NMR Structure of a Heterodimeric SAM:SAM Complex: Characterization and Manipulation of EphA2 Binding Reveal New Cellular Functions of SHIP2

Abstract

The sterile alpha motif (SAM) for protein-protein interactions is encountered in over 200 proteins, but the structural bases for its interactions is just becoming clear. Here we solved the structure of the EphA2-SHIP2 SAM:SAM heterodimeric complex by use of NMR restraints from chemical shift perturbations, NOE and RDC experiments. Specific contacts between the protein surfaces differ significantly from a previous model and from other SAM:SAM complexes. Molecular dynamics and docking simulations indicate fluctuations in the complex towards alternate, higher energy conformations. The interface suggests that EphA family members bind to SHIP2 SAM whereas EphB members may not; correspondingly we demonstrate binding of EphA1 but not of EphB2 to SHIP2 SAM. A variant of EphB2 SAM was designed that binds SHIP2. Functional characterization of a mutant EphA2 compromised in SHIP2 binding reveals two previously unrecognized functions of SHIP2 in suppressing ligand-induced activation of EphA2 and in promoting chemotactic cell migration in coordination with the receptor.

Binding and Function of Phosphotyrosines of the Ephrin A2 (EphA2) Receptor Using Synthetic Sterile α Motif (SAM) Domains

“…Adding negative charge to the EphA2 interface (which by itself is dominated by positively charged residues) would be expected to weaken binding of the negatively charged SHIP2 SAM interface. However, our recent refinement of the structure of the complex suggests that the complex can sample alternate configurations (23,40). The equilibrium between these different configurations may be shifted in the EphA2.pY921-and EphA2.pY960-SHIP2 complexes, but assessing this possibility is beyond the scope and interest of the current study.…”

“…Unphosphorylated EphA2 SAM binds SHIP2 SAM (23,31,32); phosphorylation might alter the affinity of this interaction. Unexpectedly, ITC measurements show that both the phosphorylated and unphosphorylated EphA2 SAM domains share a similar affinity for SHIP2 SAM.…”

“…The SAM domains are common protein-protein interaction modules that typically form homo-or heterodimers and are present in a diverse set of proteins (19 -21). The structures of several SAM domains have been solved, showing a relatively well conserved topology of five ␣-helices (22,23). The EphA2 SAM domain has three tyrosines, Tyr 921 , Tyr 930 , and Tyr 960 , of which Tyr 921 is absolutely conserved in Eph and many other SAM domains (Fig.…”

“…1). By contrast to most receptor tyrosine kinase phosphorylation sites, which occur in relatively unstructured protein domain linker regions or loops (23,24), the three SAM domain tyrosines are part of the folded * This work was supported, in whole or in part, by National Institutes of Health Grants R01GM092851 and R01CA152371 (to M. B.).…”

“…Biophysical studies in vitro, as reported here, can provide deeper insight and propose models for investigation at the cellular level. Specifically, the EphA2 SAM domain forms a heterodimer with the SAM domain of SH2 domain-containing inositol-5Ј-phosphatase (SHIP2) (23,30,31). Binding of EphA2 SAM to SHIP2 SAM inhibits receptor endocytosis and enhances activation of Eph kinase (31).…”