Multiple Calmodulin-binding Sites Positively and Negatively Regulate Arabidopsis CYCLIC NUCLEOTIDE-GATED CHANNEL12 Thomas A. DeFalco1, Christopher B. Marshall2, Kim Munro3, Hong-Gu Kang4, Wolfgang Moeder1, Mitsuhiko Ikura2, Wayne A. Snedden5, and Keiko Yoshioka1,6 1 2
Department of Cell & Systems Biology, University of Toronto, Toronto, ON, Canada, M5S 3B2 Department of Medical Biophysics, Campbell Family Cancer Research Institute/Princess
Margaret Cancer Centre, University of Toronto, Toronto, ON, Canada, M5G 2M9 3
Protein Function Discovery Facility, Queen’s University, Kingston, ON, Canada, K7L 3N6
4
Department of Biology, Texas State University, San Marcos, TX, 78666
5
Department of Biology, Queen’s University, Kingston, ON, Canada, K7L 3N6
6
Center for the Analysis of Genome Evolution and Function (CAGEF), University of Toronto,
Toronto, ON, Canada, M5S 3B2
Corresponding author:
Keiko Yoshioka Department of Cell & Systems Biology, University of Toronto, 25 Willcocks street, Toronto, ON, M5S 3B2 +1 416-978-3545
[email protected]
Keywords: calcium; calmodulin; immunity; CNGC; programmed cell death Short title: CaM regulation of CNGC12 The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Keiko Yoshioka (
[email protected])
SYNOPSIS: A cyclic nucleotide-gated channel that regulates plant immunity has multiple Cterminal calmodulin domains and is regulated both positively and negatively by calmodulin, challenging the current model.
1
ABSTRACT
2
Ca2+ signaling is critical to plant immunity; however, the channels involved are poorly
3
characterized. Cyclic nucleotide-gated channels (CNGCs) are non-specific, Ca2+-permeable
4
cation channels. Plant CNGCs are hypothesized to be negatively regulated by the Ca2+ sensor
5
calmodulin (CaM), and previous work has focused on a C-terminal CaM-domain (CaMBD)
6
overlapping with the cyclic nucleotide-binding domain (CNBD) of plant CNGCs. However, we
7
show that the Arabidopsis thaliana isoform CNGC12 possesses multiple CaMBDs at cytosolic
8
N- and C-termini, which is reminiscent of animal CNGCs and unlike any plant channel studied
9
to date. Biophysical characterizations of these sites suggest that apoCaM interacts with a
10
conserved isoleucine-glutamine (IQ) motif in the C-terminus of the channel, while Ca2+/CaM
11
binds additional N- and C-terminal motifs with different affinities. Expression of CNGC12 with
12
a non-functional N-terminal CaMBD constitutively induced programmed cell death, providing in
13
planta evidence of allosteric CNGC regulation by CaM. Furthermore, we determined that CaM-
14
binding to the IQ motif was required for channel function, indicating that CaM can both
15
positively and negatively regulate CNGC12. These data indicate a complex mode of plant CNGC
16
regulation by CaM, in contrast to the previously proposed competitive ligand model, and suggest
17
exciting parallels between plant and animal channels.
18 19 20
INTRODUCTION Ca2+ serves as a universal second messenger in eukaryotic signaling pathways, and
21
transient changes in cytosolic Ca2+ levels are rapidly induced by diverse stimuli in plants
22
(Sanders et al., 2002; Kudla et al., 2010). Despite such a central role for Ca2+ in plant biology,
23
relatively little is known regarding the Ca2+ channels of plants (Spalding and Harper, 2011).
24
Plant genomes examined to date lack homologs to animal voltage-gated cation channels, and
25
instead possess expanded families of ligand-gated cation channels, represented primarily by the
26
ionotropic glutamate receptor-like channel (GLR) and cyclic nucleotide-gated channel (CNGC)
27
families (Mäser et al., 2001). CNGCs are non-selective cation channels that are hypothesized to
28
function as Ca2+ channels in plants (Dietrich et al., 2010; Jammes et al., 2011; Zelman et al.,
29
2012) and are characterized by conserved structural components, including a short cytosolic N-
30
terminus, six transmembrane helices (S1-S6) with a pore-forming region between S5 and S6, and
31
a cytosolic C-terminus containing a cyclic nucleotide-binding domain (CNBD (Kaupp and
32
Seifert, 2002; Matulef and Zagotta, 2003). The CNBD mediates channel gating by cyclic
33
nucleotide monophosphates (cNMPs) such as cAMP and/or cGMP (Mäser et al., 2001; Kaupp
34
and Seifert, 2002; Matulef and Zagotta, 2003; Kaplan et al., 2007; Zelman et al., 2012), though
35
studies of this mechanism are largely restricted to animal isoforms.
36
All plant and animal CNGCs studied to date also possess at least one calmodulin (CaM)-
37
binding domain (CaMBD) (Kaplan et al., 2007; Ungerer et al., 2011). CaM is a ubiquitous
38
eukaryotic Ca2+ sensor, which binds four Ca2+ ions via EF-hand motifs arranged in N- and C-
39
terminal globular domains. Upon binding Ca2+, CaM changes conformation from a closed, Ca2+-
40
free state (apoCaM) to an extended Ca2+/CaM conformation with high affinity for a broad range
41
of target proteins (Hoeflich and Ikura, 2002; Bouché et al., 2005; DeFalco et al., 2010a;
42
Poovaiah et al., 2013). This structural flexibility, along with the ability of some proteins to
43
interact with CaM independently of Ca2+, allows CaM to regulate numerous protein targets in
44
diverse signaling pathways (Crivici and Ikura, 1995; Yamniuk and Vogel, 2004). Mammalian
45
CNGCs possess diverse sites for CaM-binding, with at least nine CaMBDs found across both the
46
N- and C-termini of the six different CNGC isoforms (Ungerer et al., 2011). However, to date,
47
experiments have provided evidence of a functional role for only the N-terminal CaMBDs (Liu
48
et al., 1994; Weitz et al., 1998; Trudeau and Zagotta, 2002; Zheng et al., 2003; Song et al.,
49
2008), particularly the N-terminal LQ site of the regulatory CNGB1 subunit (Ungerer et al.,
50
2011). Generally, CaM is hypothesized to function in the feedback regulation of CNGCs by
51
binding to one or more CaMBD(s) at elevated cytosolic Ca2+ levels and allosterically inhibiting
52
CNGC conductance (Zheng et al., 2003; Trudeau and Zagotta, 2002, 2004; Bradley et al., 2004;
53
Ungerer et al., 2011; Liu et al., 1994; Chen and Yau, 1994; Weitz et al., 1998; Bradley et al.,
54
2001; Song et al., 2008).
55
Initial studies on individual isoforms from barley (Hordeum vulgare) (Schuurink et al.,
56
1998), tobacco (Nicotiana tabacum) (Arazi et al., 1999), and Arabidopsis thaliana (Köhler and
57
Neuhaus, 2000) confirmed that plant CNGCs are also CaM-binding proteins. However, in
58
contrast to animal isoforms, a single CaMBD was mapped to a site that overlaps the C-terminal
59
α-helix (αC) of the CNBD (Arazi et al., 2000; Köhler and Neuhaus, 2000). This αC CaMBD is
60
characterized by a four residue, Trp-Arg-Thr-Trp (WRTW) motif required for CaM-binding
61
(Arazi et al., 2000), which is widely conserved across the Arabidopsis CNGC family (Chin et al.,
62
2010). Previous studies have hypothesized that the location of the CaMBD within the CNBD
63
allows CaM to compete with cNMP as a ligand in the allosteric gating of channel conductance
64
(Hua et al., 2003; Kaplan et al., 2007; Swarbreck et al., 2013). It was originally suggested that
65
this site is the only CaMBD in plant CNGCs, while sequence conservation of this region varies
66
considerably between CNGC isoforms (Zelman et al., 2012). Recently, Arabidopsis CNGC20
67
was found to bind CaM via a distinct ‘isoleucine-glutamine’ (IQ) motif adjacent to but not
68
overlapping the αC-helix (Fischer et al., 2013), suggesting that plant CNGCs, like mammalian
69
CNGCs, may possess a variety of CaMBDs. A long-standing hypothesis holds that plant
70
CNGCs, like animal isoforms, are negatively regulated by CaM (Hua et al., 2003; Kaplan et al.,
71
2007), but direct in planta evidence to support this is currently lacking.
72
Though the importance of Ca2+ signaling in immunity is well established, the molecular
73
components involved in generating and regulating Ca2+ signals during defense responses are only
74
beginning to be characterized (Seybold et al., 2014). Furthermore, despite the importance of
75
CNGCs in plant biology, as implied by the expanded size of CNGC gene families and
76
phenotypes of some CNGC mutants, very little is known regarding the structure-function of these
77
channels. Individual CNGC isoforms have been implicated in immune signaling, including the
78
positive regulator of immunity Arabidopsis CNGC12 (Yoshioka et al., 2006; Moeder et al.,
79
2011). CaM is also involved in immunity, wherein it plays both positive and negative regulatory
80
roles (Cheval et al., 2013; Poovaiah et al., 2013). Thus, CNGCs represent a poorly studied
81
junction between Ca2+, CaM, and immunity. To further understand the regulation and
82
physiological roles of CNGCs, we undertook a thorough characterization of the CaM-binding
83
properties and function of CNGC12. In this work, we demonstrate that CNGC12 has multiple
84
CaMBDs at both its cytosolic N- and C- termini, including both Ca2+-dependent and -
85
independent sites, and which mediate both positive and negative regulation of channel function,
86
revealing complexity in CNGC regulation by Ca2+.
87
RESULTS
88
At-CNGC12 contains CaMBDs at both its N- and C-termini
89
To characterize the regulation of CNGCs by CaM during immunity, we investigated the
90
CaM-binding properties of CNGC12. The region corresponding to the conventional αC CaMBD
91
within the CNBD of CNGC12 is poorly conserved relative to other members of the CNGC
92
family in Arabidopsis (Zelman et al., 2012). Thus, we empirically investigated the CaM-binding
93
properties of this region and searched for novel CaMBD(s) in CNGC12 (see Supplemental
94
Figure 1 for delineation data). A recombinant 6xHis-tagged fragment corresponding to the
95
predicted cytosolic C-terminus of the channel (CNGC12358-649) clearly bound HRP-CaM in our
96
overlay assay. Seven truncated fragments of this region were subsequently expressed and
97
assayed for CaM-binding (Supplemental Figure 1). In contrast with studies of other plant
98
CNGCs, our delineations revealed that an alternative CaMBD exists outside of the CNBD, as a
99
region C-terminal to the CNBD (CNGC12561-626) was able to bind CaM. This suggested the
100
presence of a novel CaMBD within the C-terminus of CNGC12. Our assays showed that a
101
minimal region of a.a. 595-626 contained the novel CaMBD; notably, this region had not been
102
previously characterized in any plant CNGC. Concomitant with the C-terminal delineation, the
103
cytosolic N-terminal region of CNGC12 (a.a. 1-43) was expressed as a glutathione S-transferase
104
(GST)-fusion and assayed for CaM-binding. No N-terminal CaMBDs have been previously
105
reported among plant CNGC isoforms, however, we observed clear CaM-binding in our overlay
106
assay with this N-terminal region. Following these in vitro delineations, we performed in silico
107
analyses to predict putative CaMBD motifs within these empirically derived regions. On the
108
basis of sequence, electrostatics, and helical propensity (Yap et al., 2000), two putative motifs
109
were predicted, which we named NT (CNGC1217-42) and CT (CNGC12600-623). In addition to the
110
sites identified with our binding assays, a putative IQ motif was predicted at CNGC12568-588; this
111
site is highly conserved across the entire Arabidopsis CNGC family and has been validated as a
112
CaMBD in the case of CNGC20 (Fischer et al., 2013). The positions of the NT, IQ, and CT
113
CaMBDs are shown in Figure 1A.
114
Prediction of CNGC12 secondary structure using the PSIPRED server (Jones, 1999;
115
Buchan et al., 2013) suggested that the IQ and CT motifs form helices, while the cytosolic N-
116
terminus of the channel was predicted to exist in a random coil orientation. However, given the
117
ability of CaM-binding to induce helical conformation in target sequences (Yamniuk and Vogel,
118
2004), the NT, IQ, and CT regions were all modeled as helices and visualized as helical wheels
119
(rzlab.ucr.edu/) and three-dimensional helices (PyMOL Version 1.3) (Figure 1B and 1C). The
120
NT and CT models produced amphipathic helices, with hydrophobic and basic faces
121
characteristic of Ca2+-dependent CaMBDs (Yamniuk and Vogel, 2004), while the IQ motif
122
lacked such clear amphipathic character (Figure 1B and 1C). The CNGC12 IQ motif did not bind
A 1
358
43
NT L27 L20 Y31 R24 M34 K35
V23
h
649
IQ
16-VDGKLKSVRGRLKKVYGKMKTLENW-40
B
444
CT
568-AAFFIQAAWRKHCKRKLSKTR-588
600-NLASTLYVSRFVSKALQNRRKDTA-623
W576 A569 C580 K583 Q572 L584 I572
F610 S603 A614 N617 V607 Y606 R618
h +
V30
K28
H579
R577
K613
V611
L37
K21
K586
F570
K620
T604
G32
R26 K33
+
S22 K29 T36
G25
R609
L615 S608 Q616 L605 S612 R619
A575 K581 R582 A574 F571 K578 S585
R609
C
I572
L27
L605 K613
Q573
K28
V607
D
(2)
(1)
+Ca2+
-Ca2+ 0
0.25 0.5 1.0 2.0
0 0
0.25
0.5 1.0
1.5 2.0
0.25
0.5 1.0 2.0
3.0
Molar ratio (peptide:CaM) Figure 1. Structural modelling and ND-PAGE mobility shift assay of CNGC12 CaMBD peptides. (A) Location of the CaMBDs of CNGC12 (see delineation of NT and CT sites in Supplemental Figure 1). Numbers indicate amino acid position. 1-6 = transmembrane helices, P = pore region, CNBD = cyclic nucleotide binding domain, CaMBD = calmodulin binding domain. (B) Helical wheel projections of motifs for the NT, IQ, and CT peptides (underlined sequences). Dashed lines separate proposed hydrophobic (h) and basic (+) faces of the NT and CT wheels. (C) 3D model of full-length CaMBD peptides. Specific residues used for mutant analysis of each motif in this work are indicated in each model. In both (B) and (C), hydrophobic and basic residues are coloured orange and blue, respectively, while the Q573 residue of the IQ motif is also coloured (red) due to its suspected involvement in CaM-binding. (D) Non-dissociating-polyacrylamide gel electrophoresis (ND-PAGE) mobility shift assays in the presence of 0.1 mM CaCl2 (top panels) or 2 mM EGTA (bottom panels). Closed triangles indicate the migration size of CaM alone, while open triangles indicate the migration size of CaMpeptide complex. Two distinct, IQ-CaM complexes consistently migrated separately in Ca2+/CaM assays with IQ peptide (D, top panel, numbered 2); such a pattern was never observed with NT or CT peptides, or in any apoCaM assays.
123
CaM in our overlay assays (Supplemental Figure 1), which were performed under stringent
124
conditions that may not detect relatively weak binding.
125
To characterize these sites in greater detail, we obtained synthetic peptides corresponding
126
to each site (Supplemental Table 1). Each of these peptides exhibited CaM-binding in our non-
127
dissociating polyacrylamide gel electrophoresis (ND-PAGE) assays (Figure 1D). The NT and CT
128
peptides caused a shift in the mobility of CaM only in the presence of Ca2+, while the IQ peptide
129
was able to cause a shift in the mobility of both Ca2+/CaM and apoCaM. However, we
130
consistently observed multiple complexes formed between the IQ peptide and CaM that migrated
131
separately and were observed only in the presence of Ca2+, suggesting that the IQ peptide
132
interactions with Ca2+/CaM and apoCaM have different biophysical profiles, whereby Ca2+
133
induces the formation of a higher order complex between the IQ peptide and CaM (Figure 1D),
134
though the nature of this complex remains unknown.
135 136 137
The NT, IQ, and CT motifs bind CaM with different biophysical profiles To further examine the interactions of these CaMBDs with CaM, we performed 1H-15N
138
heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR)
139
spectroscopy on uniformly 15N-labeled CaM in the absence and presence of CaMBD peptide.
140
Each of the three peptides induced chemical shift perturbations (CSPs) in the spectra of 15N-
141
Ca2+/CaM, clearly indicative of an interaction with CaM (Figure 2A, 2C, and 2D). These results
142
further corroborated our ND-PAGE findings, as both the NT and CT peptides caused minimal
143
CSPs in the spectra of 15N-apoCaM (Figure 2B and 2F), while the IQ peptide induced a clear
144
shift in the apoCaM spectra (Figure 2D). Taken together, these findings suggested that the NT
145
and CT are Ca2+-dependent CaMBDs, while the IQ motif may be a Ca2+-independent CaMBD.
146
To determine the binding affinities of each interaction, isothermal titration calorimetry
147
(ITC) was performed with each of the CaMBD peptides. In agreement with our previous results,
148
the NT, IQ, and CT peptides each exhibited high-affinity interactions with Ca2+/CaM with 1:1
149
stoichiometries (Figure 3A, 3B, and 3C). Of these three CaMBDs, the CT had the highest
150
affinity, with a measured Kd of 7 nM (Figure 3C), whereas the measured affinity for the NT
151
peptide was approximately 5-fold lower (Kd 34 nM, Figure 3A). In the case of the IQ peptide, we
152
also performed titrations into apoCaM (Figure 3D). Titration of IQ peptide into either Ca2+/CaM
153
or apoCaM each gave clear binding data with overall 1:1 molar stoichiometry, however, the IQ
115 120
105 110 115 120
ppm
110
apoCaM apoCaM + NT
15N
B
ppm
Ca2+/CaM + NT
105
15N
A Ca2+/CaM
125 125 130 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 1H ppm
110 115 120
apoCaM apoCaM + IQ
105 110 115 120
ppm
D
ppm
Ca2+/CaM + IQ
105
15N
C Ca2+/CaM
15N
10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 1H ppm
125 125
130 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 1H ppm
120
110 115 120
ppm
115
105
15N
110
apoCaM apoCaM + CT
ppm
Ca2+/CaM + CT
F
105
15N
E Ca2+/CaM
10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 1H ppm
125 125 130 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 1H ppm
10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 1H ppm
Figure 2. 1H-15N heteronuclear single quantum coherence (HSQC)-NMR spectroscopic analysis of CaM-peptide interactions. Overlaid spectra of uniformly 15N-labeled CaM in the absence (black) or presence (red) of equimolar peptide. In all assays, spectra of 0.2 mM 15N-CaM were collected in either Ca2+ buffer (10 mM HEPES, 100 mM NaCl, 5 mM CaCl2, pH 7.5) or apo buffer (10 mM HEPES, 100 mM NaCl, 1 mM EGTA, pH 7.5), as indicated. (A) Spectra Ca2+/CaM or (B) apoCaM +/- NT peptide. (C) Spectra of Ca2+/CaM or (D) apoCaM +/- IQ peptide. (E) Spectra of Ca2+/CaM or (F) apoCaM +/- CT peptide.
A
Kd 34 nM
Ca2+/CaM + NT Molar Ratio
C
Time (min)
Kd1 18nM
Kd2 84nM
Ca2+/CaM + IQ Molar Ratio
D
Time (min)
Kd 7nM
Ca2+/CaM + CT Molar Ratio
kcal/mole of injectant
µcal/sec
µcal/sec kcal/mole of injectant
Time (min)
µcal/sec
B
kcal/mole of injectant
kcal/mole of injectant
µcal/sec
Time (min)
Kd 950 nM
apoCaM + IQ Molar Ratio
Figure 3. Isothermal titration calometry (ITC) analysis of peptide-CaM interactions. For all experiments, calorimetric titrations (top panels) and the least-squares fitted model of binding are shown (bottom panels). Calculated Kd values are shown for each modeled binding site. Data shown are representative of multiple experiments. (A) Titration of 150 µM NT peptide into 16 µM CaM in the presence of 5 mM CaCl2. (B) Titration of 205 µM IQ peptide into 20 µM CaM in the presence of 5 mM CaCl2. (C) Titration titration of 150 µM CT peptide into 16 µM CaM in the presence of 5 mM CaCl2. (D) Titration of 138 µM IQ peptide into 20 µM CaM in the presence of 1 mM EGTA.
154
peptide bound Ca2+/CaM with higher affinity and the data were best fitted to a two-site model
155
(Kd1 18 nM and Kd2 84 nM), while the IQ-apoCaM data modeled as a lower affinity interaction
156
(Kd 950 nM). Parameters calculated from these ITC measurements are shown in Supplemental
157
Table 2.
158 159 160
Mutations disrupt site-specific binding of CaM to CNGC12 To determine residues required for CaM-binding within these sites, residues within the
161
NT, IQ, and CT motifs were selected for mutagenesis based on the helical wheel projections
162
shown in Figure 1B. Hydrophobic and/or basic residues were mutated to acidic residues (Glu or
163
Asp) to disrupt CaMBD interactions with the hydrophobic clefts and acidic residues of CaM.
164
In the case of the NT site, a double substitution mutant (CNGC12L27E/K28E) was designed
165
to disrupt both the hydrophobic and basic faces of the helix, and a peptide corresponding to this
166
mutant CaMBD (named NTmut) was synthesized (Supplemental Table 1). This mutant peptide
167
did not cause a shift in the mobility of CaM in our ND-PAGE assay (Figure 4A). The ability of
168
NTmut peptide and wild type (WT) NT peptide to bind CaM was further compared in detail via an
169
HSQC-NMR titration assay (Figure 4B), in which increasing amounts of each peptide were
170
incubated with CaM in the presence of Ca2+. The WT NT peptide caused chemical shift changes,
171
exhibiting slow exchange on the chemical shift time scale in the spectra of 15N-CaM, which
172
appear saturated at a 1:1 molar ratio, indicating high affinity binding. In contrast, addition of
173
increasing amounts of NTmut peptide caused much smaller chemical shift changes, which
174
exhibited fast exchange on the chemical shift time scale and did not saturate even with addition
175
of 2.5-fold molar excess amounts of NTmut peptide, indicating that the L27E/K28E mutation
176
substantially reduced CaM binding. This result was confirmed via ITC measurement with the
177
NTmut peptide (Supplemental Figure 2A).
178
A two-residue substitution was also designed to disrupt CaM-binding to the IQ motif, and
179
a synthetic peptide bearing this mutation (I564D/Q565A, referred to as IQmut) was tested for
180
CaM-binding. This IQmut peptide showed reduced CaM-binding in our ND-PAGE assays,
181
suggesting that the I564D/Q565A mutation substantially reduces both apoCaM- and Ca2+/CaM-
182
binding (Figure 5). This reduction in binding was quantified via ITC measurement, which
183
showed that the affinity of IQmut peptide was significantly lower for Ca2+/CaM compared to the
184
IQ peptide (Supplemental Figure 2B).
A
NTmut (L27E/K28E)
NT (WT)
+ Ca2+ 0
0.25 0.5 1.0 2.0
0 0.25 0.5 1.0
2.0
NT (WT) peptide G33
106.0
110
126.0
115
8.6
8.7
127.0
I27 10.0
9.8
120
128.0
ppm
105
105.0
15N
B
125 131.0
A57
130
132.0 8.6 8.4 8.2
10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0
1H
ppm 105
106.0
110
126.0
115
8.6
8.7
I27
127.0 10.0
9.8
120
128.0
125
131.0
A57
130
132.0
8.6 8.4 8.2
15N
G33
105.0
ppm
NTmut (L27E/K28E) peptide
10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0
1H
CaM : peptide 1:0 1 : 0.1
ppm
1 : 0.5
1 : 1.0
1 : 2.5
Figure 4. The L27E/K28E mutation disrupts CaM-binding to the CNGC12 NT motif. (A) NDPAGE of Ca2+/CaM with NT peptide or a peptide bearing the double mutation L27E/K28E (NTmut at molar ratios indicated. Closed triangles indicate the migration size of CaM alone, while open triangles indicate the migration size of CaM-peptide complex. (B) Overlaid 1H-15N HSQC-NMR spectra of 0.2 mM uniformly 15N-labeled CaM with increasing molar ratios of NT (as indicated under the figure). Spectra of 0.2 mM uniformly 15N-labeled CaM in the presence of increasing molar ratios of NTmut peptide. NMR samples were prepared in 10 mM Tris-Cl, 150 mM NaCl, 10 mM CaCl2 pH 7.0. The peaks corresponding to three individual CaM residues (Gly33, Ile27, and Ala57) are shown in enlarged panels.
IQmut (I572D/Q573A)
IQ (WT)
(2)
+Ca2+ (1)
-Ca2+
0
0.25
0.5
1.0
1.5
2.0
4.0
0
0.25
0.5
1.0
1.5
2.0
4.0
Molar ratio (peptide:CaM) Figure 5. The I572D/Q573A mutation disrupts CaM-binding to the CNGC12 IQ motif. NDPAGE was performed with IQ (WT sequence) or IQmut peptide (bearing the I572D/Q573A mutation). Closed and open arrows indicate the migration of free or peptide-bound CaM, respectively, while the two distinct bands formed by the IQ-Ca2+/CaM complex are numbered 1 and 2.
185
The CT site lacks a canonical CaM target motif, and thus residues Leu605, Val607,
186
Arg609, and Lys613 were each individually mutagenized to Glu to determine if any individual
187
substitutions could disrupt CaM-binding. A fragment containing the CT (CNGC12581-649) was
188
expressed as 6xHis-tagged fusion proteins, each containing a single mutation, and assayed for
189
CaM-binding in an overlay assay. Substitution of any of these individual basic or hydrophobic
190
residues to an acidic residue (Glu) reduced CaM-binding relative to WT, with the V607E
191
mutation exhibiting the most drastic reduction (Supplemental Figure 3). We further analyzed a
192
peptide containing this mutation, and observed a loss of CaM-binding in our ND-PAGE assay,
193
while ITC measurement showed this CTmut peptide had an approximately 16-fold decrease in
194
affinity for CaM relative to the CT peptide (Supplemental Figure 3).
195 196
Loss of CaM-binding to the NT site triggers CNGC12-induced programmed cell death in
197
planta
198
Previously, a chimeric channel comprising a fusion of the N-terminus of CNGC11 and the C-
199
terminus of CNGC12 (CNGC11/12) was isolated as responsible for the phenotype of the lesion-
200
mimic Arabidopsis mutant cpr22 (constitutive expressor of PR genes 22) (Yoshioka et al., 2001,
201
2006).CNGC12 is a positive regulator of immunity (Moeder et al., 2011), and CNGC11/12 was
202
hypothesized to represent a mis-regulated form of CNGC12 that constitutively induces
203
autoimmune phenotypes including hypersensitive response (HR)-like programmed cell death
204
(PCD) in a constitutive manner (Yoshioka et al., 2006; Urquhart et al., 2007; Baxter et al., 2008).
205
Activation of such autoimmunity can be assessed via Agrobacterium-mediated transient
206
expression of At-CNGC11/12 in Nicotiana benthamiana, where CNGC11/12-GFP, but not
207
CNGC12-GFP can induce PCD (Yoshioka et al., 2006). As such, we have used this system to
208
evaluate whether the novel CaMBDs of CNGC12 may have role(s) in regulating the induction of
209
PCD.
210
Interestingly, expression of CNGC12-GFP fusion constructs with a mutation disrupting
211
either the IQ motif (CNGC12I572D/Q573A) or CT (CNGC12V607E) did not induce PCD, while PCD
212
was induced by expression of the NT mutant CNGC12L27E/K28E (Figure 6A). Although induction
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of PCD by the NT mutant was slower relative to that induced by the chimeric CNGC11/12
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channel, this cell death was never observed by the expression of WT-CNGC12 (Figure 6) and
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was consistently visible at the macroscopic level 4-5 days post-infiltration (dpi), in comparison
A
11/12
NTmut
WT
WT
CTmut
E.V.
B
TEV
E.V.
E.V.
NTmut
WT
WT
11/12
11/12
11/12 L27E/K28E
WT
L27E/K28E NTmut
L27E/K28E
11/12
Δ8
Δ8
CTmut
NTmut
IQmut
E.V.
IQmut
CTmut
Δ8 GFP
48 hpi TB
C
Conductivity (µS)
96 hpi
D CNGC12 HSR203J PR1a ACTIN
Figure 6. Transient expression of NT mutants induces programmed cell death (PCD) in Nicotiana benthamiana. (A-C) E.V. : empty vector (GFP), WT: CNGC12, 11/12 : CNGC11/12, NTmut: CNGC12L27E/K28E, IQmut: CNGC12I572D/Q573A, CTmut: CNGC12V607E, Δ8:CNGC12Δ32-39. (A) Appearance of N. benthamiana leaves four dpi (scale bar = 1 cm). Areas were infiltrated with Agrobacterium carrying different constructs. Areas showing cell death are circled in red. (B) GFP confocal microscopy of leaf areas 30 h post-infiltration (scale bar = 10 µm) and trypan blue staining (TB) for cell death of N. benthamiana leaves expressing WT or mutant CNGC12 constructs at 48 and 96 hpi, as indicated (scale bar = 0.2 mm). Infiltrated and uninfiltrated leaf areas are separated by a yellow line in samples showing cell death. (C) Ion leakage of infiltrated leaf areas 4 dpi. Values shown are averages of three replicates (error bars = standard deviation; * p
