Structural and thermodynamic characterization of protein-protein interactions involved in TGF-beta signal transduction.

摘要

Transforming Growth Factor beta (TGF-beta) cytokines play central roles in embryogenesis, immunity, and tumour suppression. Signals from these cytokines are propogated through receptors which activate transcription factors called Smads. In addition to binding DNA Smad proteins form regulatory interactions with many cytoplasmic and nuclear proteins. Downregulation of various TGF-beta signalling components is mediated by ubiquitin ligases called Smad Ubiquitin Regulatory Factors (Smurf). The goal of this thesis was to increase understanding of the structural and thermodyanmic basis for Smad and Smurf function.; The Smad2 Mad Homology 2 (MH2) domain binds to many diverse proteins. NMR was used to investigate the structure of one interacting protein, the Smad Binding Domain (SBD) of Smad Anchor for Receptor Activation (SARA). The results indicate that unbound SBD is disordered and forms no stable secondary or tertiary structures. Fluorescence binding studies indicate that no region of SBD dominates the interaction between MH2 and SBD. My results are consistent with a series of hydrophobic patches on the MH2 that are able to recognize disordered regions of proteins. These findings elucidate a mechanism by which a single domain (MH2) can specifically recognize diverse proteins that are unrelated by sequence.; Smurf2 ubiquitinates the TGF-beta receptor complex to target it for degradation. Receptor recognition by Smurf2 occurs through an intermediary protein: Smad7. To probe Smurf2 specificity and recognition of Smad7, the solution structure of a complex between the third WW domain (WW3) of Smurf2 and a peptide from Smad7 containing a PPXY motif was determined. This revealed a novel interaction mode between the WW3 domain and PY motif, which allows Smurf2 to recognize a subset of PY motif containing proteins, including Smad7. This target recognition mode provides a basis for Smurf2 specificity.; Crystal structures of HECT domains from different E3 enzymes suggest that these domains undergo significant conformational change that is important for enzymatic function. To obtain samples of Smurf2 HECT suitable for NMR analysis an iterative screening approach was developed. This approach resulted in significant improvements in NMR spectra and contributes to development of screening methods for NMR. Additionally, these results have increased our understanding of HECT domain dynamics.