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 localized at the ER-Golgi network. Previous studies had shown that the biological function of these receptors critically depends on a protein-bound copper center. However, molecular processes and structures controlling assembly and in-tegration of the metal into the functional plant hormone receptor are still unknown. In the previous funding period, we have obtained novel mechanistic insights into these processes and the transmembrane structure of the copper-bound receptor. Specifically, 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 demonstrate that receptors acquire their copper cofactor by two routes - either by direct transfer from soluble chaperones ANTIOXIDANT-1 (ATX1) and COPPER TRANSPORT PROTEIN (CCH), or from copper transporter RESPONSIVE-TO-ANTAGO-NIST-1 (RAN1) preloaded with the transition metal by soluble copper carriers of the ATX1 family. We have also been able to resolve questions regarding the copper stoichiometry and structural constraints of the transmembrane sensor domain of plant ethylene receptor ETR1 by biochemical and biophysical analyses. In the current funding period, we aim to determine structure and dynamics of the different copper pathways to the receptor at the atomic and molecular level. To this end, soluble QTY receptor variants and SMA-copolymer nanodiscs will be used for crystallization of the apo-state and copper-bound holo-state of the receptors. Alternatively, cryo-EM-based structures of receptor and receptor-chaperone complexes will be determined. The structural work will be complemented by computational studies in the Gohlke group (TP A03). Based on our structural approach, the in vitro and in vivo analysis of receptor and chaperone mutations is expected to foster our understanding on how RAN1 and ETR1 exactly bind the soluble chaperones, how precisely ETR1 and RAN1 interact and how monovalent copper is transferred to the receptor target site. Further studies on conformational dynamics related to copper transfer and binding at the receptors by fluorescence spectroscopy and small-angle X-ray scattering will contribute to a comprehensive understanding of dynamics and structures in copper-routing to the ethylene receptor family.
Project leader: Prof. Dr. Georg Groth, ,
Researchers: Dominik Dluhosch M.Sc.,
Buket Uzun M.Sc.,