Heterotypic Sam-Sam association between Odin-Sam1 and Arap3-Sam: binding affinity and structural insights

摘要

Arap3 is a phosphatidylinositol 3 kinase effector protein that plays a role as GTP-ase activator (GAP) for Arf6 and RhoA. Arap3 contains a sterile alpha motif (Sam) domain that presents high sequence homology with the Sam domain of the EphA2-receptor (EphA2-Sam); both Arap3-Sam and EphA2-Sam are able to associate with the Sam domain of the lipid phosphatase Ship2 (Ship2-Sam).Recently, we have reported on a novel interaction between the first Sam domain of Odin (Odin-Sam1), a protein belonging to the ANKS (ANKyrin repeat and Sam domain containing) family, and EphA2-Sam. In the current work we apply Nuclear Magnetic Resonance (NMR) spectroscopy, Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) to characterize the association between Arap3-Sam and Odin-Sam1. We show that these two Sam domains interact with low micromolar affinity. Moreover, by means of molecular docking techniques, supported by NMR data, we demonstrate that Odin-Sam1 and Arap3-Sam may bind with a topology that is common to several Sam-Sam complexes.The unveiled structural details form the basis for the design of potential peptide-antagonists, that could be used as chemical tools to investigate functional aspects related to heterotypic Arap3-Sam associations.