Resumen de Structure, Dynamics and Complex Formation of Eukaryotic Transcriptional Regulators
Smad transcription factors are the core intracellular components of TGF-β signalling. A series of proteins involved in transcription regulation partner with the active Smad complex in the nucleus, conferring response specificity. There is a surprising lack of structural and biophysical data on Smad MH2 domain – ligand complexes and binding sites. The Smad2 MH2 domain and Smad-interacting proteins FoxH1, NCOA6 and TRIM33 have been studied in this thesis.
The sequence regions in transcription factor FoxH1 and co-activator NCOA6 that effectively interact with the Smad2 MH2 domain have been determined, plus a tendency for an α-helix secondary structure. Potential interacting sequences in TRIM33 have been pointed to. The elements required for complex crystallisation have been obtained aiming for structural data on interaction surfaces on the Smad2 MH2 domain. This information can open the door for drug design targeting Smad2 specifically, even the blocking of specific interactions. Such specificity is certainly of interest in oncologic treatments.
Additionally, the folding of FBP28 WW2 domain has been studied. This protein has long been used as a β-sheet folding model and studies has been controversial about its folding mechanisms. Interestingly, it can form amyloid-like fibrils, being of interest also in the biomedical area. The existence of an intermediate folding species has been hypothesised to occur due to slow formation of contacts defining the β-hairpin2 compared to those defining β-hairpin1. To address it, seven FBP28 WW2 single-change mutant domains were designed and NMR was used to obtain their structures. Three mutant domains presented remarkable structural characteristics: L455D, L455W and E456Y that can be linked to intermediate depletion. Additionally, temperature-induced protein denaturing experiments have been performed and the melting temperatures (Tm) values for each FBP28 WW2 mutant domain, obtained.
Experimental data was applied to perform molecular dynamics simulations. These identify additional folding scenarios for L455D and L455W not requiring an intermediate. This indicates that it is in fact slow β-turn2 formation, most likely due to hydrophobic contacts between Y450 and L455, that favour the formation of the folding intermediate implicated in fibril formation.
