BIES5>3.0.CO;2-N CAS PubMed Web of Science® Google Scholar
- 15 Tzamarias D,Struhl K. Distinct TPR motifs of Cyc8 are involved in recruiting the Cyc8-Tup1 corepressor complex to differentially regulated promoters. Genes Dev 1995; 9: 821–831.
- 16 Das AK,Cohen PW,Barford D. The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions. EMBO J 1998; 17: 1192–1199.
- 17 Scheufler C,Brinker A,Bourenkov G,Pegoraro S,Moroder L,Bartunik H,Hartl FU,Moarefi I. Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell 2000; 101: 199–210.
- 18 Lapouge K,Smith SJ,Walker PA,Gamblin SJ,Smerdon SJ,Rittinger K. Structure of the TPR domain of p67phox in complex with Rac.GTP. Mol Cell 2000; 6: 899–907.
- 19 Gatto GJ,Jr,Geisbrecht BV,Gould SJ,Berg JM. Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5. Nat Struct Biol 2000; 7: 1091–1095.
- 20 Taylor P,Dornan J,Carrello A,Minchin RF,Ratajczak T,Walkinshaw MD. Two structures of cyclophilin 40: folding and fidelity in the TPR domains. Structure 2001; 9: 431–438.
- 21 Sinars CR,Cheung-Flynn J,Rimerman RA,Scammell JG,Smith DF,Clardy J. Structure of the large FK506-binding protein FKBP51, an Hsp90-binding protein and a component of steroid receptor complexes. Proc Natl Acad Sci USA 2003; 100: 868–873.
- 22 Wu B,Li P,Liu Y,Lou Z,Ding Y,Shu C,Ye S,Bartlam M,Shen B,Rao Z. 3D structure of human FK506-binding protein 52: implications for the assembly of the glucocorticoid receptor/Hsp90/immunophilin heterocomplex. Proc Natl Acad Sci USA 2004; 101: 8348–8353.
- 23 Jinek M,Rehwinkel J,Lazarus BD,Izaurralde E,Hanover JA,Conti E. The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin α. Nat Struct Mol Biol 2004; 11: 1001–1007.
- 24 Zhang M,Windheim M,Roe SM,Peggie M,Cohen P,Prodromou C,Pearl LH. Chaperoned ubiquitylation—crystal structures of the CHIP U box E3 ubiquitin ligase and a CHIP-Ubc13-Uev1a complex. Mol Cell 2005; 20: 525–538.
- 25 Wilson CG,Kajander T,Regan L. The crystal structure of NlpI. A prokaryotic tetratricopeptide repeat protein with a globular fold. FEBS J 2005; 272: 166–179.
- 26 Wu Y,Sha B. Crystal structure of yeast mitochondrial outer membrane translocon member Tom70p. Nat Struct Mol Biol 2006; 13: 589–593.
- 27 Cliff MJ,Harris R,Barford D,Ladbury JE,Williams MA. Conformational diversity in the TPR domain-mediated interaction of protein phosphatase 5 with Hsp90. Structure 2006; 14: 415–426.
- 28 Siegel LM,Monty KJ. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta 1966; 112: 346–362.
- 29 Zeev-Ben-Mordehai T,Rydberg EH,Solomon A,Toker L,Auld VJ,Silman I,Botti S,Sussman JL. The intracellular domain of the Drosophila cholinesterase-like neural adhesion protein, gliotactin, is natively unfolded. Proteins 2003; 53: 758–767.
- 30 Bohm G,Muhr R,Jaenicke R. Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng 1992; 5: 191–195.
- 31 Andrade MA,Chacon P,Merelo JJ,Moran F. Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng 1993; 6: 383–390.
- 32 Dosztanyi Z,Csizmok V,Tompa P,Simon I. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 2005; 21: 3433–3434.
- 33 Yang ZR,Thomson R,McNeil P,Esnouf RM. RONN: the bio-basis function neural network technique applied to the detection of natively disordered regions in proteins. Bioinformatics 2005; 21: 3369–3376.
- 34 Romero P,Obradovic Z,Li X,Garner EC,Brown CJ,Dunker AK. Sequence complexity of disordered protein. Proteins 2001; 42: 38–48.
- 35 Li X,Romero P,Rani M,Dunker AK,Obradovic Z. Predicting protein disorder for N-, C-, and Internal Regions. Genome Inform Ser Workshop Genome Inform 1999; 10: 30–40.
- 36 Ferron F,Longhi S,Canard B,Karlin D. A practical overview of protein disorder prediction methods. Proteins 2006; 65: 1–14.
- 37 Garner E,Romero P,Dunker AK,Brown C,Obradovic Z. Predicting binding regions within disordered proteins. Genome Inform Ser Workshop Genome Inform 1999; 10: 41–50.
- 38 Oldfield CJ,Cheng Y,Cortese MS,Romero P,Uversky VN,Dunker AK. Coupled folding and binding with α-helix-forming molecular recognition elements. Biochemistry 2005; 44: 12454–12470.
- 39 Kabsch W,Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 1983; 22: 2577–2637.
- 40 Privalov PL. Stability of proteins. Proteins which do not present a single cooperative system. Adv Protein Chem 1982; 35: 1–104.
- 41 Dyson HJ,Rance M,Houghten RA,Wright PE,Lerner RA. Folding of immunogenic peptide fragments of proteins in water solution. II. The nascent helix. J Mol Biol 1988; 201: 201–217.
- 42 Cooper TM,Woody RW. The effect of conformation on the CD of interacting helices: a theoretical study of tropomyosin. Biopolymers 1990; 30: 657–676.
- 43 Gibney BR,Johansson JS,Rabanal F,Skalicky JJ,Wand AJ,Dutton PL. Global topology & stability and local structure & dynamics in a synthetic spin-labeled four-helix bundle protein. Biochemistry 1997; 36: 2798–2806.
- 44 Zhou NE,Kay CM,Hodges RS. Synthetic model proteins. Positional effects of interchain hydrophobic interactions on stability of two-stranded α-helical coiled-coils. J Biol Chem 1992; 267: 2664–2670.
- 45 Uversky VN. What does it mean to be natively unfolded? Eur J Biochem 2002; 269: 2–12.
- 46 Fontana A,de Laureto PP,Spolaore B,Frare E,Picotti P,Zambonin M. Probing protein structure by limited proteolysis. Acta Biochim Pol 2004; 51: 299–321.
- 47 D'Andrea LD,Regan L. TPR proteins: the versatile helix. Trends Biochem Sci 2003; 28: 655–662.
- 48
Uversky VN,Gillespie JR,Fink AL.
Why are “natively unfolded” proteins unstructured under physiologic conditions?
Proteins
2000;
41:
415–427.
10.1002/1097-0134(20001115)41:3<415::AID-PROT130>3.0.CO;2-7 CAS PubMed Web of Science® Google Scholar
- 49 Bourhis JM,Johansson K,Receveur-Brechot V,Oldfield CJ,Dunker KA,Canard B,Longhi S. The C-terminal domain of measles virus nucleoprotein belongs to the class of intrinsically disordered proteins that fold upon binding to their physiological partner. Virus Res 2004; 99: 157–167.
- 50 Jabet C,Sprague ER,VanDemark AP,Wolberger C. Characterization of the N-terminal domain of the yeast transcriptional repressor Tup1. Proposal for an association model of the repressor complex Tup1 x Ssn6. J Biol Chem 2000; 275: 9011–9018.
- 51 Main ER,Stott K,Jackson SE,Regan L. Local and long-range stability in tandemly arrayed tetratricopeptide repeats. Proc Natl Acad Sci USA 2005; 102: 5721–5726.
- 52 Croy CH,Bergqvist S,Huxford T,Ghosh G,Komives EA. Biophysical characterization of the free IκBα ankyrin repeat domain in solution. Protein Sci 2004; 13: 1767–1777.
- 53 Bradley CM,Barrick D. Limits of cooperativity in a structurally modular protein: response of the Notch ankyrin domain to analogous alanine substitutions in each repeat. J Mol Biol 2002; 324: 373–386.
- 54 Beddoe T,Bushell SR,Perugini MA,Lithgow T,Mulhern TD,Bottomley SP,Rossjohn J. A biophysical analysis of the tetratricopeptide repeat-rich mitochondrial import receptor, Tom70, reveals an elongated monomer that is inherently flexible, unstable, and unfolds via a multistate pathway. J Biol Chem 2004; 279: 46448–46454.
- 55 Cliff MJ,Williams MA,Brooke-Smith J,Barford D,Ladbury JE. Molecular recognition via coupled folding and binding in a TPR domain. J Mol Biol 2005; 346: 717–732.
- 56 Cortajarena AL,Regan L. Ligand binding by TPR domains. Protein Sci 2006; 15: 1193–1198.
- 57 Wright PE,Dyson HJ. Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. J Mol Biol 1999; 293: 321–331.
- 58 Dyson HJ,Wright PE. Coupling of folding and binding for unstructured proteins. Curr Opin Struct Biol 2002; 12: 54–60.
- 59 Dunker AK,Brown CJ,Lawson JD,Iakoucheva LM,Obradovic Z. Intrinsic disorder and protein function. Biochemistry 2002; 41: 6573–6582.
- 60 Uversky VN,Oldfield CJ,Dunker AK. Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling. JMol Recognit 2005; 18: 343–384.
- 61 Vucetic S,Brown CJ,Dunker AK,Obradovic Z. Flavors of protein disorder. Proteins 2003; 52: 573–584.
- 62 Callebaut I,Labesse G,Durand P,Poupon A,Canard L,Chomilier J,Henrissat B,Mornon JP. Deciphering protein sequence information through hydrophobic cluster analysis (HCA): current status and perspectives. Cell Mol Life Sci 1997; 53: 621–645.
- 63 Carrico PM,Zitomer RS. Mutational analysis of the Tup1 general repressor of yeast. Genetics 1998; 148: 637–644.
- 64 Lundin VF,Stirling PC,Gomez-Reino J,Mwenifumbo JC,Obst JM,Valpuesta JM,Leroux MR. Molecular clamp mechanism of substrate binding by hydrophobic coiled-coil residues of the archaeal chaperone prefoldin. Proc Natl Acad Sci USA 2004; 101: 4367–4372.
- 65 Smith RL,Johnson AD. A sequence resembling a peroxisomal targeting sequence directs the interaction between the tetratricopeptide repeats of Ssn6 and the homeodomain of α 2. Proc Natl Acad Sci USA 2000; 97: 3901–3906.
- 66 Dunker AK,Cortese MS,Romero P,Iakoucheva LM,Uversky VN. Flexible nets. The roles of intrinsic disorder in protein interaction networks. FEBS J 2005; 272: 5129–5148.
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