NMR Studies of Heterotypic Sam-Sam Domain Associations Involving EphA2 Receptor

EphA2 is a receptor tyrosine kinase that is over-expressed in many tumors and exhibits several pro-cancer activities. The process of receptor endocytosis and the consequent degradation have recently raised an increasing interest as possible routes to reduce tumor malignancy [1]. In malignant breast cancer cells the lipid phosphatase Ship2 is recruited at the receptor site by means of a heterotypic Sam (Sterile alpha motif domain)-Sam association and inhibits EphA2 endocytosis [1]. NMR and ITC studies of the interaction between Ship2-Sam and EphA2-Sam have already been performed and showed that the two domains bind with a KD value in the low micromolar range and adopt a ML (Mid Loop)/EH (End Helix) interaction model characteristic of several Sam-Sam associations [2]. Sam domains from Odin, a member of the ANKS (Ankyrin repeat and Sam domain containing) family proteins, play also a prominent inhibitory role of EphA2 endocytosis [3]. Odin contains two tandem Sam domains (Sam1 and Sam2); Odin-Sam1 shows a ~ 70% sequence homology with Ship2-Sam. We have carried out NMR and SPR experiments and demonstrated that Odin-Sam1 tightly binds to EphA2-Sam. Besides, we have identified by means of chemical shift perturbation experiments, Odin-Sam1 and EphA2-Sam reciprocal binding interfaces and shown that Ship2-Sam and Odin-Sam1 adopt a similar binding mode to EphA2-Sam. Our research further highlights the key structural features that are important for Sam-Sam associations and provides novel routes for the design of inhibitors of these interactions with potential ability to regulate EphA2 receptor endocytosis.