13C, 15N and 1H resonance assignments of receiver domain of ethylene receptor ETR1

Abstract

Ethylene plays versatile functions in regulating plant physiology. Although the high affinity ethylene receptor and its downstream regulators have been identified, the molecular recognition of the receptor interacting domains remains to be established. It has been speculated that the cytoplasmic signaling of the ethylene receptor is a two-component regulatory system involving the conserved receiver domain (RD). Here, we report the NMR chemical shift assignments for RD from Arabidopsis thaliana ethylene receptor ETR1. Nearly complete backbone and side-chain assignments were achieved at pH 6.0 and 25 °C. The assignments and backbone dynamics revealed the secondary structure and showed that ETR1-RD is a monomer in solution. These results will make it possible to monitor downstream binding partners and elucidates our understanding of phosphotransfer in the plant two-component regulatory system in the ethylene signaling pathway.

NMR Study Reveals the Receiver Domain of Arabidopsis ETHYLENE RESPONSE1 Ethylene Receptor as an Atypical Type Response Regulator

“…Fewer studies related to the properties of the ETR1-RD in solution have been performed. In addition to an X-ray diffraction study, the oligomerization status of the ETR1-RD in solution has been preliminarily investigated by NMR, and the results suggested that the RD was a monomer in solution [ 34 ]. Here, SDS-PAGE and FPLC analysis revealed that the ETR1-RD had a molecular weight of ~15 kDa in solution, further supporting the conclusion that it existed as a monomer in solution ( Fig 2 ).…”

“…These properties indicate that the ETR1-RD is a highly folded domain with high conformational stability. The assignment was finished by adopting conventional backbone assignment strategy [ 34 ]. Based on this strategy, we were able to obtain the chemical shifts for all of the backbone nuclei.…”

“…The cDNA fragment that encodes the receiver domain (RD) of Arabidopsis thaliana ETR1 (residues 605–738) was amplified by polymerase chain reaction (PCR) and cloned into the pET6H vector (modified from the pET11d vector, Novagen) with the N-terminal 10-residue His-tag M(H) 6 AMG [ 34 ]. The construct contains a total 144 residues with molecular weight of 16,228 Da.…”

Identification of Regions in the Receiver Domain of the ETHYLENE RESPONSE1 Ethylene Receptor of Arabidopsis Important for Functional Divergence

“…The crystal structure of the ETR1 receiver domain has been determined, revealing a homodimer that is stabilized by the C terminus, which forms an extended b sheet (Müller-Dieckmann et al, 1999). However, in solution, dimerization may not occur (Hung et al, 2015), and a more recent structural model of the cytosolic domains of ETR1 indicates that the receiver domain of each monomer in a receptor may not come close enough to dimerize (Mayerhofer et al, 2015). Nonetheless, the modeling shows that the C-terminal tail could have a role in interactions with other proteins, including the receiver domains of adjacent receptor dimers (Mayerhofer et al, 2015).…”

Structural Aspects of Multistep Phosphorelay-Mediated Signaling in Plants

“…In this context, it is noteworthy that phosphorylation-dependent dimerization of bacterial RDs has been shown to activate the DNA-binding domain of RRs (for a review, see Stock et al, 2000). However, ETR1 RD forms a homodimer in its unphosphorylated state (Muller-Dieckmann et al, 1999), and the recently published NMR spectra demonstrate that ETR1 RD is a monomer in solution (Hung et al, 2015). These data call into question the hypothesis stating the involvement of ETR1 RD s in phosphorylation-dependent formation of receptor clusters.…”