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Integrated Protein Array Screening
and High Throughput Validation of
70 Novel Neural Calmodulin-binding
Proteins*□
S
David J. O’Connell‡, Mikael C. Bauer§, John O’Brien‡, Winifred M. Johnson¶,
Catherine A. Divizio¶, Sara L. O’Kane‡, Tord Berggård§, Alejandro Merino‡,
Karin S. Åkerfeldt¶, Sara Linse‡§储**, and Dolores J. Cahill‡ ‡‡
Calmodulin is an essential regulator of intracellular pro-
cesses in response to extracellular stimuli mediated by a
rise in Ca
2ⴙ
ion concentration. To profile protein-protein
interactions of calmodulin in human brain, we probed a high
content human protein array with fluorophore-labeled cal-
modulin in the presence of Ca
2ⴙ
. This protein array contains
37,200 redundant proteins, incorporating over 10,000
unique human neural proteins from a human brain cDNA
library. We designed a screen to find high affinity (K
D
<1
M) binding partners of calmodulin and identified 76 human
proteins from all intracellular compartments of which 72 are
novel. We measured the binding kinetics of 74 targets with
calmodulin using a high throughput surface plasmon reso-
nance assay. Most of the novel calmodulin-target com-
plexes identified have low dissociation rates (k
off
<10
ⴚ3
s
ⴚ1
) and high affinity (K
D
<1
M), consistent with the design
of the screen. Many of the identified proteins are known to
assemble in neural tissue, forming assemblies such as the
spectrin scaffold and the postsynaptic density. We devel-
oped a microarray of the identified target proteins with
which we can characterize the biochemistry of calmodulin
for all targets in parallel. Four novel targets were verified in
neural cells by co-immunoprecipitation, and four were se-
lected for exploration of the calmodulin-binding regions.
Using synthetic peptides and isothermal titration calorime-
try, calmodulin binding motifs were identified in the potas-
sium voltage-gated channel Kv6.1 (residues 474–493), cal-
modulin kinase-like vesicle-associated protein (residues
302–316), EF-hand domain family member A2 (residues 202–
216), and phosphatidylinositol-4-phosphate 5-kinase, type I,
␥
(residues 400–415). Molecular & Cellular Proteomics 9:
1118 –1132, 2010.
High content protein arrays allow for identification of pu-
tative binding partners over all cellular compartments. The
technique may be especially valuable for identifying targets
of central signaling proteins that are known to regulate a
large number of proteins, for example calmodulin. To our
knowledge, this method has not been applied to human
calmodulin. Array screening has several advantages over
other methods, for example affinity chromatography (1).
First, affinity chromatography may lead to identification of
the more abundant proteins and the capture of secondary
proteins that bind to primary calmodulin targets. On the
protein arrays, the proteins are presented in distinct loca-
tions, and secondary targets are not likely to be identified.
Second, array screening is effective in identifying interac-
tions with transmembrane proteins, including receptors and
ion channels, which are typically not available in tissue
homogenate used for identification through affinity chro-
matography (2). A further advantage of this array system is
the ability to return to the protein-expressing clone of
an identified target protein and express it for further
characterization.
Calmodulin is present in all eukaryotic cells and consti-
tutes at least 0.1% of the total cellular protein. It is ex-
pressed at higher levels in brain, testes, and rapidly growing
cells. It participates in signaling pathways that regulate
processes such as cell proliferation, learning and memory,
growth, and movement (3–5). Regulation of these events is
exerted via direct interactions of calmodulin with a large
number of cytosolic proteins, including kinases, phospha-
tases, and cytoskeletal proteins, in response to a rise in
intracellular Ca
2⫹
concentration. In the nucleus, calmodulin
is also known to transmit Ca
2⫹
signals to a number of
transcription factors (5–7). Following an extracellular stimu-
lus, Ca
2⫹
moves into the cytosol either from the outside of
the cell via plasma membrane Ca
2⫹
channels or from intra-
cellular stores. Recently, we identified calmodulin as playing
a role in the homeostasis of intracellular Ca
2⫹
through bind-
ing to the endoplasmic reticulum transmembrane proteins
From the ‡Translational Research Centre, Conway Institute of Bio-
molecular and Biomedical Research, University College Dublin,
Belfield, Dublin 4, Republic of Ireland, §Biophysical Chemistry and
储Biochemistry, Lund University, Chemical Centre, P. O. Box 124,
SE-221 00 Lund, Sweden, and ¶Department of Chemistry, Haverford
College, Haverford, Pennsylvania 19041
Received, Nov 4, 2008, and in revised form, July 17 and December 7,
2009
Published, MCP Papers in Press, January 12, 2010, DOI 10.1074/
mcp.M900324-MCP200
Research
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.1118 Molecular & Cellular Proteomics 9.6
This paper is available on line at http://www.mcponline.org
STIM1
1
and STIM2 (8). Upon binding of Ca
2⫹
, calmodulin
undergoes a conformational change, and its binding affinity
for a number of target proteins increases. The biological
action of calmodulin is governed by its biophysical proper-
ties, including cooperative Ca
2⫹
binding (9) and structural
autonomy of two globular domains (10) that cooperate in
target binding (11–14). Previous studies have identified
many putative calmodulin-binding proteins in higher and
lower organisms (1, 15–19).
In this study, we probed a high content protein array de-
rived from human brain (20) with fluorophore-labeled calmod-
ulin. The fluorophore was attached to an engineered cysteine
residue at a surface-exposed position. By this method, we
identified 76 high affinity interaction partners of calmodulin.
The targets were validated using a high throughput surface
plasmon resonance approach. We identified high affinity cal-
modulin binding to a subset of proteins known to be engaged
in the remodeling of the molecular architecture of cells such
as the dendritic spine, which is involved in Ca
2⫹
-dependent
mechanisms of synaptic function and plasticity (21). In addi-
tion to four human proteins previously known to bind cal-
modulin, we identified 72 novel human calmodulin-binding
proteins, including receptor tyrosine kinases, cytoplasmic
scaffold proteins, signaling enzymes, and cytoskeletal ele-
ments that may be regulated in a Ca
2⫹
-dependent manner
through the concerted action of calmodulin. We demon-
strated that such high affinity networks can be characterized
using a calmodulin target microarray by examining the Ca
2⫹
sensitivity of calmodulin binding to all interacting proteins in
parallel. Four putative binding sites were produced as syn-
thetic peptides, and their binding to calmodulin was verified
using isothermal titration calorimetry. We further confirmed
the binding of calmodulin to four targets from the array screen
in neural cells using immunoprecipitation of endogenous cal-
modulin with a monoclonal antibody to the C terminus of the
protein in a mouse hippocampal cell line.
EXPERIMENTAL PROCEDURES
Calmodulin Mutant Design—Through inspection of all available
high resolution structures (x-ray and NMR structures) of calmodulin in
its Ca
2⫹
-free, Ca
2⫹
-bound, and target-bound forms, Ser-17 was
identified as a small uncharged hydrophilic residue that is solvent-
exposed in all structures. The mutant Ser-17 3Cys was designed to
allow for minimal perturbation site-specific fluorophore labeling for
array screening and site-specific immobilization for surface plasmon
resonance (SPR) studies.
Expression and Purification of Recombinant Human Calmodulin—
Full-length human calmodulin was expressed from a modified Pet3a
vector (“PetSac” with NdeI and SacI cloning sites) containing a syn-
thetic calmodulin gene that was built with the codons optimal for
expression in Escherichia coli (22). The calmodulin gene with mutation
Ser-17 3Cys was amplified by PCR from this vector using primers
containing the desired base change in two steps using standard
procedures. The PCR product was digested by NdeI and SacI and
cloned into the PetSac vector. Following transformation, the wild-type
and mutant proteins were expressed in E. coli strain BL21 Des3
pLysS star, and the wild-type protein was purified as described (22).
The mutant was purified using the same protocol except that 1 mM
DTT was included until the final desalting step to keep the protein
monomeric. Briefly, the purification procedure involved sonication of
the cells, centrifugation, pouring the supernatant into boiling buffer
and heating to 85 °C to precipitate E. coli proteins, centrifugation, ion
exchange chromatography on DEAE-cellulose in buffer containing
CaCl
2
, phenyl-Sepharose chromatography (loading and washing in
CaCl
2
followed by elution with EDTA), size exclusion chromatogra-
phy, and ion exchange chromatography on DEAE-Sephacel in buffer
with EDTA. Ca
2⫹
-free (apo) calmodulin was prepared by chelating
Ca
2⫹
with EDTA and its subsequent removal on a gel filtration col-
umn. Purity was confirmed by agarose gel electrophoresis in EDTA
and Ca
2⫹
, SDS-polyacrylamide gel electrophoresis, and
1
H NMR
spectroscopy. Titrations in the presence of Quin2 (23) confirmed that
the apoprotein was free from Ca
2⫹
(residual Ca
2⫹
less than 0.04 molar
eq, which is less than 1% of full saturation).
Alexa Fluor 488 and 546 Labeling of Calmodulin S17C—Apocal-
modulin with the S17C (or Ser-17 3Cys) mutation was dissolved in
20 mMsodium phosphate buffer, pH 8 at a concentration of 10 mg/ml.
Alexa Fluor 488 or 546 (Invitrogen) (1.2 molar eq) was added from a 5
mg/ml stock in DMSO, and the sample was allowed to react for1hin
the dark at room temperature. The labeled protein (now called CaM-
Alexa488 or CaM-Alexa546) was then separated from excess label in
water on a Sephadex G25 size exclusion column, which had been
prewashed with EDTA to remove trace metal. The collected protein
was divided into aliquots for single use and stored frozen at ⫺20 °C
and subsequently diluted for the binding experiments on the arrays.
The control proteins calbindin D9k and calbindin D28k were labeled in
the same fashion via coupling to an engineered cysteine, whereas
secretagogin and anti-His tag antibody were labeled using amine-
reactive Alexa Fluor.
Calmodulin-binding Protein Profiling on High Density Protein Ar-
rays—High density protein arrays of the human brain library (hEx1)
were obtained from the German Resource Centre for Genome Re-
search (RZPD). The PVDF arrays were soaked in 95% ethanol, rinsed
in deionized water, and washed clean of residual bacterial colonies
with 20 mMTris/HCl, 500 mMNaCl, 0.05% (v/v) Tween 20, pH 7.4
(TBST) with 0.5% (v/v) Triton X-100. For calmodulin-binding protein
profiling, the protein arrays were blocked in 2% (w/v) nonfat dry milk
powder in 20 mMTris/HCl, 150 mMNaCl, pH 7.4 (TBS) for 2 h; washed
twice in TBST; and subsequently incubated with CaM-Alexa488 at a
concentration of 1
Min TBS with 1 mMCaCl
2
. The protein arrays
were washed in TBST with 1 mMCaCl
2
six times for 10 min each. The
arrays were illuminated with long wave UV light, and the images were
taken using a high resolution charge-coupled device detection sys-
tem (Fuji). Image analysis was performed with VisualGrid (GPC Bio-
tech). Hits were counted as positive if both spots of a clone (in a
characteristic pattern around a guiding point; see Fig. 1) were signif-
icantly brighter than background.
Purification of Putative Target Proteins—The individual protein-
expressing clones that express putative calmodulin targets were cul-
tured for 20 h in 10 ml of Overnight Express culture medium (24). This
medium contains a ratio of the sugars glucose and lactose that
1
The abbreviations used are: STIM, stromal interaction molecule;
DEAE, N,N⬘-diethylaminoethyl; ITC, isothermal titration calorimetry;
NTA, nitrilotriacetic acid; PSD, postsynaptic density; SPR, surface
plasmon resonance; CaM, calmodulin; NMDA, N-methyl-D-aspartate;
NMDAR, N-methyl-D-aspartate receptor; CaMVK, CaM kinase-like
vesicle-associated protein; EFHA2, EF-hand domain family member
A2; PIP5K1C, phosphatidylinositol-4-phosphate 5-kinase, type I,
␥
;
KVGCh, potassium voltage-gated channel Kv6.1; PHD, plant home-
odomain; CaMKII, Ca
2⫹
- and calmodulin-dependent protein kinase II;
RZRD, imaGenes; CTD, C-terminal domain.
Calmodulin Neural Targets
Molecular & Cellular Proteomics 9.6 1119
supports growth of the bacteria sequentially. Once the glucose is
exhausted, the bacteria begin to metabolize the lactose, thereby
depressing the T7lac promoter resulting in expression of the recom-
binant protein from the vector cassette. The recombinant proteins
were purified from the cell pellet using metal affinity chromatography
under denaturing conditions (100 mMNaH
2
PO
4
,10mMTris/HCl, 6 M
guanidine hydrochloride, pH 8.0) utilizing the polyhistidine tag for
binding to Ni
2⫹
-NTA resin. Proteins were eluted in 1 ml of elution
buffer (100 mMNaH
2
PO
4
,10mMTris/HCl, 8 Murea, pH 4.5), desalted
and concentrated in spin columns, and washed three times with PBS
(9.5 mMphosphate buffer with 153 mMNa
⫹
, 4.1 mMK
⫹
, 140 mMCl
⫺
,
pH 7.4). In cases where there was more than one expressing clone for
the same protein, we set up protein expression and purification from
several clones. Phosphorylase kinase, used as a positive control, was
purchased from Sigma.
Surface Plasmon Resonance Studies with Immobilized Calmodu-
lin—All SPR experiments were carried out using a Biacore 3000
instrument. S17C calmodulin was immobilized using ligand thiol di-
sulfide exchange coupling. The dextran matrix of a CM5 chip was
activated by injecting 25
l of a fresh mixture of 0.05 MN-hydroxy-
succinimide and 0.2 M1-ethyl-3-(3-dimethylaminopropyl)carbodiim-
ide. A reactive disulfide group was then introduced on the sensor chip
surface by injecting 20
lof100
M2-(2-pyridinyldithioethaneamine),
0.1 Msodium borate, pH 8.5. Calmodulin was then immobilized by
injecting 100
lof10
g/ml calmodulin S17C in 10 mMHCO
2
Na
(sodium formate), pH 4.3. Finally, residual 2-(2-pyridinyldithioetha-
neamine) groups were deactivated by injecting 40
lof50mML-
cysteine, 1 MNaCl, 100 mMHCO
2
Na, pH 4.3. Blank channels for
negative control were prepared by omitting calmodulin in the coupling
step. Binding of targets was surveyed by injecting 150
l of target
protein solutions in 10 mMTris/HCl, 150 mMKCl, 1 mMCaCl
2
, 0.005%
(v/v) Tween 20, pH 7.5. The same buffer was used as running buffer.
Dissociation of target protein from calmodulin was followed for 90 min
during buffer flow. The chip was then regenerated by injecting 100
l
of 350 mMEDTA, pH 8. The flow rate was 10
l/min throughout the
experiment.
Surface Plasmon Resonance studies with Immobilized Targets—
The purified calmodulin target proteins were immobilized on NTA
sensor chips. The chip was activated by injecting 20
l of 0.5 mM
NiCl
2
, and then each protein was immobilized by injecting 150
lof
target protein solution. Calmodulin association was followed during
an injection of 150
l of 700 nMcalmodulin in 10 mMTris/HCl, 150 mM
KCl, 1 mMCaCl
2
, 0.005% (v/v) Tween 20, pH 7.5. The same buffer
was used as running buffer, and the flow rate was 10
l/min. The
dissociation of calmodulin was monitored for at least 90 min during
buffer flow, and the chip was then regenerated by injecting first 50
l
of 350 mMEDTA, pH 8 and then 100
l of 0.5% (w/v) SDS followed by
a final 50-
l injection of 350 mMEDTA.
Analysis of Surface Plasmon Resonance Data—Curve fitting to all
SPR data was performed using the BIAeval software. Dissociation
phase data were analyzed by fitting a single exponential decay func-
tion to the data,
R共t兲⫽Aexp共⫺kofft兲(Eq. 1)
where Ris the response, Ais the amplitude, and k
off
is the dissocia-
tion rate constant. The variable parameters were k
off
and A.
Association phase data were analyzed by fitting the following func-
tion to the data with a fixed value of k
off
as obtained above,
R共t兲⫽Rmax共ckon/共koff ⫹ckon兲兲共1⫺exp共⫺共koff ⫹ckon兲t兲兲 ⫹R0
(Eq. 2)
where Ris the response, cis the protein concentration in the flow, k
on
is the association rate constant, R
max
is the signal that would have
been obtained at full saturation of the immobilized targets, and R
0
is
the response resulting from injection of the protein. The variable
parameters were k
on
,R
max
, and R
0
. An estimate of K
D
was obtained
as follows.
KD⫽koff/kon (Eq. 3)
In some cases, the data could not be satisfactorily fitted without
invoking two processes with separate rates, both for the dissociation
phase (Equation 4) and association phase (Equation 5).
R共t兲⫽A1exp共⫺koff,1t兲⫹A2exp共⫺koff,2t兲(Eq. 4)
R共t兲⫽Rmax,1共ckon,1/共koff,1 ⫹ckon,1兲兲共1⫺exp共⫺共koff,1 ⫹ckon,1兲t兲兲
⫹Rmax,2共ckon,2/共koff,2 ⫹ckon,2兲兲共1⫺exp共⫺共koff,2 ⫹ckon,2兲t兲兲 ⫹R0
(Eq. 5)
Endogenous Calmodulin Co-immunoprecipitation—Confluent
monolayers of mouse hippocampal Hpl 3-4 cells (25) from 10-cm
2
tissue culture plates were lysed in 0.7 ml of lysis buffer (50 mM
Tris/HCl, 150 mMNaCl, 1% Triton X-100, 2 mMCaCl
2
with protease
inhibitors (0.068 units/ml aprotinin and 2 mMPMSF)), and cells were
disrupted with repeated aspiration through a 21-gauge needle. After
centrifugation, the supernatant was precleared of nonspecific binders
to IgG and/or agarose beads by addition of 1.0
g of mouse mono-
clonal anti-M13 IgG (27-9420-01, GE Healthcare), and 20
l Protein
A/G PLUS-agarose (sc-2003, Santa Cruz Biotechnology). After incu-
bation at 4 °C for 30 min, the suspension was centrifuged at 2,500
rpm for 5 min at 4 °C to remove the agarose beads with captured
antibody. The supernatant (⬃500
g of total cellular protein) was
transferred to a fresh microcentrifuge tube on ice to which 2
gof
mouse anti-calmodulin (ab38841, Abcam) or 2
g of mouse anti-
calbindin D9k (sc-74462, Santa Cruz Biotechnology) antibody was
added and incubated for1hat4°C.Protein A/G PLUS-agarose (20
l) was added, and the suspension was incubated for2hat4°C.The
suspension was centrifuged at 2,500 rpm for 5 min at 4 °C, and the
pellet was washed our times with lysis buffer and once with PBS with
2mMCaCl
2
. The pellet (immunoprecipitates) was resuspended in
0.125 MTris/HCl, 20% glycerol, 10% 2-mercaptoethanol, 4% SDS,
0.004% bromphenol and subjected to SDS-PAGE on either 7.5 or
12% polyacrylamide gels followed by transfer to PVDF membrane
(Amersham Biosciences). The membranes were incubated in 2%
nonfat milk in TBST buffer containing rabbit polyclonal anti-potassium
voltage-gated channel Kv6.1 (anti-KCNG1; ab49063, Abcam), anti-
glutamate (NMDA) receptor subunit
1 (anti-NMDAR1; G8913,
Sigma), or anti-CaM kinase-like vesicle-associated protein (anti-
CaMKV; ab69564, Abcam) antibody, or mouse monoclonal anti-spec-
trin
␣
chain (ab11755, Abcam) antibody followed by 3 ⫻5-min
washes. The membranes were then incubated with horseradish per-
oxidase-conjugated secondary antibodies in TBST buffer, and the
enhanced chemiluminescence was measured 1 min after adding lu-
minol ECL reagent (Pierce).
Generation of Protein Microarrays—FAST slides (Schleicher &
Schuell) were placed in a QArray System (Genetix, New Milton, UK;
humidity controlled at 60–65%) with 16 blunt-ended stainless steel
print tips with a tip diameter of 150
m that were used to generate the
protein arrays. Each identified calmodulin-binding protein was spot-
ted on these FAST slides eight times in separate spots from a stock
solution with a concentration of 0.1 mg/ml. For most of the proteins,
spotting of the undiluted purified proteins led to immobilization of 15
fmol of protein/spot on the microarray. The amount of protein was
estimated based on gel band intensity on SDS-PAGE of the spotted
solutions. Each protein microarray contained several control spots,
including mouse monoclonal anti-calmodulin IgG (Sigma-Aldrich) at
Calmodulin Neural Targets
1120 Molecular & Cellular Proteomics 9.6
0.1 mg/ml, phosphorylase kinase, and myo-inositol-1(or 4)-mono-
phosphatase. A stock solution of 200 nMCaM-Alexa546 was spotted
as an internal control for signal intensity. The microarrays were
blocked in 2% milk, TBST for 1 h and washed twice for 10 min in TBS
with 1 mMEDTA and twice in TBS with 1 mMCa
2⫹
.
CaM-Alexa Screening of Protein Microarrays—The microarrays
were incubated with 1
MCaM-Alexa546 in TBS with either 1 mM
CaCl
2
or1mMEDTA for1hatroom temperature in the dark. The
microarrays were washed in the same buffer as used in the binding
experiment with 0.05% (v/v) Tween 20 3 ⫻5 min, dried, and imaged
using a Genepix scanner (4000B, Axon Instruments). The same con-
ditions were used for all control proteins tested, including secretago-
gin (purified in our laboratory; Ref. 26), calbindin D28k (purified in our
laboratory; Ref. 27), and anti-His tag antibody (28). All experiments
were conducted at least twice.
Peptide Synthesis and Purification—The four peptides correspond-
ing to the putative calmodulin-binding sites from the CaM kinase-like
vesicle-associated protein (AQIEKNFARAKWKKA; CaMKV(302–316));
EF-hand domain family member A2 (VWKGSSKLFRNLKEKG;
EFHA2(202–216)); phosphatidylinositol-4-phosphate 5-kinase, type I,
␥
(LQSYRFIKKLEHTWKA; PIP5K1C(400–415)), and the potassium
voltage-gated channel Kv6.1 (QERVMFRRAQFLIKTKSQLS;
KVGCh(474–493)) were made by solid-phase methodology using a
standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry protocol
utilizing an Applied Biosystems 433A or a PS3 Protein Technologies
Peptide Synthesizer. For all peptides, the PAL resin was used to
furnish a C-terminal carboxamide. A mixture of O-benzotriazole-
N,N,N⬘,N⬘-tetramethyluronium hexafluorophosphate and N-hydroxy-
benzotriazole was used in the activation of the free carboxylic acids
(4–10 molar eq).

-Branched amino acids and residues immediately
following a

-branched residue were coupled twice. Prior to cleavage
of the peptide from the resin, the peptides were N-acetylated with a
solution containing 0.5 Mpyridine and 0.5 Macetic anhydride in
N,N-dimethylformamide. The cleavage of the peptide from the resin
was achieved with TFA/thioanisole/1,2-ethanedithiol/anisole in a vol-
ume ratio of 9.0:0.5:0.3:0.2 for2hatroom temperature. The solution
was then concentrated with a stream of N
2
gas, and the crude peptide
was precipitated with ice-cold diethyl ether, collected by filtration,
dissolved in a mixture of water and acetonitrile, and lyophilized. The
crude peptide was purified by HPLC using a C
4
preparative column
(Vydac) and a linear gradient of water containing 0.1% TFA by volume
(solvent A) and acetonitrile/water 9:1 containing 0.1% TFA (solvent B)
with a gradient of 0.5% increase in solvent B/min. The identity of each
peptide was verified by ESI MS performed by Texas A&M University,
College Station, TX and found to be within 0.1% of the expected
molecular weight.
Isothermal Titration Calorimetry—Isothermal titration calorimetry
(ITC) was carried out using a VP-ITC microcalorimeter (MicroCal,
Piscataway, NJ). All samples were prepared by dissolving desalted
lyophilized protein or peptide in 10 mMTris, 150 mMKCl, pH 7.50 with
either 1 mMCaCl
2
or1mMEDTA and adjustment of pH to 7.50 if
needed. Each of four synthetic peptides was titrated from a 200
M
stock solution into 10
Mwild-type human calmodulin at 25 °C. An
initial injection of 5
l was followed by 29 injections of 10
l of peptide
solution with 5-min equilibration time between injections. The con-
centrations of peptide stocks were determined by amino acid analysis
after acid hydrolysis (analysis purchased from BMC, Uppsala, Swe-
den), and the calmodulin concentration was determined by absorb-
ance using ⫽3200 liters mol
⫺1
cm
⫺1
. Data were fitted using the
Orgin 7 software (MicroCal) and a 1:1 binding model.
RESULTS
Identification of Putative Calmodulin Targets on Human
Protein Array—In this work, we used high content human
protein arrays on 22.2 ⫻22.2-cm PVDF membranes (20).
Each protein array was robotically spotted in a standard 5 ⫻
5 spotting pattern with each array containing 27,648 protein
spots in duplicate, making a total of 55,296 protein spots per
array. The protein collection of 37,200 clones was spotted as
follows: one membrane with 27,648 individual clones spotted
in duplicate and one membrane with 9,552 individual clones
spotted in duplicate multiple times. Together, the two mem-
branes held 37,200 redundant proteins of which over 10,000
were estimated to be unique (non-redundant) human proteins
based on sequencing of a subset of the clones (29). Twelve
proteins were arrayed in duplicate in the 5 ⫻5 spotting
pattern around a central ink guiding point (Fig. 1). Before use,
the membranes were washed clean of residual bacterial col-
onies, leaving the expressed proteins on the membrane. The
array was screened for calmodulin-binding proteins in the
presence of 1 mMCa
2⫹
using calmodulin labeled at a cysteine
residue with the fluorescent probe Alexa Flour 488 (CaM-
Alexa488; Fig. 2A). The cysteine was site-specifically substi-
tuted for serine 17, which is solvent-exposed in all known
structures of calmodulin, causing minimum perturbation of
the binding interactions.
We initially identified 1,200 clones as binding CaM-
Alexa488 on the high content redundant array after a 10-min
wash in 20 mMTris/HCl, 150 mMNaCl, 1 mMCaCl
2
, pH 7.5.
This set may include a number of lower affinity target proteins.
Following a series of more stringent washes (6 ⫻10 min with
20 mMTris/HCl, 500 mMNaCl, 1 mMCaCl
2
, 0.05% Tween 20,
pH 7.5) this number was reduced to 480 positives clones,
FIG.1. Scoring of human protein (hEx1) arrays with VisualGrid
software. A, scoring pattern for each of 12 clones spotted in dupli-
cate around a central guide dot. B,1
MCaM-Alexa488 binding to a
field of the human protein array with positive clones highlighted in
green squares on the blue grid that identifies each block of 24 spots.
Calmodulin Neural Targets
Molecular & Cellular Proteomics 9.6 1121
FIG.2.Equivalent fields of high content protein array incubated overnight with 1
Mprotein labeled with Alexa Fluor 488 in TBS buffer
with 1 mMCaCl
2
followed by 6 ⴛ10-min washes in TBST buffer for total of 60 min. A, calmodulin; B, secretagogin; C, calbindin D28k; D,
calbindin D9k.
Calmodulin Neural Targets
1122 Molecular & Cellular Proteomics 9.6
representing a set of high affinity binding proteins. DNA se-
quencing of the clones led to the identification of a non-
redundant set of 76 high affinity partners of human calmod-
ulin, 72 of which were not previously identified as calmodulin
binding (Table I and supplemental Table S1). For 13 of these
72 proteins, either a mouse or nematode (Caenorhabditis
elegans) homologue has been reported to bind to calmodulin
(1, 19). A subset of these proteins was identified as proteins
involved in the organization of the postsynaptic density con-
sisting of four known and eight novel calmodulin-binding pro-
teins (supplemental Table S2).
The inbuilt redundancy of arrayed proteins serves as a
useful internal control in determining protein-protein inter-
actions. This is manifested by binding of calmodulin to
many spots that correspond to the same protein. For ex-
ample, the ribosomal protein S2 was expressed by approx-
imately half of the clones whose protein product was bound
by calmodulin on the array. On the other hand, not all
existing proteins are represented on the array. The lack of
identification of some known targets may be due to factors
such as lack of full-length protein, structural constraints,
high off rates, etc.
Control experiments were performed with three Alexa Fluor
488-labeled proteins from the calmodulin superfamily of EF-
hand proteins, which all are negatively charged at neutral pH.
For the hydrophilic protein calbindin D9k, we found no puta-
tive targets on the arrays (Fig. 2D), indicating that the hits
found for calmodulin are neither mediated by a negative EF-
hand protein surface nor the Alexa Fluor 488 label. For each of
the hydrophobic EF-hand proteins, calbindin D28k (Fig. 2C)
and secretagogin (Fig. 2B), we observed a distinct set of
putative targets. None of the 13 targets for secretagogin are
shared with calmodulin, whereas one of 48 targets for calbi-
ndin D28k is shared with calmodulin (pyruvate kinase
isozymes M1/M2). This clearly shows that the putative cal-
modulin targets are not binding because of an unspecific
hydrophobic interaction or a higher abundance of the target
on the arrays. In total, 75 of the 76 putative targets are unique
to calmodulin.
Validation of Binding Partners by SPR—Using SPR assays
with either immobilized calmodulin (thiol-linked to CM5 sen-
sor chip) or linked target protein (His tag to Ni
2⫹
-NTA sensor
chip), 74 of the 76 interactions were successfully validated.
Two proteins were not validated because they failed to ex-
press in liquid culture. A range of binding affinities and rate
constants was observed for the different classes of binding
proteins as apparent from variable shapes of the binding
curves (Fig. 3 and supplemental Fig. S2). For the proteins that
were validated using thiol-linked calmodulin, the dissociation
rate constant, k
off
, was estimated from fitting Equation 1 to the
dissociation phase data and found to be in the range of
10
⫺3
–10
⫺6
s
⫺1
(supplemental Table S3). The concentration of
each target was estimated from the intensity of gel bands on
SDS-PAGE (supplemental Fig. S1), providing association rate
constants by fitting Equation 2 to the association phase data.
The k
on
values were found to be in the range of 10
2
–10
5
M
⫺1
s
⫺1
(supplemental Table S3) and are accurate to within 1
order of magnitude (⫾0.5 log
10
units). The equilibrium disso-
ciation constant, K
D
, was calculated from the estimated as-
sociation and dissociation rate constants according to Equa-
tion 3 (Table I). All targets were studied at least twice using
either one or both SPR approaches. The precision of the SPR
measurements was evaluated by using the known calmodu-
lin-binding protein, phosphorylase kinase. Using the same
method as for the novel targets, we determined a K
D
of 0.6 nM
for the interaction between calmodulin and phosphorylase
kinase (supplemental Fig. S3), whereas previously published
values for peptide fragments derived from phosphorylase ki-
nase are 1 nMor higher, i.e. within approximately 0.2 log
10
units (30). To check the reproducibility of the reported rate
and equilibrium constants, six randomly chosen proteins
(coiled-coil-helix-coiled-coil-helix domain-containing protein
2, lysophospholipid acyltransferase 7, bromodomain and
PHD finger-containing protein 3, NADH dehydrogenase, and
Znf358) were analyzed three times on different SPR chips.
Both association and dissociation rates showed little differ-
ence between experiments, and the standard deviation for the
equilibrium dissociation constants was 0.3 log
10
Kunits. To-
gether with the precision of the protein concentration deter-
mination, which is needed to estimate the association rate
constant, we thus found it appropriate to report rate constants
and dissociation constants with a precision of 1 order of
magnitude (⫾0.5 log
10
units). The complementary approach
using immobilization of the target proteins to nickel (Ni
2⫹
)-
coated NTA sensor chips may be preferred because the high
precision in the injected calmodulin concentration (700 nM)
permitted better estimation of the association rate constant
and thereby equilibrium constant for 17 proteins. For several
proteins, however, calmodulin binding after His tag immobili-
zation was ineffective, possibly because of steric hindrance of
the calmodulin binding segment.
Endogenous Calmodulin Co-immunoprecipitation—Mouse
hippocampal Hpl 3-4 cell lysates were subjected to co-immu-
noprecipitation using anti-calmodulin or mouse anti-calbindin
D9k antibody as the precipitating antibody followed by West-
ern blotting with rabbit polyclonal antibodies against gluta-
mate (NMDA) receptor subunit
1 (NMDAR1; Fig. 4A), KVGCh
(Fig. 4B), and CaMKV (Fig. 4C) and a mouse monoclonal
antibody against spectrin
␣
chain (Fig. 4D). All four targets
were detected in complex with calmodulin in the cell lysates,
whereas no target was found in complex with the negative
control protein calbindin D9k. All four proteins chosen for this
study are novel human calmodulin targets, and the potassium
voltage-gated channel Kv6.1 has not previously been found in
any organism.
Preparation and Studies of Calmodulin-binding Protein Mi-
croarray—Expressed proteins were printed onto FAST slides
and incubated with anti-His tag antibody (Fig. 5A), 1
M
Calmodulin Neural Targets
Molecular & Cellular Proteomics 9.6 1123
TABLE I
Calmodulin target proteins with accession number (Swiss-Prot), K
D
, and whether previously identified as a calmodulin-binding
protein indicated (italics)
The affinity estimated from fitting to SPR data is given as K
D
with values estimated within 1 order of magnitude (⫾0.5 log
10
units). Human
proteins that are previously known as calmodulin targets are highlighted in italics. References to earlier work are given for mouse (1), rat (59)
and C. elegans (19) homologues. ND, not determined. CTD, C-terminal domain.
Protein Accession no. K
D
Membrane proteins
Solute carrier family 16, member 8/MCT3 O95907 1 nM
Solute carrier family 7, member 5 Q01650 100 nM
Neuron-specific protein family member 2 Q9Y328 1 nM
Plasticity-related protein 2 (1) Q6T4P5 100 pM
Potassium voltage-gated channel Kv6.1 Q9UIX4 100 nM
Glutamate (NMDA) receptor subunit
1 precursor Q05586 10 nM
Tetraspanin-7 P41732 1 nM
Lysophospholipid acyltransferase 7 Q96N66 100 pM
Semaphorin 3A (1) P51805 1 nM
Transmembrane protein 9B precursor Q9NQ34 10 nM
Semaphorin 4C precursor Q9C0C4 10 nM
Cleft lip- and palate-associated transmembrane protein 1 O96005 1 nM
Receptor accessory protein 2 Q9BRK0 10 nM
Fibroblast growth factor receptor 3 precursor P22607 100 pM
Yip1-interacting factor homologue B isoform 2 Q5BJH7 10 nM
STIM1 Q13586 100 nM
Similar to double C2-like domain-containing protein

(1) Q14184 1
M
Ras-related protein Rab-11B (1) Q15907 10 nM
Syntaxin-18 Q9P2W9 10 nM
Sterol-regulatory element-binding protein cleavage-
activating protein
Q12770 1 nM

-Sarcoglycan Q16585 1 nM
Regulator of G-protein signaling 19 P49795 10 nM
EF-hand domain family, member A2 Q86XE3 10 nM
CaM kinase-like vesicle-associated (59) Q8NCB2 1 nM
Synaptosomal associated protein 29 O95721 1 nM
Serine incorporator 2 Q96SA4 1
M
Receptor-type tyrosine-protein phosphatase
precursor P23471 100 nM
Coiled-coil-helix-coiled-coil-helix domain containing
protein 2
Q9Y6H1 10 nM
CaM-kinase II
␣
chain Q9UQM7 100 nM
APLP1 P51693 10 nM
Coiled-coil domain-containing 88C Q9P219 10 pM
Nuclear proteins
ZNF527 Q8NB42 100 nM
ZNF238 Q99592 10 nM
ZNF358 Q9NW07 10 nM
ZNF330 Q9Y3S2 10 nM
Transcription factor IIIA Q92664 100 nM
Heterogeneous nuclear ribonucleoprotein D-like O14979 1
M
Zinc fingers and homeoboxes 2 Q9Y6X8 1 nM
Bromodomain and PHD finger-containing protein 3 Q9ULD4 1 nM
RNA binding motif protein 4 Q9BWF3 10 nM
RNA-binding protein 4B Q9BQ04 10 nM
Decapping enzyme, scavenger Q96C86 10 nM
RNA-binding protein 5 P52756 1 nM
G
1
to S phase transition 1 (1) P15170 10 nM
Inhibitor of growth family, member 4 (p29ING4) Q9UNL4 10 nM
DnaJ homologue subfamily C member 2/zuotin-related
factor 1
Q99543 10 nM
Ran-binding protein 3 Q9H6Z4 100 pM
Glioma tumor suppressor candidate region gene 2/p60 Q9NZM5 1 nM
TP53I11 O14683 10 nM
Phosphorylated CTD-interacting factor 1 Q9H4Z3 ND
Calmodulin Neural Targets
1124 Molecular & Cellular Proteomics 9.6
CaM-Alexa546 in 1 mMCa
2⫹
(Fig. 5B), or 1 mMEDTA (Fig. 5C).
The slides were washed with buffer at the same Ca
2⫹
or EDTA
concentration followed by imaging. In the presence of Ca
2⫹
,
strong binding was observed to the proteins on the microarray
(Fig. 5B), whereas no signal was detected in Ca
2⫹
-free buffer
(Fig. 5C), highlighting the Ca
2⫹
dependence of the interaction
between calmodulin and the arrayed proteins. We also ob-
served a differential intensity of binding of calmodulin to indi-
vidual members of the microarray that will warrant future
studies over a range of Ca
2⫹
concentrations. Control exper-
iments were performed with a 1
Mconcentration of the
EF-hand proteins secretagogin-Alexa546, calbindin D28k-
Alexa546, and calbindin D9k-Alexa546 in the presence of 1
mMCa
2⫹
. No binding of these proteins was observed to the
calmodulin target microarray, whereas all microarray proteins
were detected with the anti-His tag antibody.
Putative Calmodulin Binding Motifs—The identified cal-
modulin-binding proteins were investigated for the presence
of putative calmodulin binding motifs using the search engine
at a web-based database (31). Putative binding sites were
found for 67 of 74 proteins, and a list of the highest scoring
motif for each protein is presented in supplemental Table S4.
Based on this list, synthetic peptides were made correspond-
ing to the putative binding sites of three transmembrane pro-
teins and one cytoplasmic protein. A so-called 1-5-10 motif
was predicted at residues 302–316 of CaM kinase-like vesi-
cle-associated protein (AQIEKNFARAKWKKA). A peptide with
this sequence, CaMKV(302–316), was synthesized, purified,
and used in binding studies. For two other proteins, con-
taining no known motifs, the regions with highest scores
were selected (supplemental Table S4). Thus, peptides cor-
responding to residues 200–216 of EF-hand domain family
member A2 (VWKGSSKLFRNLKEKG; EFHA2(202–216)) and
residues 400–415 of phosphatidylinositol-4-phosphate
5-kinase, type I,
␥
(LQSYRFIKKLEHTWKA; PIP5K1C(400–
415)) were synthesized, purified, and used in binding stud-
ies. For the potassium voltage-gated channel Kv6.1, seven
putative binding regions were found by the database
search, and four of these (residues 284–303, 308–347,
361–380, and 474–493) are part of the protein expressed by
the clone on the array (residues 218–513). The highest
score was obtained for residues 361–380 (LGLQTLGLTAR-
RCTREFGLL). The score for the sequence comprising res-
idues 474–493 (QERVMFRRAQFLIKTKSQLS) was 4 times
lower but was judged more promising by manual inspection.
The 474–493 sequence was therefore chosen, and the cor-
TABLE I—continued
Protein Accession no. K
D
Cytoskeletal proteins
Tubulin folding cofactor B Q99426 10 nM
Dynein, cytoplasmic, heavy polypeptide 1 Q14204 1
M
Actin,

P60709 1 nM
Actin,
␥
1 P63261 1 nM
Centromere protein B, 80 kDa P07199 1 nM
Kinesin heavy chain isoform 5C (1) O60282 1 nM
Spectrin
␣
chain Q13813 10 nM
Cytoplasmic proteins
NLR family member X1 Q86UT6 1 nM
Elongation factor 2 P13639 1 nM
SRC-like adapter Q13239 1 nM
STIP1 homology and U-box-containing protein 1 Q9UNE7 1 nM
Pyruvate kinase isozymes M1/M2 (1) P14618 1 nM
Phosphatidylinositol-4-phosphate 5-kinase, type I,
␥
(19) O60331 1 nM
Triose-phosphate isomerase 1 variant (1) P60174 10 nM
Diphosphomevalonate decarboxylase P53602 100 nM
PDZ domain-containing 4 Q76G19 10 nM
Stathmin 1/oncoprotein 18 (1) P16949 ND
Ubiquitin-like protein FUBI P35544 10 nM
Kelch-like protein 21 Q9UJP4 1 nM
Mitochondrial proteins
NADH dehydrogenase (ubiquinone) Fe-S protein 7, 20
kDa (19)
O75251 1 nM
Protein from Golgi
Proprotein convertase subtilisin/kexin type 1 inhibitor (1) Q9UHG2 100 nM
Ribosomal proteins
40 S ribosomal protein S2 (1) P15880 1 nM
60 S ribosomal protein L9 P32969 1 nM
60 S ribosomal protein L14 P50914 1
M
Uncategorized proteins
hCG1805852 EAW73369 1 nM
Unnamed protein product BAC85305 1 nM
Calmodulin Neural Targets
Molecular & Cellular Proteomics 9.6 1125
responding synthetic peptide, KVGCh(474–493), was syn-
thesized, purified, and used in binding studies.
Calmodulin Binding to Motif Peptides by ITC—The binding
of calmodulin to the four synthetic peptides was studied in the
presence and absence of Ca
2⫹
using ITC (Fig. 6). An exothermic
process was observed for calmodulin binding to the
KVGCh(474–493) peptide in 10 mMTris/HCl, 150 mMKCl,
FIG.3.Surface plasmon resonance studies. Aand B, schematics outlining the two SPR approaches with target proteins immobilized via
His tag to Ni
2⫹
-NTA sensor chips (A) or calmodulin immobilized via a thiol linker to CM5 sensor chips (B). Shown are representative
sensorgrams from SPR studies of calmodulin-target interactions in different kinetic ranges for calmodulin binding to His tag-immobilized
ribosomal protein S2 (black), APLP1 (red), dynein (blue), and transcription factor IIIA (green)(C) and target protein binding to immobilized
calmodulin for ZHX2 (black), elongation factor 2 (red), solute carrier family 16, member 8/MCT3 (blue), and semaphorin 4C (green)(D). All data
were obtained in 10 mMTris/HCl, 150 mMKCl, 1 mMCaCl
2
, 0.005% (v/v) Tween 20, pH 7.5.
FIG.4.Western blots of immunoprecipitates from hippocampal
cell lysates using either anti-calmodulin IgG (left lane in each
panel) or anti-calbindin D9k IgG (right lane in each panel) in
immunoprecipitation (IP) step and anti-glutamate (NMDA) recep-
tor subunit
1(A), anti-potassium voltage-gated channel Kv6.1
(B), anti-CaMKV (C), or anti-spectrin
␣
chain IgG (D) in the immu-
noblotting (IB) detection step.
FIG.5.Ca
2ⴙ
sensitivity of binding of calmodulin to calmodulin-
interacting proteins on protein microarray. The microarray was
incubated with anti-RGSHis
6
and Cy3-labeled anti-mouse IgG (A), 1
MCaM-Alexa546 in 1 mMCaCl
2
(B), and 1
MCaM-Alexa546 in 1
mMEDTA (C). Lane 1, diphosphomevalonate decarboxylase; lane 2,
ribosomal protein S2; lane 3, dynein; lane 4, ZNF358; lane 5, CaM
kinase II
␣
;lane 6, buffer; lane 7, CaM-Alexa546.
Calmodulin Neural Targets
1126 Molecular & Cellular Proteomics 9.6
1mMCaCl
2
, pH 7.5 (Fig. 6a,upper panel). The negative signals
obtained indicate that the reaction produces heat, and therefore
less heat needs to be added to the sample compared with the
reference cell to keep them at constant temperature. Fitting to
the integrated data (Fig. 6a,lower panel) using a 1:1 binding
model resulted in an estimation of the equilibrium dissociation
constant K
D
of 500 nM. The signals obtained in the presence of
1m
MEDTA (Fig. 6b) were much smaller than in the presence of
FIG. 6. Shown is isothermal titration calorimetry at 25 °C for peptides titrated from 200 or 400
Msolutions into 10
Mcalmodulin in 10 mM
Tris, 150 mMKCl, pH 7. 50 with either 1 mMCaCl
2
(a,d,e, and g)or1mMEDTA (b) or peptide titrated into buffer (c). An initial injection of 5
l was followed by 29 injections of 10
l of peptide solution with 5-min equilibration time between injections. The upper panels show the raw
data. The lower panels show integrated heats versus molar ratio of peptide to protein, and the solid lines represent the best fit to the data using
a 1:1 binding model. aand b, KVGCh(474–493) titrated into calmodulin in the presence of 1 mMCaCl
2
(a)or1mMEDTA (b). c, KVGCh(474 –493)
titrated into buffer with no calmodulin. d, EFHA2(202–216) titrated into calmodulin in the presence of 1 mMCaCl
2
.e, CaMKV(302–316) titrated
into calmodulin in the presence of 1 mMCaCl
2
.d, PIP5K1C(400–415) titrated into calmodulin in the presence of 1 mMCaCl
2
.
Calmodulin Neural Targets
Molecular & Cellular Proteomics 9.6 1127
Ca
2⫹
and of the same magnitude as the friction heat observed
when water was injected into water (not shown) or the peptide
was injected into buffer (Fig. 6c).
Exothermic processes were observed in buffer with 1 mM
CaCl
2
for calmodulin binding to the three peptides
EFHA2(202–216) with a K
D
of 1.7
M(Fig. 6d), CaMKV(302–
316) with a K
D
of 740 nM(Fig. 6e), and PIP5K1C(400–415)
with a K
D
of 400 nM(Fig. 6f). Weaker binding was observed for
all three peptides in the presence of EDTA (data not shown):
EFHA2(202–216) with a K
D
of 3.2
M, CaMKV(302–316) with a
K
D
of 1.9
M, and PIP5K1C(400–415) with a K
D
of 30
M.
DISCUSSION
Specificity and sensitivity are undoubtedly the most critical
issues for high throughput protein-protein interaction screen-
ing methods. Validation of the affinities for targets that are
identified in high throughput systems using independent
quantitative methods must therefore be an integral part of the
experimental design. This consideration is of particular rele-
vance in this study where more than 90% of the identified
proteins are novel, having not been identified as human cal-
modulin binding partners in previous studies, and comprise
many different protein classes and pathways that may be
dependent on Ca
2⫹
signaling in cells.
Through the use of two complementary high throughput
SPR approaches, a high degree of confidence in a large
number of identified targets was achieved (Table I and
supplemental Table S1) with 97% (74 of 76) of the targets
confirmed. The high level of validation is particularly powerful
considering the large number of novel interacting proteins and
in particular for those proteins that have, as yet, no known
function. With the microarray, we can further investigate the
role of calmodulin in regulating neural processes and study in
parallel the differential inhibition of calmodulin targets by for
example motif peptides. The human neural calmodulin set of
binding proteins described here includes two proteins that have
not been characterized in any way beyond their sequence iden-
tification and genome location. Their interaction with calmodulin
offers a first insight into the function of these uncategorized
proteins, which may be a Ca
2⫹
-regulated process.
Screening the large arrays of human proteins with three
different control proteins from the same protein family
showed that the putative calmodulin targets are specific and
were not found due to unspecific electrostatic or hydrophobic
interactions or were not due to interaction of array proteins
with the Alexa Fluor 488 label on an EF-hand protein surface.
In array screening, there is also risk for artifacts due to the
higher abundance of some proteins on the array; however,
screening with anti-His tag antibody showed that the identi-
fied calmodulin targets are of similar abundance as other
expressed proteins on the array.
The verified targets displayed low dissociation rate con-
stants (k
off
ranging from 10
⫺3
to 10
⫺6
s
⫺1
) and high affinity (K
D
ranging from 10 pMto 1
M), consistent with the design of the
screen. The variation in binding kinetics may be related to the
biological roles of the targets in the cell. For example, tran-
scription factor IIIA has a K
D
of 100 nMand is characterized by
a high on rate and a high off rate (Fig. 3), whereas the protein
RPS2 displayed a much higher affinity for calmodulin with a
K
D
of about 1 nM, a lower on rate, and a significantly lower off
rate. These proteins may be regulated differently by calmod-
ulin in response to Ca
2⫹
oscillation in the cell.
The human proteins on the high content arrays are heter-
ologously expressed in E. coli and are therefore not post-
translationally modified; however, modifications such as
glycosylation are not expected for intracellular binding tar-
gets for calmodulin. Refolding of arrayed proteins may not
be spontaneous after induced protein expression on PVDF
membranes followed by lysis of bacterial colonies, and
some proteins may remain partially denatured on the array
membrane. For many known targets, however, the binding
region has been found to be a contiguous segment that is
unfolded when not bound to calmodulin. Previous studies
have also shown that short peptides with the target binding
sequence often retain high affinity binding to calmodulin
(32). Unfolding of the targets on the array may therefore not
significantly affect their affinity for calmodulin, which is a
key consideration in the success of our screen. For mem-
brane proteins, this feature may be especially important as
they are unlikely to be correctly folded on the arrays. How-
ever, it is recognized that the binding of calmodulin to
discontinuous epitopes is not likely to be identified using
this method.
The identified target proteins were investigated for the pres-
ence of putative calmodulin binding motifs using a web-based
database (31). As summarized in supplemental Table S4, this
procedure identified putative binding regions in most of the
targets. However, only for a few proteins are there previously
known types of target motifs. It is therefore likely that a
number of novel motifs will be identified within this set of
binding partners. For example, the calmodulin binding domain
of STIM1 and -2 is a novel motif containing hydrophobic and
positively charged residues (8) not identified by the search
engine. In addition, mutational studies have shown retained
affinity for calmodulin upon removal of positive charges or
substitution of hydrophobic residues in binding motifs (33,
34). This reflects a relatively high level of sequence variability
and heterogeneity of calmodulin target motifs, and improved
bioinformatics tools may be developed as more motif se-
quences become identified.
Four proteins were taken all the way from being identified as
putative calmodulin targets on the large arrays of human pro-
teins through validation as high affinity targets by SPR and
finally to calmodulin binding motif discovery, and one of them,
the potassium voltage-gated channel Kv6.1, was also verified
as a calmodulin binder in neural cells by co-immunoprecipita-
tion. A peptide corresponding to one of the predicted binding
Calmodulin Neural Targets
1128 Molecular & Cellular Proteomics 9.6
sites for the potassium voltage-gated channel Kv6.1 was thus
found to bind calmodulin with high affinity in a Ca
2⫹
-dependent
manner and with an equilibrium binding constant similar to the
one found for the protein by SPR. Likewise, we discovered
novel calmodulin-binding sites in EF-hand domain family mem-
ber A2; phosphatidylinositol-4-phosphate 5-kinase, type I,
␥
;
and a motif of known type (1-5-10 motif) at residues 302–316 of
the CaM kinase-like vesicle-associated protein.
Immunoprecipitation of endogenous calmodulin with a
monoclonal antibody facilitated the successful detection of
the membrane proteins potassium voltage-gated channel
Kv6.1 and NMDA receptor subunit 1 in complex with calmod-
ulin, suggesting an expanded role of calmodulin in regulation
of these ion channels in hippocampal cells. The co-immuno-
precipitation of vesicle-associated calmodulin kinase in these
cells is added confirmation of high affinity binding of calmod-
ulin to a protein that can travel to dendritic processes in the
vesicular machinery supporting synaptic plasticity. The co-
immunoprecipitation of spectrin
␣
chain from these cells with
calmodulin may serve as a starting point to study the activity
of calmodulin in organizing the spectrin scaffold. The confir-
mation of biophysical data with in-cell binding assays al-
though non-quantitative provides evidence of the significance
of these high affinity networks in neural cells.
Target Classification—We identified proteins located in the
cytosol, nucleus, mitochondria, endoplasmic reticulum, Golgi
apparatus, cytoskeleton, and intracellular and plasma mem-
branes (supplemental Table S1). These proteins include
metabolic enzymes, transcription factors, ion channels, trans-
porters, receptors, and transmembrane proteins. The identi-
fication of a protein as a calmodulin target and the measure-
ment of the affinity and exchange rate do not prove any
regulatory role of calmodulin. Nevertheless, the data pre-
sented here may serve as a starting point to investigate the
role of calmodulin in a number of critical processes, and we
will, in the following discussion, highlight the roles of some of
the identified targets. A significant number of the identified
target proteins are known to be involved in neural processes
including central nervous system development, synaptic
function, and neuroendocrine secretion. For example, four
previously known calmodulin-binding proteins, NMDAR,
spectrin, actin, and Ca
2⫹
- and calmodulin-dependent protein
kinase II
␣
(CaMKII
␣
), play a fundamental role in the organiza-
tion of the postsynaptic density (PSD) in dendritic spines (35,
36). In addition, we identified a number of novel partners for
calmodulin that are fundamentally important to this process
(supplemental Table S2). The PSD dynamically changes its
structure and composition during development and in re-
sponse to synaptic activity and is described as a huge mem-
brane-associated protein complex specialized for postsynap-
tic signaling and plasticity (37–40). The Ca
2⫹
regulation of
these processes in response to NMDA receptor activation is
exemplified by the role of CaMKII
␣
in determining the strength
of the synaptic transmission (41) and, ultimately, in storage of
information in the brain. The identification here of high affinity
calmodulin binding to several new proteins involved in PSD
architecture raises important questions as to the greater role
of calmodulin in synaptic plasticity.
Transmembrane Proteins—We identified the binding of cal-
modulin to 29 novel transmembrane proteins and two previ-
ously known partners (Table I). The previously known part-
ners, CaMKII and glutamate receptor, are integrally involved
in processes underlying learning and memory. The novel high
affinity targets may reveal new insights into the Ca
2⫹
regula-
tion of synaptic plasticity. All 31 membrane proteins were
validated, and their affinity of binding to calmodulin was es-
timated by fitting to the SPR data. The affinities for the 31
membrane proteins range from 10
6
to 10
11
M
⫺1
(K
D
values
range from 10 pMto 1
M) in the presence of Ca
2⫹
. A very
strong effect of Ca
2⫹
is illustrated by the fact that no binding
was observed to the calmodulin target microarrays in the
absence of Ca
2⫹
. The identification of this set of membrane
proteins as calmodulin targets may also provide new insights
into the role of calmodulin in regulating transport processes
over membranes. For example, we have recently indicated a
role for calmodulin in regulating store-operated Ca
2⫹
entry by
binding to the endoplasmic reticulum single pass transmem-
brane protein STIM1, a protein identified using this approach
(8). Another single pass transmembrane protein that we iden-
tified here as a calmodulin target is the EF-hand domain family
member A2 (K
D
⫽10 nM). Using ITC, a calmodulin-binding site
was found in the cytosolic portion of this protein at residues
202–216 (VWKGSSKLFRNLKEKG; K
D
⫽1.6
M). The drop in
affinity for the peptide compared with intact protein suggests
that either additional intermolecular interactions with the in-
tact target contribute to the affinity or that the binding site is
somewhat larger or shifted by a few residues.
Potassium voltage-gated channels are important compo-
nents of the dendritic spine and PSD (42). We observed high
affinity calmodulin binding (K
D
⫽100 nM) to the potassium
voltage-gated channel isoform Kv6.1 (Swiss-Prot accession
number Q9UIX4) and to a peptide corresponding to residues
474–493 (QERVMFRRAQFLIKTKSQLS; K
D
⫽500 nM). The
affinity for the peptide is of the same order of magnitude as for
the intact protein, suggesting complete coverage of this bind-
ing site. Moreover, strong Ca
2⫹
dependence was found both
for the interaction between calmodulin and Kv6.1 protein and
for the interaction between calmodulin and KVGCh(474–493)
peptide. Residues 474–493 constitute a novel calmodulin-
binding site, located in the C-terminal cytoplasmic part of
Kv6.1, which is of different length and sequence compared
with the previously identified calmodulin-binding Kv7.1–7.5
(43). Thus, calmodulin may regulate voltage-gated K
⫹
cur-
rents through several targets.
A number of other important proteins in the dendritic spine
and PSD were identified as calmodulin targets, for example
semaphorin 4C, semaphorin 3A, and dynein heavy chain.
Semaphorins are known to act as chemorepulsive molecules
Calmodulin Neural Targets
Molecular & Cellular Proteomics 9.6 1129
that guide axons during neural development. For example,
semaphorin 3A signaling pathways have been shown to play
an important role in the regulation of dendritic spine matura-
tion in cerebral cortex neurons (44, 45). Dynein heavy chain is
proposed to be a major component of PSD and is present in
dendritic spines, raising the possibility that cytoplasmic dy-
nein plays structural and functional roles in the postsynaptic
terminal (46).
Vesicle fusion and secretion are important synaptic pro-
cesses that are regulated by Ca
2⫹
. We identified the CaM
kinase-like vesicle-associated protein as a high affinity cal-
modulin target (K
D
⫽1nM). This protein contains a so called
1-5-10 motif at residues 302–316 (AQIEKNFARAKWKKA), and
we showed here that a peptide with this sequence,
CaMKV(302–316), binds to calmodulin (K
D
⫽600 nM). The
novel calmodulin target monocarboxylate transporter 2
(MCT2) is located on vesicular membranes within the
postsynaptic spine in rodent brain (47). Synaptosomal asso-
ciated protein 29 (SNAP29) was found here to bind to cal-
modulin with high affinity, whereas SNAP25 was previously
identified as a target of another Ca
2⫹
-regulatory protein of the
calmodulin superfamily, secretagogin (26). The protein similar
to double C2-like domain-containing protein

(DOC2-

) be-
longs to a family of proteins identified in mouse brain to
regulate synaptic vesicle docking (48).
Cytoplasmic Proteins—The 12 cytoplasmic proteins are all
novel. The binding of calmodulin to PIP5K1C (K
D
⫽1nM) may
be of particular interest as a potential new link between two
signaling pathways of central importance, Ca
2⫹
signaling and
phosphoinositide signaling (5). PIP5K1C acts to phosphor-
ylate phosphatidylinositol 4-phosphate to generate phos-
phatidylinositol 4,5-bisphosphate. Phosphatidylinositol 4,5-
bisphosphate regulates several cellular processes, including
actin assembly and vesicle trafficking, processes that seem to
involve calmodulin at several levels. A calmodulin-binding site
in human PIP5K1C was identified around residues 400–415
(LQSYRFIKKLEHTWKA; K
D
⫽400 nM). A C. elegans homo-
logue of PIP5K1C has previously been found to bind calmod-
ulin in a screening study (19). Besides the novel interaction
between calmodulin and PIP5K1C, the Ca
2⫹
and phosphoi-
nositide signaling pathways are linked at several levels. For
example, inositol 1,4,5-trisphosphate is a major trigger of
Ca
2⫹
release in the cell though binding to the inositol 1,4,5-
trisphosphate receptor, which in turn is regulated by calmod-
ulin (49). Calmodulin also binds to and regulates phos-
phatidylinositol 3-kinase and inositol-1,4,5-trisphosphate
3-kinase (50). Additional links are provided by calbindin D28k,
which activates myo-inositol-1(or 4)-monophosphatase (51),
and frequenin (neuronal calcium sensor-1), which modulates
the activity of phosphatidylinositol 4-kinase (52). Among the
other 11 cytoplasmic proteins found in the screen are two
proteins that may play a role in the assembly of macromolecular
complexes in PSD in response to Ca
2⫹
signaling, i.e. the PDZ
domain-containing protein 4 and SRC-like adapter protein.
Nuclear Proteins—All 19 nuclear proteins are novel binding
partners of calmodulin, and 18 were validated by SPR tech-
nology, including transcription factor IIIA; zinc finger proteins
ZNF330, ZNF527, ZNF358, ZNF238, ZHX2, and bromodo-
main and PHD finger-containing protein 3; RNA binding motif
proteins RBM4 and RBM5; and inhibitor of growth family,
member 4 (ING4). The related zinc finger protein 268 has been
described in mouse hippocampus as having a role in regula-
tion of structural plasticity (53).
Cytoskeletal Proteins—Two of the seven cytoskeletal pro-
teins, spectrin and

-actin, have previously been character-
ized as human calmodulin-binding proteins.
␥
-Actin has also
been found to be involved in the assembly of these complexes
and binds to human calmodulin with equally high affinity as

-actin. The spectrin scaffold in neurons binds to cytoplasmic
domains of NMDA receptor subunits (41) and associates with
other cytoskeletal structures, protein kinases, and phospha-
tases enriched in synapses (35, 36, 54, 55). It is fascinating to
speculate that calmodulin may be involved in regulating the
postsynaptic machinery by linking members of these assem-
blies in a Ca
2⫹
-dependent fashion.
Ribosomal Proteins—Among the three ribosomal proteins
are two novel targets (L9 and L14), whereas the mouse ribo-
somal protein S2 was previously identified as a calmodulin
binder by affinity chromatography (1). Future studies may be
designed to find out whether calmodulin binds only to free
ribosomal proteins, as verified in the present study, or also to
these proteins when present in the intact ribosome. Interest-
ingly, translation machinery components, e.g. polyribosomes,
have been detected in dendrite shafts and dendritic spines
(56–58).
Mitochondrial and Golgi Apparatus Proteins—The mito-
chondrial protein NADH dehydrogenase (ubiquinone) Fe-S
protein 7 is a component of the mitochondrial respiratory
chain. A homologue of the proprotein convertase subtilisin/
kexin type I inhibitor was previously identified as a calmodulin
target in mouse brain (1). Conversion of prohormones and
precursor proteins into biologically active peptides and
proteins involves the concerted action of a number of con-
vertases and post-translation modification enzymes. The reg-
ulation of these enzymes is not well characterized, but the
Ca
2⫹
-dependent regulation by calmodulin in neural tissues is
a potential mechanism by which these processes are
controlled.
Unclassified Proteins—Identification and validation of two
unclassified proteins as calmodulin targets is intriguing and
motivates further studies of the occurrence and function of
these proteins.
Concluding Remarks—The present work provides a large
number of validated novel neural calmodulin-interacting pro-
teins. Our results suggest that Ca
2⫹
/calmodulin regulate a
significantly higher number of proteins involved in structural
plasticity than previously identified. This serves as a starting
point for biochemical and biological studies toward a deeper
Calmodulin Neural Targets
1130 Molecular & Cellular Proteomics 9.6
understanding of Ca
2⫹
signaling in the brain. Particularly in-
teresting is the very large number of membrane proteins that
are found to bind calmodulin in the presence of Ca
2⫹
. The set
of human neural calmodulin targets provides an exciting new
foundation to explore the involvement of calmodulin in regu-
lating macromolecular assemblies such as the postsynaptic
density and ultimately synaptic strength and potentiation. The
calmodulin target microarray provides a versatile tool for in-
vestigating factors that regulate the interactions of calmodulin
with a large number of targets in parallel.
Acknowledgments—The help with purification of calmodulin S17C
by Eva Thulin is gratefully acknowledged. We are grateful to Professor
Takashi Onodera, University of Tokyo for the gift of hippocampal cell
line Hpl 3-4.
* This work was supported by the Swedish Research Council
(Vetenskapsrådet); the Royal Physiographic Society, Lund, Swe-
den; Science Foundation Ireland and its Walton Visitor Award; and
the Koshland Integrated Science Center at Haverford College.
□SThis article contains supplemental Tables S1–S4 and Figs.
S1–S3.
** To whom correspondence may be addressed. Tel.:
46462228246; Fax: 46462224116; E-mail: sara.linse@biochemistry.
lu.se.
‡‡ To whom correspondence may be addressed. Tel.:
353861725572; E-mail: dolores.cahill@ucd.ie.
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