Βιοχημικός και δομικός χαρακτηρισμός του τμήματος TPR επαναλήψεων του μεταγραφικού παράγοντα Ssn6 του Saccharomyces cerevisiae

Abstract

The tetratrico peptide repeat (TPR) is a 34 amino acid protein motif. Over 10000 TPRs have been identi¬fied in several species from archeobacteria to human. TPRs are protein-protein interaction medi¬a¬tors. Each TPR adopts a helix-turn-helix conformation in the form of α hairpin. Tandem TPRs stack toge¬ther producing a right handed curved superhelix, with a convex and a concave surface. The Ssn6-Tup1 protein complex is a general transcriptional co-repressor in yeast. Homologous comple¬xes have been identified in many organisms. The Ssn6 protein contains 10 tandem TPRs. TPRs 1-3 interact with the N-terminus (aa: 1-72) of Tup1 to form the complex. Using 3D-mode¬ling and mutagenesis data, our group has previously shown that the structural integrity of TPR 1 and its cor¬rect positioning relatively to TPR 2 are crucial for Tup1 binding. In the present study we investigate the structural stability of the Tup1-binding domain of Ssn6 and the importance of the Ssn6 N-terminus, which contains a polyglutamine sequence. For the pur¬pose of the study we used limited proteolysis mapping of Ssn6 and biochemical chara¬cte¬rization and circular dichroism spectroscopy of specific Ssn6 and Tup1 segments and their complexes, followed by order/disorder predictions and molecular dynamics simulations. Our data suggest that TPRs 1 and 2 of Ssn6 are partially unfolded with the helix B of TPR 1 being highly dynamic, exposing an apolar surface to the solvent. However, TPRs 1-3 seem to adopt the TPR-typically expected secondary structure when the Ssn6 segment is in asso¬cia¬tion with Tup1. In addition there are strong evi¬dence that a conformational change occurs upon complex formation, consisting of acquisition of a-helical structure with simultaneous sta¬bi¬li¬zation of coiled coil configuration. The presence of the Ssn6 N-terminus prevents the aggregation of the TPR 1-4 segment in the absence of Tup1. According to the above we propose that: Ssn6 is a member of the partially unfolded proteins group. The lack of typical conformation and the structural flexibility of TPR 1 serve the Tup1 recognition and binding. Complex formation followed by TPR 1 and 2 folding is consistent with the coupled folding to binding mechanism, reported on other bin¬ding proteins. The Ssn6-Tup1 complex formation is driven by hydrophobic interactions, in which mainly the convex surface of the TPR-superhelix takes part. The later two points reveal a novel interaction mechanism of the TPR motives.