MS04-P06 Crystal structures of chimeric sh3 domains forming 3d-domain swapping in presence of ureaAmyloid forming proteins are the main cause of many neurodegenerative diseases. However, the mechanism of their formation at molecular level is not known. Tackling this task is difficult because the limitations to obtain high resolution structural information. We have constructed several chimeric proteins of the SH3 domains of the tyrosine kinases c-Src, Abl and Fyn as model proteins to tackle these studies. In those SH3 domains the RT and n-Src loops of each domain have been interchanged. Previous studies have demonstrated that some of these chimeric proteins are able to form oligomers by 3D domain-swapping (3D-DS), and the interchage of secondary structure elements results in the formation of dimers1,2. Structural studies performed with proteins under conditions near denaturation might help to understand the initial steps which drives misfolding in proteins. Considering this approach, we have studied the binding of the chemical denaturant urea to several chimeric constructions of the c-Src SH3 domain. We have crystallized these proteins in presence of different concentrations of urea to determine its interaction. Previously, we have analyzed the unfolding of the chimeric proteins in presence of urea at pH 5.0 and pH 7.0 by means of the intrinsic fluorescence of the protein. Some chimeras of the Src-SH3 where the RT and n-Src loops have been interchanged by those present in the Fyn SH3 domain (SF chimeras) does not fit a two-state model, which might indicate the presence of some intermediate in the unfolding process as it has been described in other SH3 domains3. Most of the crystals grown in presence of urea diffracted to atomic resolution, which allowed us to model the urea molecules interacting with residues at the surface of the protein. In all the structures solved the urea molecules were modelled replacing first-shell water molecules, which would modify the water-water hydrogen bond network. We have analyzed the results considering structural changes due to urea binding: in the position of the protein main chain; rotamer side chain changes; and, displacement of buried water molecules. In some structures a urea molecule is placed interacting with Glu106, which is critical in the nucleation of the folding process of the c-Src-SH3 domain by forming a hydrogen bond with Ser123.
1. Camara-Artigas, A., (2016). Archives of Biochemistry and Biophysics, 602, p. 116-126.
2. Bacarizo, J., et al., (2014). PLoS One, 9(12), p. e113-224.
3. Dasgupta, A., et al., (2014). The journal of Physical Chemistry. 118(24) p. 6380-6392.Keywords: 3-D domain swapping, SH3-domain, Unfolding