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

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

doi:10.1039/d2ra00604a

Cited by 5 publications

(6 citation statements)

References 37 publications

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“…While the structure of the cytoplasmic part of the ETR1 receptor has been characterized experimentally and also by homology modeling [8][9][10], only recently we described the first ab initio model of the transmembrane domain of the protein, showing that two copper ions in the +1 oxidation state are bound with high affinity to the receptor [11]. This structural model is supported by a recent site-directed spin labeling and electron paramagnetic resonance spectroscopy study [12]. Although residues C65/H69 and copper are required for ethylene binding, and it is generally assumed that ethylene interacts with the copper ion, these interactions have yet to be directly observed.…”

Section: Introductionmentioning

confidence: 58%

Spectroscopic and QM/MM studies of the Cu(I) binding site of the plant ethylene receptor ETR1

Cutsail¹,

Schott‐Verdugo²,

Müller³

et al. 2022

Biophysical Journal

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Section: Introductionmentioning

confidence: 58%

Spectroscopic and QM/MM studies of the Cu(I) binding site of the plant ethylene receptor ETR1

Cutsail¹,

Schott‐Verdugo²,

Müller³

et al. 2022

Biophysical Journal

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“…While the structure of the cytoplasmic part of the ETR1 receptor has been characterized experimentally and also by homology modeling [8–10], only recently we described the first ab initio model of the transmembrane domain of the protein, showing that two copper ions in the +1 oxidation state are bound with high affinity to the receptor [11]. This structural model is supported by a recent site-directed spin labeling and electron paramagnetic resonance spectroscopy study [12]. Although residues C65/H69 and copper are required for ethylene binding, and it is generally assumed that ethylene interacts with the copper ion, these interactions have yet to be directly observed.…”

Section: Introductionmentioning

confidence: 60%

Spectroscopic and QM/MM studies of the Cu(I) binding site of the plant ethylene receptor ETR1

Cutsail

Schott‐Verdugo

Müller

et al. 2022

Preprint

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Herein, we present the first spectroscopic characterization of the Cu(I) active site of the plant ethylene receptor ETR1. The X-ray absorption (XAS) and extended X-ray absorption fine structure (EXAFS) spectroscopy presented here establish that ETR1 has a low-coordinate Cu(I) site. The EXAFS resolves a mixed first coordination sphere of N/O and S scatterers at distances consistent with potential histidine and cysteine residues. This finding agrees with the coordination of residues C65 and H69 to the Cu(I) site, which are critical for ethylene activity and well-conserved. Further, the Cu K-edge XAS and EXAFS of ETR1 exhibit spectroscopic changes upon addition of ethylene that are attributed to modifications in the Cu(I) coordination environment, suggestive of ethylene binding. Results from umbrella sampling simulations of the proposed ethylene binding helix of ETR1 at a mixed quantum mechanics/molecular mechanics (QM/MM) level agree with the EXAFS fit distance changes upon ethylene binding, particularly in the increase of the distance between H69 and Cu(I), and yield binding energetics comparable to experimental dissociation constants. The observed changes in the copper coordination environment might be the triggering signal for the transmission of the ethylene response.

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“…Mutation of Asp25 to Ala (D25A) abolishes ethylene binding by the receptor when analyzed in a heterologous yeast expression system and also confers dominant ethylene insensitivity when expressed in Arabidopsis ( 20 ). Computational modeling places Asp25 of helix I in proximity to Cys65 and His69 of helix II ( 24 , 27 ), suggesting that it could play a role in coordinating the copper cofactor. Interestingly, although Asp is found in 93.55% of the sequences examined, in some cases (3.67%) it is substituted by an Asn residue, most commonly in cyanobacteria but also in several plants, Pyrus communis (Pear) and Cajanus cajan (Pigeon pea).…”

Section: Resultsmentioning

confidence: 99%

“…The etr1-1 mutation confers dominant ethylene insensitivity on plants because of this inability to perceive the ethylene signal. Additional missense mutations in the receptor have further refined our understanding of ethylene binding and signal transduction ( 20 ), as has computational modeling and tryptophan scanning mutagenesis ( 24 , 27 ).…”

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confidence: 99%

Basis for high-affinity ethylene binding by the ethylene receptor ETR1 of Arabidopsis

Azhar

Abbas

Aman

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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The gaseous hormone ethylene is perceived in plants by membrane-bound receptors, the best studied of these being ETR1 from Arabidopsis. Ethylene receptors can mediate a response to ethylene concentrations at less than one part per billion; however, the mechanistic basis for such high-affinity ligand binding has remained elusive. Here we identify an Asp residue within the ETR1 transmembrane domain that plays a critical role in ethylene binding. Site-directed mutation of the Asp to Asn results in a functional receptor that has a reduced affinity for ethylene, but still mediates ethylene responses in planta. The Asp residue is highly conserved among ethylene receptor-like proteins in plants and bacteria, but Asn variants exist, pointing to the physiological relevance of modulating ethylene-binding kinetics. Our results also support a bifunctional role for the Asp residue in forming a polar bridge to a conserved Lys residue in the receptor to mediate changes in signaling output. We propose a new structural model for the mechanism of ethylene binding and signal transduction, one with similarities to that found in a mammalian olfactory receptor.

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

Abstract: The ethylene receptor 1 transmembrane domain was site-directedly spin labelled to obtain distance restraints.

Cited by 5 publications

References 37 publications

Spectroscopic and QM/MM studies of the Cu(I) binding site of the plant ethylene receptor ETR1

Spectroscopic and QM/MM studies of the Cu(I) binding site of the plant ethylene receptor ETR1

Spectroscopic and QM/MM studies of the Cu(I) binding site of the plant ethylene receptor ETR1

Basis for high-affinity ethylene binding by the ethylene receptor ETR1 of Arabidopsis

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