Basis for high-affinity ethylene binding by the ethylene receptor ETR1 of Arabidopsis.

The gaseous hormone ethylene is perceived in plants by membrane-bound receptors, the best studied of these being ETR1 from Arabidopsis. Ethylene receptors can mediate a response to ethylene concentrations at less than one part per billion; however, the mechanistic basis for such high-affinity ligand binding has remained elusive. Here we identify an Asp residue within the ETR1 transmembrane domain that plays a critical role in ethylene binding. Site-directed mutation of the Asp to Asn results in a functional receptor that has a reduced affinity for ethylene, but still mediates ethylene responses in planta. The Asp residue is highly conserved among ethylene receptor-like proteins in plants and bacteria, but Asn variants exist, pointing to the physiological relevance of modulating ethylene-binding kinetics. Our results also support a bifunctional role for the Asp residue in forming a polar bridge to a conserved Lys residue in the receptor to mediate changes in signaling output. We propose a new structural model for the mechanism of ethylene binding and signal transduction, one with similarities to that found in a mammalian olfactory receptor.

Mapping the helix arrangement of the reconstituted ETR1 ethylene receptor transmembrane domain by EPR spectroscopy.

The plant ethylene receptor ETR1 is a key player in the perception of the phytohormone and subsequent downstream ethylene signal transmission, crucial for processes such as ripening, senescence and abscission. However, to date, there is sparse structural knowledge about the transmembrane sensor domain (TMD) of ETR1 that is responsible for the binding of the plant hormone and initiates the downstream signal transmission. Sequence information and ab initio modelling suggest that the TMD consists of three transmembrane helices. Here, we combined site-directed spin labelling with electron paramagnetic resonance spectroscopy and obtained distance restraints for liposome-reconstituted ETR1_TMD on the orientation and arrangement of the transmembrane helices. We used these data to scrutinize different computational structure predictions of the TMD.

Soluble and membrane-bound protein carrier mediate direct copper transport to the ethylene receptor family.

The plant hormone ethylene is a key regulator of plant growth, development and stress adaption. Ethylene perception and response are mediated by a family of integral membrane receptors (ETRs) localized at the ER-Golgi network. The biological function of these receptors relies on a protein-bound copper cofactor. Nonetheless, molecular processes and structures controlling assembly and integration of the metal into the functional plant hormone receptor are still unknown. Here, we have explored the molecular pathways of copper transfer from the plant cytosol to the ethylene receptor family by analyzing protein-protein interactions of receptors with soluble and membrane-bound plant copper carriers. Our results suggest that receptors primarily acquire their metal cofactor from copper transporter RESPONSIVE-TO-ANTAGONIST-1 (RAN1) which has been loaded with the transition metal beforehand by soluble copper carriers of the ATX1-family. In addition, we found evidence for a direct interaction of ETRs with soluble chaperones ANTIOXIDANT-1 (ATX1) and COPPER TRANSPORT PROTEIN (CCH) raising the possibility of a direct copper exchange between soluble chaperones and receptors.