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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:743840 |
| Date | January 2018 |
| Creators | Ning, Jia |
| Contributors | Walkinshaw, Malcolm ; Ball, Kathryn |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/31145 |
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