Structural study of the intracellular domains of the ethylene receptor ETR1 from Arabidopsis thaliana

Zuzana Jaseňáková, Agnieszka Szmitkowska, Blanka Pekarová, Jan Hejátko, Lukáš Žídek

 Central European Institute of Technology (CEITEC) and National Centre for Biomolecular Research, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic

Ethylene acts as a gaseous hormone that controls numerous aspects of plant growth and development. In Arabidopsis, the ethylene signaling cascade is initiated upon ethylene binding to the ethylene receptors ETR1, ERS1, ETR2, ERS2 and EIN4, localized at the endoplasmic reticulum, and proceeds via the pathway containing the Raf-like serin/threonine (S/T) kinase CTR1 acting upstream of EIN2 and EIN3. However, ETR1 receptor possesses all sequence motifs of canonical histidine kinase (HK) domains including HK activity, necessary for the signal transduction via parallel multistep phosphorely (MSP) pathway, mediating a wide spectrum of responses including plant hormones cytokinins. Accumulating evidence suggests a role of ETR1 in integrating ethylene recognition with MSP mediated signaling.

The main objective of our work is the structural determination and understanding of ETR1 features which will help us to elucidate the structural aspects and HK activity of ETR1 in the ethylene/MSP cross-talk. We  prepared 15N- labelled protein samples of ETR1 HK and receiver domain (RD) at high concentrations necessary for nuclear magnetic resonance (NMR) spectroscopy. Titration trials with divalent ions (Mg2+, Mn2+) were performed in order to determine whether the ions affect the ETR1RD structure. Minor changes were observed in the presence of Mg2+ in several residues, but in region distant from the phosphorylation site.

Furthermore, we investigated possible interactions between the receiver and HK domains. Our preliminary results from NMR measurements indicate complex formation and undergoing structural changes on RD including its phosphorylation site. Because of the large complex size of approximate 75 kDa, expression and purification must be optimized for structural studies applicable to large proteins (segmental labelling, deuteration).

Supported by 13-25280S and LQ1601.