Biochem. J. (2012) 444, 261–267 (Printed in Great Britain)
261
doi:10.1042/BJ20111447
Cyanide is an adequate agonist of the plant hormone ethylene for studying
signalling of sensor kinase ETR1 at the molecular level
Melanie M. A. BISSON and Georg GROTH1
Institute for Biochemical Plant Physiology, Heinrich-Heine University, Universitätsstrasse 1, 40225 Düsseldorf, Germany
The plant hormone ethylene is involved in many developmental
processes and responses to environmental stresses in plants.
Although the elements of the signalling cascade and the receptors
operating the ethylene pathway have been identified, a detailed
understanding of the molecular processes related to signal
perception and transfer is still lacking. Analysis of these processes
using purified proteins in physical, structural and functional
studies is complicated by the gaseous character of the plant
hormone. In the present study, we show that cyanide, a πacceptor compound and structural analogue of ethylene, is a
suitable substitute for the plant hormone for in vitro studies with
purified proteins. Recombinant ethylene receptor protein ETR1
(ethylene-resistant 1) showed high level and selective binding
of [14 C]cyanide in the presence of copper, a known cofactor in
ethylene binding. Replacement of Cys65 in the ethylene-binding
domain by serine dramatically reduced binding of radiolabelled
cyanide. In contrast with wild-type ETR1, autokinase activity
of the receptor is not reduced in the ETR1-C65S mutant upon
addition of cyanide. Additionally, protein–protein interaction with
the ethylene signalling protein EIN2 (ethylene-insensitive 2) is
considerably sustained by cyanide in wild-type ETR1, but is not
affected in the mutant. Further evidence for the structural and
functional equivalence of ethylene and cyanide is given by the
fact that the ethylene-responsive antagonist silver, which is known
to allow ligand binding but prevent intrinsic signal transduction,
also allows specific binding of cyanide, but shows no effect on
autokinase activity and ETR1–EIN2 interaction.
INTRODUCTION
that is provided by the copper-transporting P-type ATPase RAN1
(responsive to antagonist 1) [16]. Cys65 and His69 in the second
transmembrane helix of ETR1 function as the metal-co-ordinating
residues, as the substitution of the two residues led to a complete
loss of copper binding [15]. Silver, a metal cation that was shown
to inhibit ethylene signal transduction, but not ethylene binding,
can replace the Cu2 + ion in the ethylene-binding pocket [15,17].
In spite of the knowledge gained about ethylene binding in the last
few years, the molecular structure of the ethylene-binding pocket
and the binding mode of the plant hormone are still unclear.
Likewise, the molecular mechanism related to signal perception
and signal transfer is not yet resolved.
Signal transfer downstream of ETR1 is mediated by the negative
regulator CTR1 (constitutive triple response 1), a Raf-like
serine/threonine kinase. Interaction of the N-terminal domain of
CTR1 with the kinase domain of ETR1 was demonstrated by yeast
two-hybrid studies and in vitro protein–protein binding assays
[18]. Owing to the similarity of CTR1 with MAPKs (mitogenactivated protein kinases), it is supposed that CTR1 marks the
starting point of a multi-step phosphorylation-dependent MAPK
cascade [19–21]. In the absence of ethylene, CTR1 represses
further downstream components of the ethylene signalling
pathway by the inhibition of EIN2, a transmembrane protein
which is also located at the ER network and associates with the
ethylene receptor family [22,23]. EIN2 is an important positive
regulator of ethylene signalling and the only gene of all of the
components involved in the ethylene pathway whose loss-offunction mutation leads to complete ethylene-insensitivity [24].
In the absence of ethylene, EIN2 is rapidly degraded by the
26S proteasome which prevents further ethylene signalling via
EIN3 and EIL (EIN3-like) transcription factors. In the presence
The small unsaturated hydrocarbon plant hormone ethylene has
a wide range of essential functions in plant growth and development, including seed germination, organ senescence and fruit
ripening, as well as leaf and petal abscission. Ethylene is also
well known as a common mediator of responses to biotic or
abiotic stress [1,2]. Owing to its gaseous character, ethylene
stimulation occurs not only between cells and adjacent tissues,
but also between distant parts within the same or neighbouring
plants, which is unique to plant hormones. However, this gaseous
character of the plant hormone also complicates analysis of
ethylene-related responses in biochemical and biophysical studies
on the protein level.
Signal perception and response to the plant hormone ethylene
have been extensively studied in the model plant Arabidopsis
thaliana. These studies have identified a number of mutants
and the related molecular components involved in ethylene
signalling [3,4]. Phenotype, molecular and biochemical analyses
suggest that perception and signal transduction of ethylene are
mediated by a family of integral membrane receptors consisting
of ETR (ethylene-resistant) 1 and its four isoforms ERS (ethyleneresponse sensor) 1, ETR2, ERS2 and EIN (ethylene-insensitive)
4 [5–10]. The receptor proteins show a modular structure
resembling bacterial two-component histidine kinases and form
homo- and hetero-dimers at the ER (endoplasmic reticulum)
membrane [11–13]. ETR1, the prototype of the receptor family,
is characterized by an N-terminal transmembrane domain, an
extramembranous canonical histidine kinase domain and a
C-terminal receiver domain [14]. Binding of ethylene at the Nterminal sensor domain is mediated by a copper cofactor [14,15]
Key words: cyanide, ethylene-insensitive 2 (EIN2), ethylene receptor, ligand binding, plant hormone signalling, protein–protein
interaction.
Abbreviations used: CTR1, constitutive triple response 1; EIN, ethylene-insensitive; ER, endoplasmic reticulum; ERS, ethylene-response sensor; ETR,
ethylene-resistant; MAPK, mitogen-activated protein kinase.
1
To whom correspondence should be addressed (email [email protected]).
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262
M. M. A. Bisson and G. Groth
of ethylene, EIN2 was shown to accumulate in the cell, resulting
in typical ethylene responses [25]. Tight interaction of EIN2 with
the sensor kinase ETR1 was shown to depend on the autokinase
activity of the receptor [23]. This activity in turn is controlled by
ethylene itself, as ethylene was shown to efficiently turn off the
intrinsic kinase activity of purified recombinant ETR1 [26].
Biochemical and biophysical studies on isolated purified
proteins such as quantitative protein–protein interaction studies
or structural studies by NMR or X-ray crystallography have the
potential to resolve the detailed molecular processes of ethylene
binding, signal perception and signal transduction. However,
for these experiments, application of controlled amounts of a
gaseous ligand such as ethylene is laborious and complicated
in contrast with genetic screens on plant seedlings where
this application is straightforward. The non-gaseous ethylene
structural analogue cyanide was shown to efficiently replace
ethylene by inhibiting the autokinase activity of ETR1 [26] and
affecting the ETR1–EIN2 interaction in vitro [23]. Furthermore,
previous in vivo experiments have demonstrated that cyanide
mimics ethylene responses [27] and competes with ethylene
binding in living cyanide-resistant tissues [28]. Nevertheless,
cyanide is a negatively charged molecule and ethylene is an
uncharged non-polar gas, and specific binding of cyanide to the
hydrophobic ethylene-binding pocket of the receptor proteins
has not yet been shown. In the present study, we demonstrate
that cyanide effectively binds to purified recombinant ETR1.
We demonstrate further that the binding of cyanide mimics the
ethylene-induced signal transduction of ETR1 and the interaction
of the receptor with the downstream ethylene signalling protein
EIN2. In addition, we show that replacement of the essential
copper cofactor in the ethylene-binding site by silver, a known
inhibitor of ETR1, still allows ligand binding, but prevents intraand inter-molecular signal transfer of ETR1. These results of our
in vitro studies fit very well with the observed physiological effects
of ethylene. In summary, the present study reveals that cyanide is
an adequate agonist of ethylene for biochemical and biophysical
studies on purified ethylene receptor proteins.
EXPERIMENTAL
Cloning, expression and purification of the Arabidopsis ethylene
receptor protein ETR1
In order to generate an ETR1 mutant deficient in
ethylene binding, Cys65 was replaced by serine by sitedirected mutagenesis using the protocol described in [29].
Plasmid pET-16b-ETR1 was used as a template in the
first amplification reaction. Mutagenic antisense primer
5 -AAGATGAGTTGCTCCAGAAAGAACGATA-3 and T7promotor sense primer 5 -TAATACGACTCACTATAGGG-3
were used to substitute serine for Cys65 . PCR was carried out
in a BioMetra Gene Cycler using Pfu polymerase. Then T7terminator antisense primer 5 -GCTAGTTATTGCTCAGCGG-3
was used in a second PCR to obtain the fragment encoding
full-length ETR1. The amplified fragment was agarose-gelpurified and digested with NdeI and BamHI and ligated into
the equivalent sites of vector pET-15b (Novagen) appropriately
pre-digested with the same restriction enzymes. Plasmid DNA
was sequenced in order to confirm the C65S mutation and the
absence of additional mutations. The resulting plasmid pET-15bETR1-C65S was transformed in Escherichia coli C43(DE3) cells,
and recombinant protein was expressed and purified in the same
way as for wild-type ETR1. Wild-type ETR1 was cloned from
A. thaliana, heterologously expressed in E. coli and purified
as described previously [26,30,31]. Purification was verified by
c The Authors Journal compilation c 2012 Biochemical Society
SDS/PAGE [32], followed by silver staining of the gel [33]. As
described in [26], only a single protein band was detected on
the silver-stained protein gel. Identity of the protein band was
verified by antibodies directed against ETR1 and by antibodies
directed against the N-terminal histidine tag as described in
[26]. Moreover, purity and identity of the recombinant protein
was verified by MS, which confirmed that the purified
protein band corresponds to ETR1 and revealed that no additional
contaminating proteins from the expression host are present in
the purified sample (Supplementary Figure S1 at http://www.
BiochemJ.org/bj/444/bj4440261add.htm). The amount of purified
recombinant proteins was determined using the bicinchoninic acid
assay (Thermo Scientific).
Binding of [14 C]cyanide to recombinant receptor proteins
For binding assays, purified recombinant wild-type and mutant
ETR1 were dissolved in a buffer containing 50 mM Tris/HCl
(pH 8), 100 mM potassium chloride, 0.1 % β-dodecylmaltoside
and 0.002 % PMSF at a final concentration of 1 mg/ml.
Background readings were obtained from samples containing only
buffer but no protein. Samples were pre-incubated with 200 μM
copper chloride or 300 μM silver nitrate for 15 min to provide the
metal cofactor for ethylene binding to the recombinant proteins,
except for controls where cations were omitted to avoid functional
ethylene binding. Potassium [12 C]cyanide was mixed with 4 μCi
of [14 C]cyanide (Hartmann Analytic) to a final concentration of
280 μM, a concentration corresponding to the dissociation
constant (K d ) of purified ETR1 for cyanide. This pre-mixture
was added to the protein, and samples were incubated for 20 min
at room temperature. Non-bound radioactivity was removed by
gel filtration on PD-10 columns (GE Healthcare). Radioactivity
incorporated into purified recombinant ETR1 was quantified
by a liquid-scintillation counter (Beckman LS Analyzer) in
c.p.m. In order to demonstrate specific and saturable binding of
cyanide to the receptor, samples were pre-incubated with 100or 1000-fold molar excess of non-labelled ligand ([12 C]cyanide),
before the radiolabelled cyanide was added. A constant
[12 C]cyanide/[14 C]cyanide ratio and cyanide concentrations up
to 10 mM were used in the binding assays. The apparent K d
value of ETR1 for cyanide was determined from the radioactivity
(detected c.p.m.) recorded at different cyanide concentrations by
non-linear least-squares fitting of the experimental data to a model
that assumes a single class of ethylene-binding sites in the receptor
by the program GraFit (Erithacus Software).
Autokinase activity of recombinant receptor proteins
Kinase activity of purified receptor proteins was analysed using
the method described in [26]. Briefly, recombinant receptor
proteins were dissolved in 50 mM Tris/HCl (pH 8), 100 mM
potassium chloride, 10 mM manganese chloride and 0.1 %
β-dodecylmaltoside and pre-incubated with 200 μM copper
chloride. Kinase activity of the recombinant proteins was tested
by the addition of radiolabelled [γ -32 P]ATP. In experiments
addressing the effect of cyanide on the receptor activity, 200 μM
potassium cyanide was added to the reaction medium. For
background controls, the ETR1-C65S mutant was denatured by
SDS before the kinase reaction was started with [γ -32 P]ATP.
Intensity of autoradiographic signals was quantified by Image
Gauge software (Fujifilm).
In vitro interaction assay of recombinant ethylene signalling
protein ETR1 and EIN2
For quantitative analysis of the ETR1–EIN2 complex, a
tryptophan-less mutant of the C-terminal part of EIN2
Cyanide can mediate ethylene-responses in ETR1 in vitro
263
[EIN2479–1294 (W)] was expressed and purified as described in
[22]. Steady-state fluorescence measurements on purified ETR1
and EIN2 were carried out using an LS55 spectroluminometer
(PerkinElmer) at a controlled temperature of 20 ◦ C using an
excitation wavelength of 295 nm and an emission wavelength
of 345 nm as described in [22]. ETR1 proteins were preincubated with 200 μM copper chloride or 300 μM silver nitrate
respectively as described in [15]. In order to analyse the effect
of cyanide on ETR1–EIN2 complex formation, ETR1 proteins
were additionally treated with 100 μM cyanide before titration
with EIN2479–1294 (W). Fluorescence data were analysed with the
program GraFit, and K d values were calculated from a fit of
the experimental data to a model expecting a 1:1 stoichiometry
of the two binding partners.
RESULTS
Sensor kinase ETR1 selectively binds cyanide
The π-acceptor compound cyanide shows high structural analogy
to the plant hormone ethylene. We showed previously that cyanide
inhibits autokinase activity of purified ethylene receptor ETR1
[26]. The resulting non-phosphorylated state of the receptor kinase
increases the affinity for ethylene signalling protein EIN2 at
the ER membrane [23]. To determine further whether decreased
autophosphorylation and increased association to EIN2 are based
on specific binding of cyanide to the ethylene-binding site of the
receptor kinase, we performed radioactive binding studies using
[14 C]cyanide. Purified recombinant wild-type ETR1 was used to
address this question. Binding assays were performed with assay
buffer alone (Figure 1A, white bar) and in the absence (Figure 1A,
grey bar) or presence of the copper cofactor (Figure 1A, black
bar) that is essential for ethylene binding [15]. Non-specific
binding of cyanide was addressed by adding unlabelled cyanide
in 100-fold (Figure 1A, hatched bar) and 1000-fold (Figure 1A,
dotted bar) molar excess before addition of the radiolabelled
ligand. The buffer control, the binding assay lacking the ethylenebinding metal cofactor and the binding assays containing a
large excess of unlabelled ligand showed only low radioactivity.
However, when 200 μM copper chloride was added to the purified
recombinant receptor, a 10-fold increase in radioactivity was
observed corresponding to a substantial increase in cyanide
binding under these conditions. In summary, the binding assays
indicate a specific copper-dependent binding of cyanide to the
purified recombinant protein. Immunoblotting and MS analysis of
the purified protein emphasize that this binding is solely attributed
to ETR1. In order to determine the K d value for cyanide, ligand
concentration was increased stepwise and [14 C]cyanide binding
was plotted against the ligand concentration. Figure 1(B) shows
the results of these radioligand-binding experiments. The broken
line represents the binding curve fitted to the experimental data
assuming a single class of ligand-binding sites in the ETR1
protein. The fitted curve corresponds to an apparent K d value
of 280 +
− 50 μM.
Cyanide binding is prevented in the ETR1-C65S mutant
The data presented above demonstrate that cyanide can
specifically bind to the purified wild-type receptor. To analyse
further the cyanide-binding site in the purified receptor and to
confirm that ethylene and cyanide compete for the same binding
site in the N-terminal transmembrane domain of ETR1, we used
the ETR1-C65S mutant which is deficient in ethylene binding
[15]. When the ETR1-C65S mutant was tested for cyanide
binding, similar basal radioactivity levels were obtained with
Figure 1
Analyses of [14 C]cyanide binding to purified ETR1
(A) Specific binding of cyanide to ETR1 is expressed by the high level of [14 C]cyanide that is
incorporated into the protein in the presence of the cofactor copper. In the absence of the essential
cofactor or when non-labelled ligand was applied (100× or 1000× corresponds to a 100- or
1000-fold molar excess of [12 C]cyanide), only low levels of protein-associated radioactivity were
found that compare with background activities detected in a sample containing only buffer but no
protein. (B) Determination of the apparent K d value of ETR1 for cyanide. The binding curve was
fitted to the protein-associated radioactivity obtained when purified ETR1 was incubated with
different concentrations of the radiolabelled ligand as described in the Experimental section.
The binding curve corresponds to a K d value of 280 +
− 50 μM.
the buffer control (Figure 2A, white bar) and in the absence
of the copper cofactor (Figure 2A, grey bar) as those of wildtype ETR1 (Figure 1A). However, in contrast with wild-type
ETR1, no increase in cyanide binding was observed when the
essential cofactor was added to the recombinant ETR1-C65S
(Figure 2A, black bar). Radioactivity remained essentially in the
range of the buffer control. Furthermore, cyanide binding was not
affected by adding a 100- or 1000-fold molar excesses of the nonlabelled ligand (Figure 2A, hatched and dotted bars). Together
with the strict dependence of cyanide binding on the presence
of the copper cofactor, these results indicate that binding of the
π-acceptor compound cyanide takes places at the same site as
ethylene binding in ETR1.
Previous in vitro studies from our laboratory have shown that
cyanide inhibits autophosphorylation of wild-type ETR1 [26],
but not of ETR1165–738 , a truncated form of the receptor lacking
the transmembrane ethylene-binding domain [26]. These data
indicate that ligand binding in the sensor domain is correlated
with molecular processes in the catalytic domain of the receptor.
In order to pinpoint that binding of cyanide to the ethylene binding
is directly related to the autokinase activity of the receptor, we
have analysed autokinase activity of the ETR1 wild-type and
the ETR1-C65S mutant in the presence and in the absence of
cyanide. In the absence of cyanide, autophosphorylation activity
of the ETR1-C65S mutant is comparable with that of wild-type
(Figure 2B, dark grey bars). However, in contrast with the wildtype which shows a clear reduction in autokinase activity in
c The Authors Journal compilation c 2012 Biochemical Society
264
M. M. A. Bisson and G. Groth
Figure 3
Effect of cyanide on ETR1-C65S–EIN2 complex formation
Titration of purified ETR1-C65S mutant with the tryptophan-less C-terminal part of EIN2
[EIN2479–1294 (W)]. Experiments were performed in the absence (䉫) and in the presence
(•) of cyanide. Fluorescence quenching data were processed using the method described in
[22]. All experiments were carried out in the presence of copper chloride. The broken line
represents the best-fit curve for experiments in the absence of cyanide. The K d value calculated
for these experiments is 415 +
− 51 nM. The continuous line indicates the best-fit curve for
measurements taken in the presence of cyanide. Complex formation of ETR1 and EIN2 under
+
these conditions is described by a K d value of 391 +
− 55 nM. Results are means − S.E.M. for
three independent measurements.
Figure 2
Cyanide binding is prevented in the ETR1-C65S mutant
(A) In the ETR1-C65S mutant, only basal incorporation of [14 C]cyanide corresponding to the
buffer control is observed under all conditions. In contrast with wild-type ETR1, addition of
copper has no effect on cyanide incorporation. (B) Autokinase activities of wild-type ETR1 and
the ETR1-C65S mutant observed in the absence (dark grey bars) and in the presence (light
grey bars) of cyanide. All experiments were carried out as described in the Experimental section
in the presence of copper chloride. Radiographic signals resulting from incorporation of 32 P
into purified ETR1 were quantified using the program Image Gauge (Fujifilm) and related to
the protein concentration in the sample. Maximum phosphorylation of wild-type ETR1 in the
absence of cyanide was set to 100 %. Pre-incubation of wild-type ETR1 with cyanide results in
a 50 % inhibition of autokinase activity. No effect on autophosphorylation was observed for the
ETR1-C65S mutant after cyanide treatment.
the presence of cyanide (Figure 2B, left-hand side, light grey
bar), phosphorylation activity is not affected by cyanide in the
ETR1-C65S mutant (Figure 2B, right-hand side, light grey bar).
Both recombinant receptor proteins were pre-incubated with
copper chloride in all measurements. For negative controls,
receptor proteins were chemically denatured before incubation
with [γ -32 P]ATP. Only 10 % of the activity associated with the
non-denatured proteins was found in these controls (results not
shown).
Formation of the ETR1-C65S–EIN2 complex is independent of
cyanide
The phosphorylation status in the kinase domain of ETR1
was shown to modulate the interaction with the downstream
signalling element EIN2 [23]. Previous studies have shown that
this interaction is affected by the addition of ethylene or cyanide
which is reflected in a decrease of K d values from 400 to 100 nM
[23]. In order to test whether this interaction is mediated by
the binding of cyanide to the N-terminal transmembrane ethylenebinding domain, we have analysed the interaction between the
ETR1-C65S mutant and EIN2 as a function of cyanide. We
used the tryptophan fluorescence-based approach described in
[22] to quantify the ETR1-C65S–EIN2 interaction and monitored
c The Authors Journal compilation c 2012 Biochemical Society
the tryptophan fluorescence of the ETR1-C65S mutant after
incremental titration with a tryptophan-less mutant of the Cterminal part of EIN2 [EIN2479–1294 (W)]. Interaction of the
two proteins results in a fluorescence quench which was used
to calculate the K d values of the complex. Experiments run
in the absence of cyanide gave a K d value of 415 +
− 51 nM
(Figure 3) corresponding to the K d values obtained with wild-type
ETR1 carrying a functional ethylene-binding site but no ethylene.
Figure 3 shows that experiments in the presence of cyanide give
almost the same K d value (391 +
− 55nM), which is in contrast
with the results observed with wild-type ETR1 showing a 4-fold
increase in complex affinity (K d value of 100 nM) in the presence
of cyanide [23]. The fact that the ethylene analogue cyanide has
no effect on complex formation in the ethylene-binding mutant
emphasizes further that ethylene binding in the sensor domain is
linked directly to catalytic processes in the extramembranous part
of the receptor such as phosphorylation in the kinase domain and
to the interaction with essential downstream signalling elements
such as EIN2.
The ethylene-response antagonist silver has no effect on cyanide
binding, autokinase activity and ETR1–EIN2 interaction
Binding studies in transgenic yeast expressing ETR1 have
identified that Ag + ions can substitute for the essential copper
cofactor in the transmembrane sensor domain [15]. On the other
hand, silver salts have been known as antagonists that block
ethylene responses in plants [17] and have been used for a
long time as efficient inhibitors to extend the vase life of cut
flowers [34]. Thus silver is supposed to prevent conformational
changes in ETR1 required for signal transmission [15]. In the
present study, we tested the impact of silver on the binding
of the ethylene structural analogue cyanide, on the autokinase
activity of the recombinant receptor protein, and on its interaction
with its downstream signalling partner EIN2. Binding studies with
radiolabelled cyanide and the recombinant purified ETR1 protein
using silver nitrate instead of copper chloride demonstrate that the
Cyanide can mediate ethylene-responses in ETR1 in vitro
265
ethylene analogue does bind in the presence of silver. Figure 4(A)
(black bar) shows that the binding level obtained in the presence
of silver nitrate is approximately 60 % of the maximal binding
observed in the presence of copper chloride (Figure 1A). Buffer
control (Figure 4A, white bar) and binding assays obtained in
the absence of silver (Figure 4A, grey bar) are in the same range
as observed in previous experiments when the effect of copper
on cyanide binding was tested. In the presence of Ag2 + ions,
binding of radiolabelled cyanide was quenched by the addition
of non-radiolabelled ligand as found with the copper cofactor. In
summary, these data clearly emphasize that silver can substitute
for copper for specific and saturable binding of cyanide to ETR1.
Next, we tested the effect of silver on the autokinase activity of the
ETR1 sensor kinase. Figure 4(B) shows that autophosphorylation
is not affected by the presence of silver nitrate and shows the same
activity as observed with copper chloride. However, cyanide fails
to block autokinase activity when silver is applied as a cofactor
(see Figure 4B, right-hand side, light grey bar), even though it was
shown to reduce phosphorylation of the recombinant receptor
in the presence of copper. Finally, we addressed the effect of
silver on the interaction of ETR1 with EIN2. For quantitative
analysis, wild-type ETR1 was pre-incubated with silver nitrate
before the incremental titration with tryptophan-less EIN2. From
these experiments, a K d value of 491 +
− 50 nM was calculated
(Figure 4C) which is similar to the K d value obtained in the
presence of the essential cofactor copper chloride [23]. However,
the K d value of the ETR1–EIN2 interaction was not affected by the
addition of cyanide. Figure 4(C) illustrates that, in the presence of
cyanide, a K d value of approximately 441 +
− 94 nM was obtained.
This is in contrast with the situation with copper [23]. Similar
to the kinase activity, the structural analogue cyanide had no
effect on the ETR1–EIN2 interaction when applied in the presence
of the ethylene-response antagonist silver. Taken together, these
data reveal that Ag + ions can occupy the ethylene-binding site
and interact with the plant hormone or with the ethylene agonist
cyanide, but fail to induce changes in autokinase and in the ETR1–
EIN2 interaction that are observed when the receptor is complexed
with its native cofactor copper.
DISCUSSION
Previous studies have indicated that cyanide, a structural analogue
of the plant hormone ethylene, mimics ethylene responses
in planta [27] and competes with ethylene binding in cyanideresistant tissues [28]. Thus cyanide might serve as a suitable
molecular substitute for ethylene in physical, structural and
functional studies on isolated ethylene signalling proteins which
are complicated by the gaseous character of the plant hormone
ethylene. Beyond any doubt, this kind of study on isolated
individual proteins or on isolated signalling complexes of the
ethylene signalling pathway will provide a detailed understanding
of the molecular structure and the signalling mechanism of these
signalling proteins. In the present study, we provide the first
clear evidence that cyanide specifically binds to the ethylenebinding site in the transmembrane sensor domain of ETR1. Using
purified recombinant wild-type ETR1, we have determined an
apparent K d value of 280 μM for cyanide which is clearly higher
that the nanomolar binding of ethylene obtained in ethylenebinding studies using transgenic yeast [15]. However, similar
K d values in the upper micromolar range were also found in
in vitro displacement assays with purified ionotropic glutamate
receptor GluA2 showing a K d value of 460 μM for the IDRA-21
ligand [35]. Moreover, the discrepancy might be due to the high
Figure 4 The ETR1 inhibitor silver permits cyanide binding to the
transmembrane sensor domain, but prevents intra- and inter-molecular
signalling
(A) Cyanide-binding capacity of wild-type ETR1 in the presence of 300 μM silver nitrate.
Protein-associated radioactivity detected after [14 C]cyanide treatment of purified ETR1 with
silver as the metal cofactor. Only basal levels of radioactivity were observed in the absence of
Ag + ions or after pre-saturation of purified ETR1 with 100- and 1000-fold molar excess
of [12 C]cyanide. (B) Autokinase activity of wild-type ETR1 with copper chloride or silver
nitrate as cofactor. Data were processed as described in the Experimental section and in
the legend to Figure 2. Dark grey bars represent the kinase activity detected in the absence
of cyanide, but in the presence of copper or silver respectively. Addition of cyanide (light
grey bars) causes inhibition of the receptor autokinase activity of approximately 50 % when
copper was used as a cofactor. On the other hand, cyanide was irrelevant for kinase activity
when copper was replaced by silver. (C) Quantitative fluorescence interaction assay with
purified EIN2479–1294 (W) and wild-type ETR1 in dependence of silver nitrate and cyanide.
Experiments were carried out with 300 μM silver nitrate, but in the absence and in the
presence of the ethylene agonist cyanide. Measurements without cyanide correspond to a
K d value of 491 +
− 50 nM (broken line). The continuous line represents the best-fit curve for
experiments obtained in the presence of cyanide. Complex formation under these conditions is
+
described by a K d value of 441 +
− 91 nM. Results are means − S.E.M. for three independent
measurements.
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266
M. M. A. Bisson and G. Groth
solubility of ethylene in hydrophobic systems such as biological
membranes in contrast with the charged cyanide which is wellsoluble in aqueous systems, but not in non-polar hydrophobic
environments. Furthermore, our experiments were performed
with functional detergent-solubilized receptor [22,23,26] rather
than with an intact cellular system or with isolated transgenic
yeast membranes [15]. Thus the efficient concentration of cyanide
at the receptor-detergent micelle might be substantially lower
than the bulk cyanide concentration and therefore the actual K d
value might compare better with the in vivo ethylene binding
studies. No matter of the exact K d values of ETR1 for cyanide,
our studies clearly emphasize that cyanide is a suitable substitute
for ethylene that can be used due to its simple manageability and
good solubility in aqueous solution in quantitative protein–protein
interaction studies or in structural studies using NMR or X-ray
crystallography.
On the basis of their labelling studies in transgenic yeast,
Bleecker et al. [14] hypothesized that the binding site of the
plant hormone is formed by a receptor dimer which co-ordinates
an essential copper cofactor. Recent studies from our laboratory
have shown that purified recombinant ETR1 as used in the present
in vitro study also forms homodimers [36], emphasizing further
that in vitro experiments on purified isolated proteins can compare
well with in vivo physiological data. The labelling experiments in
transgenic yeast of Bleecker and colleagues using radiolabelled
ethylene also revealed that the ETR1-C65S mutant in the
transmembrane sensor domain of the receptor shows strongly
reduced ethylene-binding capacity [15]. Our binding experiments
using radiolabelled cyanide agree with these data and reveal that
Cys65 is also essential for cyanide binding. Furthermore, our
experiments demonstrate that mutation of this residue is sufficient
to abolish inhibition of the ETR1 autokinase activity by cyanide
that is observed with the wild-type protein [26].
Hence we have not only demonstrated that cyanide binds at the
ethylene-binding site in the sensor domain, but also shown that
cyanide is a suitable agonist to mimic in vitro effects of ethylene.
Besides its effect on intramolecular signalling shown by the
reduced autokinase activity, cyanide also affects intermolecular
signalling of the receptor as indicated by the observed increased
affinity of ETR1 to the downstream signalling protein EIN2 in
the presence of the ethylene agonist [23]. Cyanide, which
substantially increases the affinity between wild-type ETR1 and
the C-terminal domain of EIN2, does not affect complex formation
with EIN2 in the ETR1-C65S mutant. As previous studies have
indicated that the ETR1–EIN2 interaction is localized at the
kinase domain of the receptor [23], failure of cyanide to affect
the complex formation in the mutant has to be ascribed to the
observed failure in binding to the ethylene-binding site in
the mutant transmembrane sensor domain and the in turn
unaffected autokinase activity of the receptor. As a consequence
of the sustained autokinase activity, interaction with EIN2 is not
changed in the ETR1-C65S mutant.
Equivalence of ethylene and cyanide as signalling molecules
in in vitro assays is supported further by our studies with the
ethylene-response antagonist silver. In vivo studies revealed that
silver supports ethylene binding to ETR1, but inhibits ethylene
responses in plant tissues [15,17]. Experiments with purified
ETR1 and the ethylene agonist cyanide in the present study
agree with these results. Wild-type ETR1 showed a reduced, but
clear, binding of cyanide when the essential copper cofactor was
replaced by silver in the labelling studies. Hence binding of the
ethylene analogue is only slightly affected in the presence of silver.
In accordance with the absence of ethylene responses in planta
when copper is replaced by silver, we found that substitution of
silver completely abolished the intramolecular and intermolecular
c The Authors Journal compilation c 2012 Biochemical Society
signalling events normally observed with the purified receptor
protein. These results support further the equivalence of cyanide
and ethylene for studies on ethylene signalling and provide
compelling evidence for the structural and functional equivalence
of ethylene and cyanide.
AUTHOR CONTIBUTION
Melanie Bisson planned and performed research, analysed data and wrote the paper. Georg
Groth designed and led the project, analysed results and wrote the paper.
ACKNOWLEDGEMENTS
We thank Dr Nicole Linka (Heinrich-Heine University) for her help with the radioactive
binding assay and Dr Melanie Brocker [Biotechnology (IBG-1), Forschungszentrum Jülich,
Jülich, Germany] for MS determinations. We are also grateful to Dr Daniel Schlieper
(Heinrich-Heine University) for a critical reading of the paper before submission.
FUNDING
This work was supported by the Deutsche Forschungsgemeinschaft within the
Sonderforschungsbereich 590 ‘Inhärente und adaptive Differenzierungsprozesse’ at
the Heinrich-Heine University Düsseldorf.
REFERENCES
1 Kende, H. (1993) Ethylene biosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44,
283–307
2 Johnson, P. R. and Ecker, J. R. (1998) The ethylene gas signal transduction pathway: a
molecular perspective. Annu. Rev. Genet. 32, 227–254
3 Bleecker, A. B. and Kende, H. (2000) Ethylene: a gaseous signal molecule in plants. Annu.
Rev. Cell Dev. Biol. 16, 1–18
4 Stepanova, A. N. and Ecker, J. R. (2000) Ethylene signalling: from mutants to molecules.
Curr. Opin. Plant Biol. 3, 353–360
5 Bleecker, A. B., Estelle, M. A., Somerville, C. and Kende, H. (1988) Insensitivity to ethylene
conferred by a dominant mutation in Arabidopsis thaliana . Science 241, 1086–1089
6 Chang, C., Kwok, S. F., Bleecker, A. B. and Meyerowitz, E. M. (1993) Arabidopsis ethylene
response gene ETR1: similarity of product to two component regulators. Science 262,
539–544
7 Hua, J., Chang, C., Sun, Q. and Meyerowitz, E. M. (1995) Ethylene sensitivity conferred
by Arabidopsis ERS gene. Science 269, 1712–1714
8 Hua, J., Sakai, H., Nourizadeh, S., Chen, Q. G., Bleecker, A. B., Ecker, J. R. and
Meyerowitz, E. M. (1998) EIN4 and ERS2 are members of the putative ethylene receptor
family in Arabidopsis . Plant Cell 10, 1321–1332
9 Sakai, H., Hua, J., Chen, Q. G., Chang, C., Medrano, L. J., Bleecker, A. B. and Meyerowitz,
E. M. (1998) ETR2 is an ETR1-like gene involved in ethylene signalling in Arabidopsis .
Proc. Natl. Acad. Sci. U.S.A. 95, 5812–5817
10 Hua, J. and Meyerowitz, E. M. (1998) Ethylene responses are negatively regulated by a
receptor gene family in Arabidopsis thaliana . Cell 94, 261–271
11 Grefen, C., Städele, K., Rzicka, K., Obrdlik, P., Harter, K. and Horák, J. (2008) Subcellular
localization and in vivo interactions of the Arabidopsis thaliana ethylene receptor family
members. Mol. Plant 1, 308–320
12 Chen, Y. F., Gao, Z., Kerris, 3rd, R. J., Wang, W., Binder, B. M. and Schaller, G. E. (2010)
Ethylene receptors function as components of high-molecular-mass protein complexes in
Arabidopsis . PLoS ONE 5, e8640
13 Gao, Z., Wen, C. K., Binder, B. M., Chen, Y. F., Chang, J., Chiang, Y. H., Kerris, 3rd, R. J.,
Chang, C. and Schaller, G. E. (2008) Heteromeric interactions among ethylene receptors
mediate signalling in Arabidopsis . J. Biol. Chem. 283, 23801–23810
14 Bleecker, A. B., Esch, J. J., Hall, A. E., Rodrı́guez, F. I. and Binder, BM. (1998) The
ethylene-receptor family from Arabidopsis : structure and function. Phil. Trans. R. Soc.
London Ser. B 353, 1405–1412
15 Rodrı́guez, F. I., Esch, J. J., Hall, A. E., Binder, B. M., Schaller, G. E. and Bleecker, A. B.
(1999) A copper cofactor for the ethylene receptor ETR1 from Arabidopsis . Science 238,
996–998
16 Hirayama, T., Kieber, J. J., Hirayama, N., Kogan, M., Guzman, P., Nourizadeh, S., Alonso,
J. M., Dailey, W. P., Dancis, A. and Ecker, J. R. (1999) RESPONSIVE-TO-ANTAGONIST1, a
Menkes/Wilson disease-related copper transporter, is required for ethylene signalling in
Arabidopsis . Cell 97, 383–393
Cyanide can mediate ethylene-responses in ETR1 in vitro
17 Beyer, E. M. (1976) A potent inhibitor of ethylene action in plants. Plant Physiol. 58,
268–271
18 Clark, K. L., Larsen, P. B., Wang, X. and Chang, C. (1998) Association of the Arabidopsis
CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors. Proc. Natl. Acad. Sci.
U.S.A. 95, 5401–5406
19 Kieber, J. J., Rothenberg, M., Roman, G., Feldmann, K. A. and Ecker, J. R. (1993) CTR1, a
negative regulator of the ethylene response pathway in Arabidopsis , encodes a member of
the raf family of protein kinases. Cell 72, 427–441
20 Huang, Y., Li, H., Hutchison, C. E., Laskey, J. and Kieber, J. J. (2003) Biochemical and
functional analysis of CTR1, a protein kinase that negatively regulates ethylene signalling
in Arabidopsis . Plant J. 33, 221–233
21 Yoo, S. D., Cho, Y. H., Tena, G., Xiong, Y. and Sheen, J (2008) Dual control of nuclear
EIN3 by bifurcate MAPK cascades in C2 H4 signalling. Nature 451, 789–795
22 Bisson, M. M., Bleckmann, A., Allekotte, S. and Groth, G. (2009) EIN2, the central
regulator of ethylene signalling, is localized at the ER membrane where it interacts with
the ethylene receptor ETR1. Biochem. J. 424, 1–6
23 Bisson, M. M. and Groth, G. (2010) New insight in ethylene signalling: autokinase activity
of ETR1 modulates the interaction of receptors and EIN2. Mol. Plant 3, 882–889
24 Alonso, J. M., Hirayama, T., Roman, G., Nourizadeh, S. and Ecker, J. R. (1999) EIN2, a
bifunctional transducer of ethylene and stress responses in Arabidopsis . Science 284,
2148–2152
25 Qiao, H., Chang, K. N., Yazaki, J. and Ecker, J. R. (2009) Interplay between ethylene,
ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in
Arabidopsis . Genes Dev. 23, 512–521
26 Voet-van-Vormizeele, J. and Groth, G. (2008) Ethylene controls autophosphorylation of
the histidine kinase domain in ethylene receptor ETR1. Mol. Plant 1, 380–387
267
27 Sisler, E. C. (1977) Ethylene activity of some π-acceptor compounds. Tob. Sci. 21, 43–45
28 Solomos, T. and Laties, G. G. (1974) Similarities between the actions of ethylene and
cyanide in initiating the climacteric and ripening of avocadoes. Plant Physiol. 54,
506–511
29 Cormack, B. (1992) Site directed mutagenesis by the polymerase chain reaction. In Short
protocols in Molecular Biology (Struhl, K., ed.), pp. 8–25, John Wiley and Sons, New York
30 Scharein, B., Voet van Vormizeele, J., Harter, K. and Groth, G. (2008) Ethylene signalling:
identification of a putative ETR1–AHP1 phosphorelay complex by fluorescence
spectroscopy. Anal. Biochem. 377, 72–76
31 Voet-van-Vormizeele, J. and Groth, G. (2003) Heterologous expression and single-step
purification of the ethylene receptor protein ETR1 from Arabidopsis thaliana . Protein
Expression Purif. 32, 89–94
32 Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227, 680–685
33 Heukeshoven, J. and Dernick, R. (1988) Improved silver staining procedure for fast
staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels.
Electrophoresis 9, 28–32
34 Veen, H. (1979) Effects of silver on ethylene synthesis and action in cut carnations. Planta
145, 467–470
35 Krintel, C., Frydenvang, K., Olsen, L., Kristensen, M. T., de Barrios, O., Naur, P., Francotte,
P., Pirotte, B., Gajhede, M. and Kastrup, J. S. (2012) Thermodynamics and structural
analysis of positive allosteric modulation of the ionotropic glutamate receptor GluA2.
Biochem. J. 441, 173–178
36 Scharein, B. and Groth, G. (2011) Phosphorylation alters the interaction of the
Arabidopsis phosphotransfer protein AHP1 with its sensor kinase ETR1. PLoS ONE 6,
e24173
Received 8 August 2011/16 February 2012; accepted 5 March 2012
Published as BJ Immediate Publication 5 March 2012, doi:10.1042/BJ20111447
c The Authors Journal compilation c 2012 Biochemical Society
Biochem. J. (2012) 444, 261–267 (Printed in Great Britain)
doi:10.1042/BJ20111447
SUPPLEMENTARY ONLINE DATA
Cyanide is an adequate agonist of the plant hormone ethylene for studying
signalling of sensor kinase ETR1 at the molecular level
Melanie M. A. BISSON and Georg GROTH1
Institute for Biochemical Plant Physiology, Heinrich-Heine University, Universitätsstrasse 1, 40225 Düsseldorf, Germany
Figure S1 Mascot search results for protein identification of ETR1 protein
using MALDI–TOF-MS/MS
Trypsin digestion of purified recombinant ETR1 and sample preparation for MS was carried
out using the method described in [1]. MALDI–TOF-MS/MS (matrix-assisted laser-desorption
ionization–time-of-flight tandem MS) was performed using an Ultraflex III TOF/TOF mass
spectrometer (Bruker Daltonics). For protein identification, raw MS/MS data were searched
using the Mascot algorithm (Matrix Science, version 2.2.0) against an in-house database
containing more than 200 user-added proteins and against an E. coli database (NCBI) with the
following search parameters: variable modifications due to methionine oxidation. In addition, a
maximum of one missed cleavage site, in case of incomplete trypsin hydrolysis, was considered
for database searches. The molecular mass search (MOWSE) scoring scheme [2] was used for
unequivocal identification of proteins and peptides.
REFERENCES
1 Schultz, C., Niebisch, A., Schwaiger, A., Viets, U., Metzger, S., Bramkamp, M. and Bott, M.
(2009) Genetic and biochemical analysis of the serine/threonine protein kinases PknA,
PknB, PknG and PknL of Corynebacterium glutamicum : evidence for non-essentiality and
for phosphorylation of OdhI and FtsZ by multiple kinases. Mol. Microbiol. 74, 724–741
2 Pappin, D. J., Hojrup, P. and Bleasby, A. J. (1993) Rapid identification of proteins by
peptide-mass fingerprinting. Curr. Biol. 3, 327–332
Received 8 August 2011/16 February 2012; accepted 5 March 2012
Published as BJ Immediate Publication 5 March 2012, doi:10.1042/BJ20111447
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society