Ethylene-regulated dimeric ETR1 kinase reveals multi-site EIN2 docking targeted by peptides that delay climacteric ripening.

Abstract

Ethylene receptors are ER membrane histidine kinases that repress ethylene responses, and the central transducer ETHYLENE INSENSITIVE 2 (EIN2) docks to receptors through its C-terminal tail. Here we express full-length <i>Arabidopsis</i> ETR1 in <i>Pichia pastoris</i> and show that ethylene selectively suppresses the autokinase activity of the dimer, establishing a ligand-responsive receptor preparation. Bio-layer interferometry shows that EIN2-C binds both full-length ETR1 and an isolated transmembrane-GAF module with low-micromolar affinity. NMR mapping of EIN2-C titrated with ETR1-GAF identifies three discrete docking segments, including the known NOP-1 octapeptide and two additional peptides (DP1 and DP2). Synthetic peptides engage ETR1-GAF with distinct kinetics and affinities, and docking models place DP2 in a hydrophobic pocket at the GAF dimer interface. When applied as surface coatings to tomato and mango, these peptides delay ripening in proportion to binding strength. Multi-site competitive inhibition at the EIN2-ETR1 interface thus provides a tunable strategy to modulate ethylene signaling and extend climacteric fruit shelf life.