Investigating the structural stability of the Tup1-interaction domain of Ssn6: evidence for a conformational change on the complex.
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 alpha-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.
Palaiomylitou
Maria
M
Institute of Biology, National Centre for Scientific Research Demokritos, 15310 Ag. Paraskevi Attikis, Greece.
Tartas
Athanassios
A
Vlachakis
Dimitrios
D
Tzamarias
Dimitris
D
Vlassi
Metaxia
M
D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
C065455:CYC8 protein, S cerevisiae; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C066502:TUP1 protein, S cerevisiae