Crystallization of Ethylene Plant Hormone Receptor-Screening for Structure.

The plant hormone ethylene is a key regulator of plant growth, development, and stress adaptation. Many ethylene-related responses, such as abscission, seed germination, or ripening, are of great importance to global agriculture. Ethylene perception and response are mediated by a family of integral membrane receptors (ETRs), which form dimers and higher-order oligomers in their functional state as determined by the binding of Cu(I), a cofactor to their transmembrane helices in the ER-Golgi endomembrane system. The molecular structure and signaling mechanism of the membrane-integral sensor domain are still unknown. In this article, we report on the crystallization of transmembrane (TM) and membrane-adjacent domains of plant ethylene receptors by Lipidic Cubic Phase (LCP) technology using vapor diffusion in meso crystallization. The TM domain of ethylene receptors ETR1 and ETR2, which is expressed in E. coli in high quantities and purity, was successfully crystallized using the LCP approach with different lipids, lipid mixtures, and additives. From our extensive screening of 9216 conditions, crystals were obtained from identical crystallization conditions for ETR1 (aa 1-316) and ETR2 (aa 1-186), diffracting at a medium-high resolution of 2-4 Å. However, data quality was poor and not sufficient for data processing or further structure determination due to rotational blur and high mosaicity. Metal ion loading and inhibitory peptides were explored to improve crystallization. The addition of Zn(II) increased the number of well-formed crystals, while the addition of ripening inhibitory peptide NIP improved crystal morphology. However, despite these improvements, further optimization of crystallization conditions is needed to obtain well-diffracting, highly-ordered crystals for high-resolution structural determination. Overcoming these challenges will represent a major breakthrough in structurally determining plant ethylene receptors and promote an understanding of the molecular mechanisms of ethylene signaling.

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

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.

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

Herein, we present, to our knowledge, 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) spectroscopies 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. Furthermore, 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 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 with experimental dissociation constants. The observed changes in the copper coordination environment might be the triggering signal for the transmission of the ethylene response.

Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects.

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.75 ± 1.22 µM. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

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

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.

Interfering Peptides Targeting Protein-Protein Interactions in the Ethylene Plant Hormone Signaling Pathway as Tools to Delay Plant Senescence.

Interfering peptides (iPs) have been recognized as valuable substances to specifically target protein-protein interactions (PPIs) in senescence and disease. Although the concept of iPs has been validated for several PPIs in medical and pharmaceutical research, little attention so far has been paid to the enormous potential iPs that may provide to target and control plant growth and developmental processes or plant environmental responses. However, recent research on PPIs in the ethylene signaling pathway has identified the synthetic peptide NOP-1 derived from the nuclear localization signal of ethylene regulator EIN2 as an efficient inhibitor of typical ethylene responses such as ripening, aging, and senescence. Biophysical and biochemical studies on purified recombinant proteins of the ethylene receptor family from various plant species demonstrate that the synthetic peptide binds in the nM-µM range at the plant target. Here, we describe methods to evaluate and quantify the effect of the NOP-1 peptide on flower senescence as a typical ethylene response in the intact plant system. This approach will help to systematically advance our technological capability to delay plant ethylene responses and to expand shelf-life or vase life of fruits and flowers.

Novel insights into the transfer routes of the essential copper cofactor to the ethylene plant hormone receptor family.

The plant hormone ethylene is a key regulator of growth, development and stress adaptation at all stages of the plant life cycle. Signal perception and response to the plant hormone are mediated by a family of receptor kinases localized at the ER-Golgi network which gain their high affinity and specificity for the chemically simple ethylene molecule by an essential copper cofactor bound at their transmembrane domain. Transfer of this cofactor from the plant plasma membrane to the ER-localized receptors requires secured cellular transport of the reactive transition metal. In a recent study, we disclosed the transport proteins involved in the copper transfer to the receptors and identified that cytoplasmic chaperones of the ATX1-family and a membrane-bound P-type ATPase are involved in copper routing. Strictly speaking, our data show that receptors can acquire their copper load by different routes and adopt the metal ion from the plasma membrane either by sequential transfer from soluble chaperones of the ATX1-family via the ER-bound copper-transporting ATPase RAN1 or by direct transfer from the soluble chaperones. Here, we have studied the properties of the soluble plant copper chaperone isoforms, ATX1 and CCH, in more detail. Our data support different cellular functions of these isoforms on copper mobilization.

Soluble and membrane-bound protein carrier mediate direct copper transport to the ethylene receptor family.

The plant hormone ethylene is a key regulator of plant growth, development and stress adaption. Ethylene perception and response are mediated by a family of integral membrane receptors (ETRs) localized at the ER-Golgi network. The biological function of these receptors relies on a protein-bound copper cofactor. Nonetheless, molecular processes and structures controlling assembly and integration of the metal into the functional plant hormone receptor are still unknown. Here, we have explored the molecular pathways of copper transfer from the plant cytosol to the ethylene receptor family by analyzing protein-protein interactions of receptors with soluble and membrane-bound plant copper carriers. Our results suggest that receptors primarily acquire their metal cofactor from copper transporter RESPONSIVE-TO-ANTAGONIST-1 (RAN1) which has been loaded with the transition metal beforehand by soluble copper carriers of the ATX1-family. In addition, we found evidence for a direct interaction of ETRs with soluble chaperones ANTIOXIDANT-1 (ATX1) and COPPER TRANSPORT PROTEIN (CCH) raising the possibility of a direct copper exchange between soluble chaperones and receptors.

Molecular Analysis of Protein-Protein Interactions in the Ethylene Pathway in the Different Ethylene Receptor Subfamilies.

Signal perception and transmission of the plant hormone ethylene are mediated by a family of receptor histidine kinases located at the Golgi-ER network. Similar to bacterial and other plant receptor kinases, these receptors work as dimers or higher molecular weight oligomers at the membrane. Sequence analysis and functional studies of different isoforms suggest that the ethylene receptor family is classified into two subfamilies. In Arabidopsis, the type-I subfamily has two members (ETR1 and ERS1) and the type-II subfamily has three members (ETR2, ERS2, and EIN4). Whereas subfamily-I of the Arabidopsis receptors and their interactions with downstream elements in the ethylene pathway has been extensively studied in the past; related information on subfamily-II is sparse. In order to dissect the role of type-II receptors in the ethylene pathway and to decode processes associated with this receptor subfamily on a quantitative molecular level, we have applied biochemical and spectroscopic studies on purified recombinant receptors and downstream elements of the ethylene pathway. To this end, we have expressed purified ETR2 as a prototype of the type-II subfamily, ETR1 for the type-I subfamily and downstream ethylene pathway proteins CTR1 and EIN2. Functional folding of the purified receptors was demonstrated by CD spectroscopy and autokinase assays. Quantitative analysis of protein-protein interactions (PPIs) by microscale thermophoresis (MST) revealed that ETR2 has similar affinities for CTR1 and EIN2 as previously reported for the subfamily-I prototype ETR1 suggesting similar roles in PPI-mediated signal transfer for both subfamilies. We also used in planta fluorescence studies on transiently expressed proteins in Nicotiana benthamiana leaf cells to analyze homo- and heteromer formation of receptors. These studies show that type-II receptors as well as the type-I receptors form homo- and heteromeric complexes at these conditions. Notably, type-II receptor homomers and type-II:type-I heteromers are more stable than type-I homomers as indicated by their lower dissociation constants obtained in microscale thermophoresis studies. The enhanced stability of type-II complexes emphasizes the important role of type-II receptors in the ethylene pathway.

Structural Model of the ETR1 Ethylene Receptor Transmembrane Sensor Domain.

The structure, mechanism of action and copper stoichiometry of the transmembrane sensor domain of the plant ethylene receptor ETR1 and homologs have remained elusive, hampering the understanding on how the perception of the plant hormone ethylene is transformed into a downstream signal. We generated the first structural model of the transmembrane sensor domain of ETR1 by integrating ab initio structure prediction and coevolutionary information. To refine and independently validate the model, we determined protein-related copper stoichiometries on purified receptor preparations and explored the helix arrangement by tryptophan scanning mutagenesis. All-atom molecular dynamics simulations of the dimeric model reveal how ethylene can bind proximal to the copper ions in the receptor, illustrating the initial stages of the ethylene perception process.

The NOP-1 peptide derived from the central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers.

The plant hormone ethylene was identified as important triggering factor and primary regulator of flower senescence in many species. Consequently, application of chemical inhibitors of ethylene biosynthesis and action is used to extend the longevity of ethylene-sensitive flowers. Here, we show that the peptide NOP-1, a biological derived from the nuclear localization signal of ethylene regulator EIN2 tightly binds to the ethylene receptor of carnation plants - a model to study flower senescence. When applied on cut flowers the peptide biological delays petal senescence similar to previously identified and currently used chemical inhibitors, but offers significant advances to these chemicals in biodegradability, sustainability and ecotoxicity. Our bioinformatic analysis of a wide range of ethylene receptors indicates complete sequence conservation of the anticipated NOP-1 binding site in flower species supporting a widespread use of the peptide on flowering ornamentals to delay senescence and decay in cut flowers. We anticipate our innovative approach to extend flower longevity by a new class of biomolecules such as peptides, peptide analogues and peptide mimetics will significantly advance our technological capability to delay flower senescence and expand vase-life of cut flowers in a sustainable and environmentally friendly manner.

Influence of the ethylene-related signal-inhibiting octapeptide NOP-1 on postharvest ripening and quality of 'Golden Delicious' apples.

BACKGROUND: Processes extending the shelf life of climacteric fruit play an important role in terms of a sustainable global food supply. In a previous study, a synthetic octapeptide (NOP-1) was shown to inhibit the interaction between ethylene receptor (ETR) and ethylene insensitive-2 (EIN2), and in consequence delay tomato ripening. We investigated for the first time the effect of NOP-1 on inhibiting the ripening of apples ('Golden Delicious') during postharvest. RESULTS: Using purified recombinant proteins from a bacterial expression system, we demonstrate here that EIN2 also interacts tightly (Kd = 136 ± 29 nmol L-1 ) with the corresponding apple ETR MdETR1. In line with previous binding studies on tomato ETRs, the ripening-delaying peptide NOP-1 clearly binds to the purified apple ETR. An NOP-1 solution (1000 µmol L-1 ) was applied with a brush or microdispenser and compared with apples treated with 1-methylcyclopropene (1-MCP) (SmartFresh™, Agrofresh) applied as gaseous treatment or untreated control fruits. NOP-1 inhibited colour development and chlorophyll degradation during shelf life. These effects were more pronounced with the brush application (surface film) than with microdroplets application (mimicking a sprayable formulation). NOP-1 did not alter ethylene release or respiration rate, whereas 1-MCP expectedly strongly suppressed both. There were no differences in quality parameters evaluated. CONCLUSION: Our study shows that NOP-1 binds to MdETR1 which results in delaying of ethylene-dependent ripening developments of skin colour and chlorophyll. Besides application methods, possible reasons for the weak effect of NOP-1 in comparison with previous tomato experiments could be different receptor affinity and penetration differences. © 2019 Society of Chemical Industry.

Structure of the γ-ε complex of cyanobacterial F1-ATPase reveals a suppression mechanism of the γ subunit on ATP hydrolysis in phototrophs.

F1-ATPase forms the membrane-associated segment of F0F1-ATP synthase - the fundamental enzyme complex in cellular bioenergetics for ATP hydrolysis and synthesis. Here, we report a crystal structure of the central F1 subcomplex, consisting of the rotary shaft γ subunit and the inhibitory ε subunit, from the photosynthetic cyanobacterium Thermosynechococcus elongatus BP-1, at 1.98 Šresolution. In contrast with their homologous bacterial and mitochondrial counterparts, the γ subunits of photosynthetic organisms harbour a unique insertion of 35-40 amino acids. Our structural data reveal that this region forms a ß-hairpin structure along the central stalk. We identified numerous critical hydrogen bonds and electrostatic interactions between residues in the hairpin and the rest of the γ subunit. To elaborate the critical function of this ß-hairpin in inhibiting ATP hydrolysis, the corresponding domain was deleted in the cyanobacterial F1 subcomplex. Biochemical analyses of the corresponding α3ß3γ complex confirm that the clinch of the hairpin structure plays a critical role and accounts for a significant interaction in the α3ß3 complex to induce ADP inhibition during ATP hydrolysis. In addition, we found that truncating the ß-hairpin insertion structure resulted in a marked impairment of the interaction with the ε subunit, which binds to the opposite side of the γ subunit from the ß-hairpin structure. Combined with structural analyses, our work provides experimental evidence supporting the molecular principle of how the insertion region of the γ subunit suppresses F1 rotation during ATP hydrolysis.

Targeting HSP90 dimerization via the C terminus is effective in imatinib-resistant CML and lacks the heat shock response.

Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38-) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.

Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1.

Synthetic peptides derived from ethylene-insensitive protein 2 (EIN2), a central regulator of ethylene signalling, were recently shown to delay fruit ripening by interrupting protein-protein interactions in the ethylene signalling pathway. Here, we show that the inhibitory peptide NOP-1 binds to the GAF domain of ETR1 - the prototype of the plant ethylene receptor family. Site-directed mutagenesis and computational studies reveal the peptide interaction site and a plausible molecular mechanism for the ripening inhibition.

Novel Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for Ripening Control.

Ethylene signaling is decisive for many plant developmental processes. Among these, control of senescence, abscission and fruit ripening are of fundamental relevance for global agriculture. Consequently, detailed knowledge of the signaling network along with the molecular processes of signal perception and transfer are expected to have high impact on future food production and agriculture. Recent advances in ethylene research have demonstrated that signaling of the plant hormone critically depends on the interaction of the ethylene receptor family with the NRAMP-like membrane protein ETHYLENE INSENSITIVE 2 (EIN2) at the ER membrane, phosphorylation-dependent proteolytic processing of ER-localized EIN2 and subsequent translocation of the cleaved EIN2 C-terminal polypeptide (EIN2-CEND) to the nucleus. EIN2 nuclear transport, but also interaction with the receptors sensing the ethylene signal, both, depend on a nuclear localization signal (NLS) located at the EIN2 C-terminus. Loss of the tight interaction between receptors and EIN2 affects ethylene signaling and impairs plant ethylene responses. Synthetic peptides derived from the NLS sequence interfere with the EIN2-receptor interaction and have utility in controlling plant ethylene responses such as ripening. Here, we report that a synthetic peptide (NOP-1) corresponding to the NLS motif of Arabidopsis EIN2 (aa 1262-1269) efficiently binds to tomato ethylene receptors LeETR4 and NR and delays ripening in the post-harvest phase when applied to the surface of sampled green fruits pre-harvest. In particular, degradation of chlorophylls was delayed by several days, as monitored by optical sensors and confirmed by analytical methods. Similarly, accumulation of ß-carotene and lycopene in the fruit pulp after NOP-1 application was delayed, without having impact on the total pigment concentration in the completely ripe fruits. Likewise, the peptide had no negative effects on fruit quality. Our molecular and phenotypic studies reveal that peptide biologicals could contribute to the development of a novel family of ripening inhibitors and innovative ripening control in climacteric fruit.

Circular Dichroism and Fluorescence Spectroscopy to Study Protein Structure and Protein-Protein Interactions in Ethylene Signaling.

Circular dichroism (CD) spectroscopy is an invaluable technique to analyze secondary structure and functional folding of recombinant purified proteins. CD spectroscopy can also be applied to detect changes in protein secondary structure related to the pH or redox conditions found in different cellular compartments or to the interaction with other molecules. Another biophysical technique to monitor conformational changes and interaction with small molecule ligands or biological macromolecules is protein fluorescence spectroscopy making use of the aromatic amino acid tryptophan as a sensitive intrinsic fluorescent probe. Here, we describe the application of CD and tryptophan fluorescence spectroscopy to study soluble and membrane proteins of the ethylene signaling pathway.

Peptides interfering with protein-protein interactions in the ethylene signaling pathway delay tomato fruit ripening.

The plant hormone ethylene is involved in the regulation of several processes with high importance for agricultural applications, e.g. ripening, aging and senescence. Previous work in our group has identified a small peptide (NOP-1) derived from the nuclear localization signal of the Arabidopsis ethylene regulator ETHYLENE INSENSITIVE-2 (EIN2) C-terminal part as efficient inhibitor of ethylene responses. Here, we show that NOP-1 is also able to efficiently disrupt EIN2-ETR1 complex formation in tomato, indicating that the NOP-1 inhibition mode is conserved across plant species. Surface application of NOP-1 on green tomato fruits delays ripening similar to known inhibitors of ethylene perception (MCP) and ethylene biosynthesis (AVG). Fruits treated with NOP-1 showed similar ethylene production as untreated controls underlining that NOP-1 blocks ethylene signaling by targeting an essential interaction in this pathway, while having no effect on ethylene biosynthesis.

Design and biological testing of peptidic dimerization inhibitors of human Hsp90 that target the C-terminal domain.

BACKGROUND: Small molecules targeting the dimerization interface of the C-terminal domain of Hsp90, a validated target for cancer treatment, have yet to be identified. METHODS: Three peptides were designed with the aim to inhibit the dimerization of Hsp90. Computational and biophysical methods examined the α-helical structure for the three peptides. Based on the Autodisplay technology, a novel flow cytometer dimerization assay was developed to test inhibition of Hsp90 dimerization. Microscale thermophoresis was used to determine the K(D) of the peptides towards the C-terminal domain of Hsp90. RESULTS: MD simulations and CD spectroscopy indicated an α-helical structure for two of the three peptides. By flow cytometer analysis, IC(50) values of 2.08 µM for peptide H2 and 8.96 µM for peptide H3 were determined. Dimer formation of the C-terminal dimerization domain was analyzed by microscale thermophoresis, and a K(D) of 1.29 nM was determined. Furthermore, microscale thermophoresis studies demonstrated a high affinity binding of H2 and H3 to the C-terminal domain, with a K(D) of 1.02 µM and 1.46 µM, respectively. CONCLUSIONS: These results revealed the first peptidic inhibitors of Hsp90 dimerization targeting the C-terminal domain. Furthermore, it has been shown that these peptides bind to the C-terminal domain with a low micromolar affinity. GENERAL SIGNIFICANCE: These results can be used to design and screen for small molecules that inhibit the dimerization of the C-terminal domain of Hsp90, which could open a new route for cancer therapy.

Targeting Plant Ethylene Responses by Controlling Essential Protein-Protein Interactions in the Ethylene Pathway.

The gaseous plant hormone ethylene regulates many processes of high agronomic relevance throughout the life span of plants. A central element in ethylene signaling is the endoplasmic reticulum (ER)-localized membrane protein ethylene insensitive2 (EIN2). Recent studies indicate that in response to ethylene, the extra-membranous C-terminal end of EIN2 is proteolytically processed and translocated from the ER to the nucleus. Here, we report that the conserved nuclear localization signal (NLS) mediating nuclear import of the EIN2 C-terminus provides an important domain for complex formation with ethylene receptor ethylene response1 (ETR1). EIN2 lacking the NLS domain shows strongly reduced affinity for the receptor. Interaction of EIN2 and ETR1 is also blocked by a synthetic peptide of the NLS motif. The corresponding peptide substantially reduces ethylene responses in planta. Our results uncover a novel mechanism and type of inhibitor interfering with ethylene signal transduction and ethylene responses in plants. Disruption of essential protein-protein interactions in the ethylene signaling pathway as shown in our study for the EIN2-ETR1 complex has the potential to guide the development of innovative ethylene antagonists for modern agriculture and horticulture.