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

Eph-Ephrin are powerful membrane-anchored signaling molecules that form dimers, tetramers, and tetramer superclusters at sites of cell-cell contact. The tetramerization mechanism - central to how these receptor-ligand molecules become activated - has remained poorly understood. We find EphrinB2 functions as a proteinaceous chelator that binds to two juxtaposed arginine-rich electropositive pockets on the surface of EphB receptors when presented as two precisely aligned EphB-EphrinB2 dimers. This snaps the two low-affinity dimers into the very high-affinity circular tetramer, activating the molecules to begin transducing their forward and reverse signals into the cells they are expressed on. Contrary to assumptions that Ephrins do not interact with metals, we show EphrinB2 does indeed bind copper ions at very low micromolar concentrations, a feature consistent with Ephrin ancestry to Cupredoxins. This explains why metal chelators such as EDTA, EGTA, and 8-hydroxyquinoline are potent tetramer inhibitors, as they can bind the EphB arginine-rich copper-like electropositive pocket to compete with EphrinB2 binding. 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.