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

Abstract

The gaseous plant hormone ethylene, recognized by plant ethylene receptors, plays a pivotal role in various aspects of plant growth and development. ETHYLENE RESPONSE1 (ETR1) is an ethylene receptor isolated from Arabidopsis and has a structure characteristic of prokaryotic two-component histidine kinase (HK) and receiver domain (RD), where the RD structurally resembles bacteria response regulators (RRs). The ETR1 HK domain has autophosphorylation activity, and little is known if the HK can transfer the phosphoryl group to the RD for receptor signaling. Unveiling the correlation of the receptor structure and phosphorylation status would advance the studies towards the underlying mechanisms of ETR1 receptor signaling. In this study, using the nuclear magnetic resonance technique, our data suggested that the ETR1-RD is monomeric in solution and the rigid structure of the RD prevents the conserved aspartate residue phosphorylation. Comparing the backbone dynamics with other RRs, we propose that backbone flexibility is critical to the RR phosphorylation. Besides the limited flexibility, ETR1-RD has a unique γ loop conformation of opposite orientation, which makes ETR1-RD unfavorable for phosphorylation. These two features explain why ETR1-RD cannot be phosphorylated and is classified as an atypical type RR. As a control, phosphorylation of the ETR1-RD was also impaired when the sequence was swapped to the fragment of the bacterial typical type RR, CheY. Here, we suggest a molecule insight that the ETR1-RD already exists as an active formation and executes its function through binding with the downstream factors without phosphorylation.

Conformational dynamics are a key factor in signaling mediated by the receiver domain of a sensor histidine kinase from Arabidopsis thaliana

“…also substantially different from another plant hybrid HKs, ETR1 RD , crystallizing with the inactive closed conformation of the ␤3-␣3 loop and exhibiting negligible conformational exchange in solution. This is in line with the recently described phosphorylation-independent mode of action of ETR1 RD (22).…”

“…The loop adopts different orientations in the crystal structure (21) from those observed in the CKI1 RD X-ray structures. Furthermore, no severe NMR line-broadening was reported, suggesting its lower conformational flexibility in comparison with RDs of bacterial HKs (22). We confirmed the published results by acquiring CPMG data for ETR1 RD at 950 MHz.…”

ETR1 Integrates Response to Ethylene and Cytokinins into a Single Multistep Phosphorelay Pathway to Control Root Growth

“…However, there is also evidence speaking against the role of ETR1 in directly mediating transphosphorylation through the MSP pathway. In comparison with bacterial and other Arabidopsis RDs, ETR1 RD shows important structural differences (Hung et al, 2016;Pekarova et al, 2016), raising questions about its ability to mediate an ETR1-triggered phosphorelay. Accordingly, the tobacco homolog of ETR1, NtHK2, is able to phosphorylate myelin basic protein but not its own RD (Zhang et al, 2004).…”

Ethylene signaling in plants

“…The cytosolic domains of ETR1 have been structurally characterized (42,43,68). This has led to a model of the ETR1 dimer where the DHp domain of the histidine kinase domain dimerizes with the DHp of the other monomer (Fig.…”

“…The orientation of the receiver domain in relationship to the remainder of the protein is predicted to be different from prokaryotic histidine kinases suggesting that this domain may be diverged in function from prokaryotes (68). Additionally, structural studies show that the γ-loop of ETR1, which is part of the catalytic region of receiver domains, is in a different orientation from characterized prokaryote receiver domains (42,68). No structural information is published characterizing the ethylene-binding domain, but a computational model is available (69).…”