Proteinaceous Metal-Binding Eph-Ephrin Tetramerization is Modulated by Copper and Chelators

Eph-Ephrin are powerful signaling molecules that form dimers, tetramers, and tetramer superclusters. The tetramerization mechanism - central to how these receptor-ligand molecules become activated - remains poorly understood. We find EphrinB2 is a proteinaceous chelator that binds to two juxtaposed arginine-rich electropositive pockets on the surface of EphB1 and EphB2 receptors when presented as two precisely aligned EphB-EphrinB2 dimers. This snaps the two low-affinity dimers into a high-affinity circular tetramer. We show EphrinB2 binds copper, consistent with ancestry to Cupredoxins, explaining why metal chelators such as EDTA, EGTA, and 8-hydroxyquinoline are potent tetramer inhibitors: they bind the EphB arginine-rich, copper-like pocket to compete with EphrinB2. Our findings reveal how small molecules, pH, salts, and copper dynamically modulate the kinetics of Eph-Ephrin binding and provide a mechanistic framework for therapeutic targeting of the tetramer.