Mapping the helix arrangement of the reconstituted ETR1 ethylene receptor transmembrane domain by EPR spectroscopy.

Anandi Kugele,Lena Müller,Holger Gohlke,Malte Drescher,Stephan Schott-Verdugo,Buket Uzun,Georg Groth

doi:10.1039/d2ra00604a

Abstract

The plant ethylene receptor ETR1 is a key player in the perception of the phytohormone and subsequent downstream ethylene signal transmission, crucial for processes such as ripening, senescence and abscission. However, to date, there is sparse structural knowledge about the transmembrane sensor domain (TMD) of ETR1 that is responsible for the binding of the plant hormone and initiates the downstream signal transmission. Sequence information and ab initio modelling suggest that the TMD consists of three transmembrane helices. Here, we combined site-directed spin labelling with electron paramagnetic resonance spectroscopy and obtained distance restraints for liposome-reconstituted ETR1_TMD on the orientation and arrangement of the transmembrane helices. We used these data to scrutinize different computational structure predictions of the TMD.

Highlights

Plant hormones are the key players in integrating developmental signals and responses to the environment
To obtain suitable ETR1_TMD constructs for thiol-mediated spin labelling, native cysteines were replaced in the A. thaliana ETR1 receptor by serine residues (ETR1_TMD_C4S/C6S/ C65S/C99S, referred to as ETR1_DC in the following)
New cysteines for site-directed spin labelling (SDSL) were installed at strategically positioned sites (Fig. 1C, 3C and S1, Electronic supplementary information (ESI)†)

Summary

Introduction

Plant hormones (phytohormones) are the key players in integrating developmental signals and responses to the environment. To date, there is sparse structural knowledge about the transmembrane sensor domain (TMD) of ETR1 that is responsible for the binding of the plant hormone and initiates the downstream signal transmission. We combined site-directed spin labelling with electron paramagnetic resonance spectroscopy and obtained distance restraints for liposome-reconstituted ETR1_TMD on the orientation and arrangement of the transmembrane helices.

Results

Conclusion