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
Supported by 13-25280S and LQ1601.