Structural Mapping of the EIN2–EIN3 Interaction Core and Its Integration with ENAP1 in Ethylene Signaling

Ethylene regulates diverse developmental processes, yet the molecular function of its central regulator, ETHYLENE INSENSITIVE 2 (EIN2), has remained unclear. Although EIN2 nuclear import is mediated by the Importin-α/β pathway, the molecular events initiated by EIN2 after nuclear entry were unknown. Here we show that EIN2 directly engages the transcription factor EIN3, establishing a mechanistic link between EIN2 nuclear accumulation and transcriptional activity. Microscale thermophoresis, yeast two-hybrid analysis and in planta FLIM-FRET consistently support this interaction. Domain mapping identifies EIN3 residues 86-173 as the core EIN2-binding region, and structural modeling refines the interface to a conserved segment within residues 86-120 that contacts a conserved region near the N-terminus of the EIN2-CEND fragment. In planta , EIN2 residues 1042-1214 are sufficient for EIN3 binding, revealing multiple interaction-competent surfaces with distinct affinities. The chromatin-associated protein ENAP1 also binds EIN2 and competes with EIN3, indicating a dynamic, concentration-dependent regulatory mechanism rather than a stable ternary complex. These findings define the molecular basis of the EIN2-EIN3 interaction and provide a mechanistic framework for EIN2-dependent transcriptional control in ethylene signaling.