Functional ETHYLENE RESPONSE 1 (ETR1) from Pichia pastoris: Dimer-dependent coupling of Cu(I)/ethylene sensing to kinase output

Ethylene is a crucial gaseous phytohormone that regulates diverse aspects of plant growth and development. In Arabidopsis thaliana , perception of ethylene is mediated primarily by ETHYLENE RESPONSE 1 (ETR1), a prototypic membrane protein that couples ligand sensing to signal transduction through a transmembrane copper cofactor-dependent ethylene-binding site and a cytoplasmic histidine kinase domain. Previous work established that ETR1 binds copper and forms covalent homodimers, but the functional significance of its dimeric architecture in signal transduction has remained elusive. In this study, we expressed and purified full-length ETR1 from Pichia pastoris, enabling the systematic dissection of its oligomeric state, copper redox chemistry, and ligand binding effects. Using an in vitro luminescent kinase assay, we demonstrate that the Cu(I)-ethylene complex strongly suppresses ETR1 autophosphorylation, but only when the receptor is in its disulfide-linked dimer form. Monomeric ETR1 generated by reductive cleavage retained basal kinase activity yet was insensitive to ethylene. Furthermore, the ethylene antagonists Ag + and 1-MCP reversed the ligand-induced suppression. Mutational analysis revealed that the H353Q variant lacked kinase activity, while the C65S/H69A double mutant retained basal activity but was resistant to ethylene-induced inhibition, implicating both the catalytic histidine residue and the copper-binding site in the regulatory mechanism. Together, these findings establish that ETR1 functions as a dimer-dependent ethylene sensor, in which covalent dimerization is essential for allosteric coupling between the ligand-binding and kinase domains. This work thus provides a definitive biochemical model for ethylene perception and offers a robust platform for future structural and mechanistic studies of plant membrane-receptor signaling.