The tetratricopeptide repeat (TPR) motif contains 34 amino acids forming a helix-turn-helix structure. Different numbers of tandem TPR motifs assemble to form a TPR domain, thereby generating a polypeptide-binding interaction surface. The TPR domain provides a scaffold for mediating protein-protein interactions. Proteins that contain TPR domains exist in a broad range of organisms. These proteins have various functions. Cyclophilin 40 (Cyp40) and C-terminal Hsc70 interaction protein (CHIP) are two typical members of the family of TPR-containing proteins. Both proteins have the ability to bind the molecular chaperones Hsp70 and Hsp90. In most cases, TPR domains act as a scaffold to link chaperone and substrate or multi-protein complexes. Recent evidence suggests that Hsp90 binding to TPR domains can change the overall protein conformation but the allosteric mechanism triggered by ligand binding to the TPR domain remained unknown. This study focuses on using biophysical methods on the two TPR domain containing proteins Cyp40 and CHIP. In particular, this study reveals how the binding of the molecular chaperones Hsp70/90 to the TPR domains of Cyp40 and CHIP influences protein conformation and function. Here we show how conformational changes of the TPR domains affect structure and activity of Cyp40 and CHIP. By using biophysical methods, including thermal denaturation assay (TDA), differential scanning calorimetry (DSC), hydrogen deuterium exchange with mass spectrometry (HDX-MS) and small angle X-ray scattering (SAXS), together with enzymatic assays, we showed that (1) heat shock proteins allosterically affect the enzyme activity of both Cyp40 and CHIP, (2) heat shock proteins bind to the TPR domains of both Cyp40 and CHIP; (3) the binding increases the thermostability of both proteins. Further, by mutating an essential lysine in the TPR1 domain of both proteins (K30 for CHIP, and K227 for Cyp40) to alanine, the thermostability was significantly affected. The SAXS data showed in addition of the SRMEEVD peptide reduced the flexibility of CHIP. HDX-MS experiments suggest that the dynamic alteration due to binding with the Hsp90 peptide or the mutations further reduce the flexibility of the catalytic domains of both proteins. The results imply that the allosteric effects on the enzymatic activity are consequences of dynamic changes of the TPR domains. Hsp70 was also found to bind less tightly to CHIP-K30A than to wild-type CHIP, and thus showed less inhibition of enzymatic activity. These results further confirmed the discovery, that the dynamics of TPR domains allosterically affect enzymatic activity.

Upon ligand binding, the aryl hydrocarbon receptor (AhR) translocates into the nucleus and dimerizes with its partner Ah receptor nuclear translocator (Arnt). The AhR/Arnt heterodimer binds to the enhancer element DRE to regulate target gene expression. It is known that the formation of the ligand-dependent AhR/Arnt/DRE complex requires protein factors in vitro. The first aim is to determine whether two other Hsp90-associated proteins present in rabbit reticulocyte lysate (RRL), namely CyP40 and Hsp70, play any role in forming the AhR/Arnt/DRE complex. Fractionation and immunodepletion experiments revealed that Hsp70 is not necessary for the formation of this complex. In contrast, CYP40 is involved in forming the complex since (1) immunodepletion of CyP40 from a RRL fraction reduces the intensity of the AhR-Arnt-DRE complex by 48% and (2) recombinant human CyP40 alone causes the formation of this complex. In addition, CyP40-interacting proteins appear to be essential for the full CyP40 effect on the AhR gel shift complex. The second aim is to determine the role of β-tubulin in Amt-dependent signaling pathways. From the insect Sf9 cytosol, β-tubulin enriched fraction (F5) was isolated which suppresses the AhR/Arnt/DRE complex formation in a gel shift assay. Tubulin enriched from pig brain had a similar inhibition of the AhR gel shift complex, suggesting that β-tubulin in F5 is likely responsible for the action. Using the TALON resin, β-tubulin was co-precipitated with the baculovirus 6His-Arnt, showing that β-tubulin interacts with Arnt. β-tubulin was examined to decide its role in the hypoxia inducible factor-1α (HIF-1α) signaling which is also Arnt-dependent. Gel shift data using HIF-1α and Arnt showed that F5 suppressed the formation of the HIF-1α/Arnt/HRE complex. Subsequently the Sf9 β-tubulin was cloned and about 95% of its full-length sequence was identified. The amino acid sequence of Sf9 β-tubulin shares high sequence identity with human β-tubulin. Upon transient transfection of a plasmid containing a human β-tubulin cDNA into MGF7 or Hep3B cells, the HRE-driven luciferase activity was clearly suppressed. In conclusion, we have evidence supporting that β-tubulin inhibits the Arnt-dependent signaling and the mechanism may involve the interaction between Arnt and β-tubulin.