Structure, Dynamics and Complex Formation of Eukaryotic Transcriptional Regulators

The common scope of this thesis is the application of structural biology and biophysics to obtain novel data on nuclear proteins that modulate DNA transcription. The proper regulation and functioning of these elements are essential to ensure organism development, homeostasis and survival. Provided that the biological mechanisms under study in this work are not directly related, the thesis is presented as two independent projects. Binding Determinants on the Smad Surface and in Smad Partners Smad transcription factors, as the core intracellular components of TGF- β signalling, are involved in a myriad of cellular activities and gene transcription programs and is usually affected in proliferative and autoimmune diseases. For its correct activity, a series of proteins involved in transcription regulation partner with the active Smad complex in the nucleus, conferring response specificity. Smad-interacting proteins bind to either the MH2 domain or the linker region between domains, with the former being the most commonly-reported binding site. Nevertheless, there is a surprising lack of related structural and biophysical data. As a consequence, any new details revealed on how the interactions of Smad2 (and in extension, any other Smad) with its nuclear partners occur at the atomic label and the physico-chemical details governing these interactions can likely have an impact on the basic and applied research. 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, and likely interacting sequences in TRIM33 have been pointed to. Moreover, a tendency for an α-helix secondary structure has been identified by NMR. The elements required for complex crystallisation have been obtained with the aim to obtain structural data regarding interaction surfaces on the Smad2 MH2 domain. If specificity-defining sites can be determined, it opens a door for drug design targeting Smad2 specifically, even the blocking of specific interactions. Such specificity is certainly of interest in oncologic treatments. Mutation of Residues in the Third β-strand of the FBP28 WW2 Domain Results in Alternative Folding Trajectories The main secondary structure typology of amyloid plaques is anti-parallel β-sheet. This structure is acquired following an alternative folding trajectory. The second WW domain of Formin binding protein 28 (FBP28) has been long used as a β-sheet folding model. Moreover, its folding kinetics and its propensity to form amyloid-like fibrils are the reason for an ongoing controversy regarding its folding mechanism The working hypothesis to explain the folding mechanism behind the appearance of the intermediate species is that the formation of contacts defining the β-hairpin2 occurs at a slower rate than 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. In L455D, the orientation of the D455 side chain suggests the existence of a novel water-mediated bond with Y450. L455W presents the peculiarity of a solvent-exposed W455 side-chain yet not heavily affecting the overall domain stability. E456Y, where the inclusion of the bulky Y456 successfully manages to force a rearrangement of surrounding side chains by π-π stacking with W436. 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