proteins STRUCTURE O FUNCTION O BIOINFORMATICS Investigating the structural stability of the Tup1-interaction domain of Ssn6: Evidence for a conformational change on the complex Maria Palaiomylitou, 1 Athanassios Tartas, 1 Dimitrios Vlachakis, 1 Dimitris Tzamarias, 2 and Metaxia Vlassi 1 * 1 Institute of Biology, National Centre for Scientific Research ‘‘Demokritos’’, 15310 Ag. Paraskevi Attikis, Greece 2 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, 71110 Heraklion, Greece INTRODUCTION Transcriptional repression of a wide variety of gene families in Saccharomyces cerevisiae requires the function of two physically asso- ciated proteins, Ssn6 and Tup1. The Ssn6-Tup1 complex consists of one Ssn6 and four Tup1 molecules 1 and is recruited to target pro- moters by interacting with gene-specific DNA-binding proteins. 2–5 Transcription regulators resembling Ssn6 and Tup1, both in amino acid sequence and function, have also been found in worms, flies, plants, and mammals, suggesting that transcriptional repression by an Ssn6-Tup1-like system is evolutionary conserved. 2,6 The N-terminal domain of the 966 amino acid Ssn6 protein contains 10 tandem tetratricopeptide repeats, known as TPRs (Fig. 1). The TPR constitutes a degenerate recurrent sequence motif, the consensus sequence of which is defined by a pattern of small and large hydropho- bic amino acids. 7,11 TPR mediated protein–protein interactions are involved in a diverse spectrum of cellular functions. 12–14 On the basis of the functional diversity and importance of TPR containing proteins, Blatch and Lassle 14 proposed that the TPR motif may represent an an- cient general protein–protein interaction module adopted by function- ally different proteins and adapted for specific functions. The minimal Tup1 interaction domain of Ssn6 includes its three N-terminal TPRs (Fig. 1), whereas various combinations of TPR4 to TPR10 mediate interactions with structurally distinct DNA-binding repressor proteins specific for each gene family regulated by the Ssn6-Tup1 complex. 15 The structure of Ssn6 is unknown. However, the crystal structures of several natural 16–26 as well as of designed TPR containing pro- teins 8 have been determined so far demonstrating that each TPR motif is highly a-helical composed of a pair of antiparallel a-helices, termed helices A and B, as first determined by the crystal structure of the three-TPR containing protein Pp5. 16 The packing of helices within and between adjacent TPR motifs is identical so that each a-helix interacts with its two immediate a-helix neighbors 8,16 in a The Supplementary Material referred to in this article can be found online at http://www. interscience.wiley.com/jpages/0887-3585/suppmat/ Grant sponsor: General Secretariat for Research and Technology of Greece, NCSR ‘‘Demoritos’’. Maria Palaiomylitou and Athanassios Tartas contributed equally to this work. *Correspondence to: Metaxia Vlassi, Institute of Biology, National Centre for Scientific Research ‘‘Demokritos’’, 15310 Ag. Paraskevi Attikis, Greece. E-mail: meta@bio.demokritos.gr Received 30 December 2006; Revised 23 February 2007; Accepted 6 March 2007 Published online 16 July 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.21489 ABSTRACT Ssn6, a tetratricopeptide repeat (TPR) containing protein, associates with the Tup1 repressor to form a global transcriptional co-repressor com- plex, 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 muta- genesis 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 sim- ulations and disorder/order predictions. This com- bined 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. Subse- quent CD spectroscopy on this domain complexed with a Tup1 fragment comprising its Ssn6 binding region provided strong evidence for a conforma- tional change consisting of acquisition of a-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 par- ticular TPR-mediated complex. Proteins 2008; 70:72–82. V V C 2007 Wiley-Liss, Inc. Key words: tetratricopeptide repeat; structural flexi- bility; coupled folding and binding; circular dichro- ism; limited proteolysis; molecular dynamics. 72 PROTEINS V V C 2007 WILEY-LISS, INC.