Interactions of SH3 domains in adaptor proteins

This thesis explores the interactions between adaptor proteins containing Src homology domain 3 (SH3) domains and their potential binding partners. Biochemical, biophysical and in vivo studies were used to investigate the structure, function and dynamics of two SH3-containing adaptors CIN85 and Grb2 in apo and ligand bound states. The project aimed to understand the role of CIN85 and Grb2 as key modulators in signalling cascades and the significance of their interactions with other proteins at the physiological level. The interaction between CIN85 and Cbl/Cbl peptides was investigated. The global architecture and dynamics of CIN85 in apo and Cbl bound states were reported for the first time. NMR studies reported that all three SH3 domains of CIN85 binds to Cbl derived peptides in CIN85ABC, however the interaction have a complex mechanism due to existence of complex dynamics between the SH3 domains and linker regions. However, the study identifies that the SH3 domains in CIN85 works in cooperation with each other with the linkers contributing towards the dynamics and global conformation of CIN85 in the presence and absence of Cbl, which is yet to be understood. The interaction between CIN85 and ubiquitin was investigated by in vitro and in vivo approaches. Ubiquitin binds to all three isolated SH3 domains of CIN85 with diverse affinities in vitro. However the complex between CIN85ABC and ubiquitin was weak and physiologically insignificant. No interactions were observed in cell studies between CIN85ABC and CIN85FL with ubiquitin. Finally, the complex between Grb2 SH3C domain with FGF receptor 2 peptide shows a novel mode of interaction investigated using NMR and docking studies. This work shows that SH3-adaptors have discrete preference for ligands and cannot be generalised as linkers in signalling cascades. Each SH3 domain in adaptors displays their function and subtle regulatory character that is interwoven with their structure and dynamics