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INTRODUCTION |
Calmodulin is a multifunctional signaling protein that elicits
myriad effects in cells by modulating the function of target proteins
(1-3). A diverse array of proteins are regulated by Ca2+/calmodulin, ranging from the classic kinases (such as
myosin light chain kinase and the Ca2+/calmodulin kinase
family) (1) to ion channels and anthrax (4, 5). The calmodulin targets
have short (~14-26 amino acid residues) regions to which calmodulin
binds. Although these domains exhibit little sequence conservation,
many adopt an amphiphilic
-helical conformation (6). In addition to
these Ca2+-dependent targets, proteins that
bind to calmodulin in the absence of Ca2+ were subsequently
identified (7, 8). These targets contain a sequence called the IQ motif
(9). Initially described in neuromodulin and unconventional myosins
(9), examination of the Pfam data base reveals IQ motifs in over 100 human proteins. The IQ motif comprises 20-25 amino acids, with the
core fitting the consensus IQXXXRGXXXR (where
X is any amino acid) (9-11). IQ motifs frequently appear in
tandem repeats that bind multiple calmodulin molecules with highest
affinity in the absence of Ca2+ (12).
IQGAP1, a ubiquitous 190-kDa protein, contains several protein
recognition motifs through which it interacts with targets (13, 14).
Proteins that bind to and are regulated by IQGAP1 include E-cadherin
(15, 16),
-catenin (15, 17), Cdc42 (18-21), and actin (19, 22, 23).
In addition, calmodulin binds to the IQ region of IQGAP1 both in the
presence and absence of Ca2+ (19, 24). In contrast to most
IQ-containing proteins, IQGAP1 exhibits an affinity for
Ca2+/calmodulin ~2-fold higher than for apocalmodulin
(19, 24). Investigation of the functional sequelae of the interaction
reveals that calmodulin modulates the binding of IQGAP1 to its other
targets (16, 17, 19, 23, 24). Interestingly, calmodulin attenuates some
of the IQGAP1-target interactions only in the presence of Ca2+ (19, 23). To elucidate the molecular mechanism by
which Ca2+/calmodulin and apocalmodulin differentially
regulate IQGAP1 function, we have generated a series of constructs of
IQGAP1 with selected point mutations in each of the four tandem IQ
motifs. Analysis of the binding of these constructs to calmodulin
provides insight into our understanding of the mode of interaction
between calmodulin and IQ motifs.
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EXPERIMENTAL PROCEDURES |
IQGAP1 Plasmid Construction--
A Myc-tagged human IQGAP1 in
pcDNA3 vector (24) was used. Construction of IQGAP1
CHD (residues
35-265 deleted), IQGAP1
WW (residues 643-744 deleted), IQGAP1
IQ
(residues 699-905 deleted), and IQGAP1
GRD (residues 1122-1324
deleted) mutants was described previously (16, 20). All deletion
mutants migrated to the expected position on SDS-PAGE (see Fig.
1A). To perform site-directed mutagenesis, a PacI
linker was inserted into pBluescript KS at an SspI site to
produce pBluescript-PacI. An ~2-kilobase
PacI-ClaI fragment containing the IQ region of
IQGAP1 was isolated from pcDNA3-IQGAP1 and inserted into
pBluescript-PacI digested with PacI and
ClaI to produce pBluescript-IQ. Site-directed mutagenesis was performed with the QuikChange site-directed mutagenesis kit (Stratagene). After mutagenesis, the PacI-ClaI
fragment of pBluescript-IQ was re-inserted into pcDNA3-IQGAP1 from
which the wild type IQ region had been removed. The sequence of all
constructs was confirmed by DNA sequencing. Plasmids were purified with
a QIAprep Spin Miniprep kit (Qiagen) according to the manufacturer's instructions.
Cell Culture and Transient Transfection--
COS and
MCF-7 cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% (v/v) fetal bovine serum in a 37 °C
humidified incubator with 5% CO2. Transient transfections
of wild type or mutant IQGAP1 constructs were performed with FuGENE 6 (Roche Molecular Biochemicals) as instructed by the manufacturer. Briefly, cells were grown to 70-80% confluence in 100-mm dishes. Five
micrograms of plasmid DNA was mixed with 15 µl of FuGENE 6 and added
to the cells. After 24 to 48 h, cells were harvested, lysed, and
processed as described below.
Binding Analysis--
Mutant and wild type IQGAP1 cDNAs were
subcloned into the pGEX-2T vector. Glutathione
S-transferase
(GST)1 fusion constructs of
wild type and the indicated mutant constructs of IQGAP1 were expressed
in Escherichia coli and isolated by glutathione-Sepharose essentially as described previously (19). All GST-IQGAP1 constructs were >90% pure (data not shown). Cells were lysed in 500 µl of buffer A (150 mM NaC1, 1% Triton X-100, and 50 mM Tris, pH 7.4) containing 1 mM
CaC12 or 1 mM EGTA. Equal amounts of protein
lysate were precleared with glutathione-Sepharose beads for 1 h at
4 °C. Lysates were then incubated with 500 ng of GST-IQGAP1 on
glutathione-Sepharose beads for 3 h at 4 °C. In all cases, GST
alone was used as control. After sedimentation by centrifugation,
samples were washed, resolved by SDS-PAGE, and transferred to
polyvinylidene difluoride membrane. The resultant Western blots were
probed with anti-Myc (16) and anti-calmodulin (25) primary antibodies,
followed by the appropriate horseradish peroxidase-conjugated secondary
antibody and developed by enhanced chemiluminescence (ECL). Where
indicated, GST fusion proteins were incubated with 2 µg of
calmodulin. Following SDS-PAGE, the gel was cut in half; the top
portion (containing IQGAP1) was stained with Coomassie Blue
and the bottom half was processed by blotting for calmodulin.
Binding to calmodulin was evaluated by calmodulin-Sepharose
chromatography as described (24). Briefly, after preclearing with
Sepharose beads for 1 h at 4 °C, equal amounts of protein lysate were incubated with 40 µl of calmodulin-Sepharose (~20 µg
of calmodulin) (or Sepharose without calmodulin as control) and
incubated on a rotator at 4 °C for 3 h. The
calmodulin-Sepharose was washed five times in buffer A containing
either 1 mM CaC12 or 1 mM EGTA as
appropriate and resuspended in SDS-PAGE sample buffer (20 mM Tris-HCl, pH 7.5, 2% (w/v) sodium dodecyl sulfate, 2%
(v/v)
-mercaptoethanol, 0.01% (w/v) bromphenol blue, 0.25 M sucrose, and 2 mM EDTA). Samples were heated
at 100 °C for 5 min and processed by immunoblotting as described above.
Miscellaneous--
Densitometry of ECL signals was analyzed with
UNSCAN-IT software (Silk Scientific Corp.). Statistical analysis was
performed by Student's t test, using InStat software
(GraphPad Software, Inc.). Protein concentrations were measured with
the DC protein assay (Bio-Rad).
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RESULTS |
Binding of Mutant IQGAP1 Constructs to Calmodulin--
Initial
analysis was performed with four deletion mutant IQGAP1 constructs. As
previously demonstrated with endogenous IQGAP1 (24),
transfected wild type IQGAP1 bound readily to calmodulin in the absence
of Ca2+ (Fig. 1A).
Deletion of the CHD or the WW domains from IQGAP1 did not significantly
change its affinity for calmodulin. By contrast, deletion of all four
IQ motifs (IQGAP1
IQ) essentially eliminated binding (Fig. 1).
Surprisingly, IQGAP1
GRD had significantly reduced binding to
calmodulin, although the region deleted is more than 200 amino acids
distal to the IQ domain. The reason is not known, but is presumably
because of effects on the tertiary conformation of IQGAP1. The reduced
binding is not caused by total disruption of IQGAP1 structure
because both IQGAP1
IQ (17) and IQGAP1
GRD (data not shown) bind to
-catenin with an affinity similar to that of wild type IQGAP1.
Interestingly, the conformational "cross-talk" between the IQ and
GRD regions seems to be reciprocal. We previously observed that
deletion of the IQ motifs prevents Cdc42, which binds to the GRD and
adjacent residues in the C-terminal half of the molecule (13), from
co-immunoprecipitating with IQGAP1 (20). Binding was specific
for calmodulin as wild type IQGAP1 did not bind to Sepharose alone
(Fig. 1A). Essentially identical findings were observed in
the presence of Ca2+ (data not shown).

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Fig. 1.
Deletion of selected domains of IQGAP1
attenuates calmodulin binding. Cells were transiently transfected
with wild type IQGAP1 (WT) or deletion mutants IQGAP1 CHD
( CHD), IQGAP1 WW ( WW), IQGAP1 IQ
( IQ), or IQGAP1 GRD ( GRD). After 48 h, cells were lysed in buffer containing 1 mM EGTA and
equal amounts of protein were incubated with calmodulin-Sepharose or
Sepharose alone. Both unfractionated lysates (Lysate) and isolated
complexes (CaM-Sepharose or Sepharose) were resolved by SDS-PAGE and
transferred to polyvinylidene difluoride. Western blots were probed
with anti-Myc antibody (to detect the Myc-tagged IQGAP1), followed by
horseradish peroxidase-conjugated secondary antibodies and developed by
ECL (panel A). B, the relative amount of
Myc-IQGAP1 that bound calmodulin was quantified by laser scanning
densitometry and corrected for the amount of IQGAP1 in the lysate.
Results are expressed relative to wild type IQGAP1 (clear
bar) and represent the mean ± S.E. from three independent
experimental determinations. *, significantly different from wild type
(p < 0.01); **, significantly different from wild type
(p < 0.0001).
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Selected Point Mutations of Basic Residues in the IQ Motifs of
IQGAP1 Decrease Binding to Apocalmodulin, but Not to
Ca2+/Calmodulin--
Because deletion of all
four IQ motifs abrogated the binding of IQGAP1 to calmodulin, point
mutations were generated of individual residues in each IQ motif. A
prior publication (26) indicated that substitution of Gln for the
conserved Arg residues in the IQ motif of Ras-GRF prevented calmodulin
binding. Moreover, binding of light chains to the IQ region of scallop
myosin demonstrates that the conserved Arg residues in the IQ form
important hydrogen bonds (27). For these reasons, we chose to mutate to
Gln the Arg residues in the IQ motifs of IQGAP1. IQ1, IQ3, and IQ4 are complete IQ motifs, whereas IQ2 is incomplete as it lacks Arg at
position 11 (11) (Fig. 2). Therefore, for
IQ1, IQ3, and IQ4 we mutated the two Arg (at positions 6 and 11) to
Gln, whereas for IQ2 the single Arg and proximal Gln (at position 2)
were replaced by Gln and Ala, respectively (Fig. 2). These constructs
were termed the IQR series, because the Arg residues were mutated.

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Fig. 2.
IQGAP1 R series mutations. A,
schematic representation of IQGAP1 with illustration of protein
recognition motifs. WW, poly-proline-binding domain;
IQ, four tandem calmodulin-binding motifs; GRD,
RasGAP-related domain. B, alignment of the four IQ motifs.
The Arg (R) residues depicted in bold and labeled
with an asterisk were mutated to Gln (Q) and the
underlined Gln (Q) residue was replaced by Ala
(A). This is termed the IQR series.
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Replacement of the charged Arg residues in all four IQ motifs
(IQ1,2,3,4R) essentially eliminated binding to apocalmodulin (Fig.
3). To ascertain the relative
contribution of individual IQ motifs to overall calmodulin binding,
analysis was also performed using constructs with point mutations in
single or multiple IQ motifs. Interestingly, replacement of the Arg
residues in only IQ3 and IQ4 (IQ3,4R) yielded a lack of binding
essentially identical to that seen with mutation of all four IQ motifs
(Fig. 3). By contrast, mutation of both IQ1 and IQ2 (IQ1,2R) resulted
in binding to calmodulin that was not significantly different from that
of wild type IQGAP1. The constructs with mutation of IQ3 alone (IQ3R) or IQ4 alone (IQ4R) bound calmodulin with an affinity ~50% of that
seen with wild type IQGAP1, whereas mutation of IQ1 alone (IQ1R) had no
effect on binding (Fig. 3). These data imply that apocalmodulin binds
only to IQ3 and IQ4, with approximately equal affinity for each of the
two IQ motifs. This interpretation is supported by the observation that
mutation of IQ1, IQ2, and IQ4 (IQ1,2,4R) yields a construct that binds
calmodulin indistinguishably from IQ4R (Fig. 3), indicating that IQ1
and IQ2 do not bind apocalmodulin.

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Fig. 3.
Selected point mutations of Arg residues in
the IQ motifs of IQGAP1 decrease binding to apocalmodulin. COS
cells were transiently transfected with wild type IQGAP1
(WT) or the point mutants indicated. After 48 h, cells
were lysed in buffer containing 1 mM EGTA and equal amounts
of protein were incubated with calmodulin-Sepharose (CaM-Sepharose).
Isolated complexes were resolved by SDS-PAGE and Western blots were
probed with anti-Myc antibody (panel A). B, the
relative amount of Myc-IQGAP1 that bound calmodulin was quantified by
laser scanning densitometry and corrected for the amount of IQGAP1 in
the lysate. Results are expressed relative to wild type IQGAP1 and
represent the mean ± S.E. from three or four independent
experimental determinations. *, significantly different from wild type
(p < 0.001).
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A markedly different effect was observed with
Ca2+/calmodulin. Mutation of Arg residues in one, two,
three, or all four IQ motifs had no significant effect on the
interaction of IQGAP1 with Ca2+/calmodulin (Fig.
4). Ca2+/calmodulin bound to
IQ1,2,3,4R IQGAP1 with the same affinity as to wild type IQGAP1. The
binding properties of IQ1,2,3,4R led us to rename this construct
IQGAP1·apoCaM(
).

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Fig. 4.
Selected point mutations of Arg residues in
the IQ motifs of IQGAP1 do not attenuate binding to
Ca2+/calmodulin. COS cells were transiently
transfected with wild type IQGAP1 (WT) or the point mutants
indicated. After 48 h, cells were lysed in buffer containing 1 mM CaC12 and equal amounts of protein were
incubated with calmodulin-Sepharose. Both unfractionated lysates
(Lysate) and isolated complexes (CaM-Sepharose)
were resolved by SDS-PAGE and Western blots were probed with anti-Myc
antibody (panel A). B, the relative amount of
Myc-IQGAP1 that bound calmodulin was quantified by laser scanning
densitometry and corrected for the amount of IQGAP1 in the
lysate. Results are expressed relative to wild type IQGAP1 and
represent the mean ± S.E. from three independent experimental
determinations.
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Mutation of Hydrophobic Residues in the IQ Motifs of
IQGAP1 Attenuates Calmodulin Binding--
Because replacing
the basic charged Arg residues in the IQ motifs with Gln failed to
reduce binding to Ca2+/calmodulin, an alternative strategy
was adopted. Based on the mechanism by which calmodulin recognizes
target peptides in the presence of Ca2+, hydrophobic amino
acids in the IQ motifs were targeted (Fig. 5A). The prediction was that
one of the lobes of calmodulin would interact with the H-1
residues, whereas the other calmodulin lobe would bind to the H-2
residues. Therefore, selected hydrophobic residues distal to the Gln
(Q) of the IQ motif were mutated to Asp (Fig. 5A). All the
H-1 and the distal H-2 (at position 14) residues were mutated. These
point mutant constructs are termed the QH series (H for hydrophobic).
Replacement of these hydrophobic residues (at positions 5, 8, and 14)
in all four IQ motifs prevented binding to apocalmodulin (Fig.
5B).

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Fig. 5.
Mutation of selected hydrophobic residues in
the IQ motifs of IQGAP1 modulates calmodulin binding.
A, the four IQ motifs of IQGAP1 are aligned. The consensus
sequence is at the bottom with hydrophobic (H)
residues in bold. The residues depicted in bold
and labeled with an asterisk in each IQ motif were mutated
to Asp (D). B, COS cells were transiently
transfected with wild type IQGAP1 (WT) or the point mutant
constructs indicated. After 48 h, cells were lysed in buffer
containing 1 mM EGTA and equal amounts of protein were
incubated with calmodulin-Sepharose or Sepharose alone. Both
unfractionated lysates (Lysate) and isolated complexes
(CaM-Sepharose or Sepharose) were resolved by
SDS-PAGE and Western blots were probed with anti-Myc antibody.
C, cells were transfected and processed as described for
panel B, but cells were lysed in buffer containing 1 mM CaC12. Representative experiments are shown.
Relative intensities of the CaM-Sepharose images in panels B
and C cannot be compared with one another because they are
from separate exposures adjusted to demonstrate the relative
intensities of wild type and mutant constructs.
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Unexpectedly, the IQGAP1 construct with all four IQ motifs mutated as
described (QH) still bound to Ca2+/calmodulin, although the
affinity was attenuated (Fig. 5C). Therefore, an additional
point mutation was performed in the residue immediately proximal to the
Gln (the "I" of the IQ). Leu-752 in IQ1 was changed to Glu, whereas
the Ile in IQ2, IQ3, and IQ4 (Ile-782, Ile-812, and Ile-842,
respectively) were converted to Asp (Fig.
6A). These constructs are
termed the IQH series. Analysis of the IQ1,2,3,4H mutant with all four
IQ motifs mutated revealed a complete absence of binding to
apocalmodulin (Fig. 6B). Similarly, mutation of all four IQ
motifs abrogated binding to Ca2+/calmodulin. The IQ1,2,3,4H
was renamed IQGAP1·CaM(
) because it does not bind
calmodulin regardless of whether Ca2+ is absent or present.
As previously observed (24), more wild type IQGAP1 bound to
Ca2+/calmodulin than to apocalmodulin (Fig. 6, compare
lane 1 (WT) in calmodulin-Sepharose pull-downs in
panels B and C).

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Fig. 6.
Mutation of hydrophobic residues
in the IQ motifs of IQGAP1 attenuates calmodulin binding.
A, the four IQ motifs of IQGAP1 are aligned. The consensus
sequence is at the bottom with hydrophobic (H)
residues in bold. The residues depicted in bold
and labeled with an asterisk in each IQ motif were mutated
to Asp (D), except Lys-752, which was replaced with Glu
(E). B, COS cells were transiently transfected
with wild type IQGAP1 (WT), the point mutant constructs
indicated, or IQGAP1 IQ ( IQ). After 48 h, cells
were lysed in buffer containing 1 mM EGTA and equal amounts
of protein were incubated with calmodulin-Sepharose. Both
unfractionated lysates (Lysate) and isolated complexes
(CaM-Sepharose) were resolved by SDS-PAGE and Western blots
were probed with anti-Myc antibody. C, cells were
transfected and processed as described for panel B, but
cells were lysed in buffer containing 1 mM
CaC12. Data are representative of at least three separate
experimental determinations.
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Binding of Calmodulin to Purified IQGAP1--
To verify these
findings, the ability of calmodulin to bind to purified IQGAP1
was evaluated. GST fusion proteins of wild type,
IQGAP1·apoCaM(
), and IQGAP1·CaM(
) were
expressed in E. coli and their ability to bind
endogenous calmodulin was evaluated. As seen with the
calmodulin-Sepharose analysis, purified IQGAP1·CaM(
)
was unable to bind calmodulin regardless of whether Ca2+
was present or absent (Fig.
7A). Moreover, consistent with
the data obtained with calmodulin-Sepharose analysis, purified
IQGAP1·apoCaM(
) was unable to interact with calmodulin
in the absence of Ca2+, but readily bound to
Ca2+/calmodulin. The presence of the GST-IQGAP1 construct
in each sample was validated by probing blots for IQGAP1 (data not
shown).

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Fig. 7.
Point mutations in the IQ motifs of IQGAP1
selectively modulate binding to calmodulin. GST fusion constructs
of wild type IQGAP1 (WT), IQGAP1·CaM( )
(CaM( )), IQGAP1·apoCaM( )
(apo( )), or GST alone bound to
glutathione-Sepharose were incubated with equal amounts of protein
lysate from MCF-7 cells (panel A) or purified calmodulin
(panel B) in the presence of 1 mM
CaC12 or 1 mM EGTA. Complexes were pelleted by
centrifugation, washed, and resolved by SDS-PAGE. A, after
transfer to polyvinylidene difluoride, Western blots were probed for
calmodulin and developed by ECL. B, after SDS-PAGE, the gel
was cut into two pieces; the top portion (containing IQGAP1) was
stained with Coomassie Blue, whereas the bottom half was transferred to
polyvinylidene difluoride and probed for calmodulin. Representative
experiments of two to four determinations are shown. Relative
intensities of the images for Ca2+ and EGTA samples cannot
be compared with one another because they are from separate exposures
adjusted to demonstrate the relative intensities of wild type and
mutant constructs.
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Analysis was also performed in vitro with purified proteins.
Pure calmodulin was incubated with GST fusion proteins of wild type and
mutant IQGAP1 in the presence or absence of Ca2+. Congruent
with the previous results, IQGAP1·CaM(
) bound no
calmodulin, whereas IQGAP1·apoCaM(
) bound
Ca2+/calmodulin but not apocalmodulin (Fig. 7B).
These data validate our findings and indicate that other proteins in
the cell are not responsible for the altered calmodulin binding to the
IQGAP1 mutants.
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DISCUSSION |
IQ motifs, first recognized as calmodulin-binding domains in
neuromodulin (7, 8), have been identified in many proteins with a
diverse array of functions (10). One of these is the scaffolding
protein IQGAP1. In this paper, we generated two different series of
point mutant constructs of the four tandem IQ motifs of IQGAP1, namely
the IQR (Arg residues mutated) and IQH (hydrophobic residues mutated)
series. Analysis of these constructs reveals that apocalmodulin binds
to only IQ3 and IQ4, whereas Ca2+/calmodulin binds to all
four IQ motifs of IQGAP1 (note that the stoichiometry of CaM:IQGAP1 is
4:1 (23)). We previously documented that Ca2+/calmodulin
and apocalmodulin bind to fusion proteins of the IQ region of
IQGAP1 (19). A low affinity binding site for
Ca2+/calmodulin, but not apocalmodulin, was also identified
in the calponin homology domain (CHD). The CHD appears to make a small contribution to total calmodulin binding as deletion of this region did
not significantly reduce the binding of IQGAP1 to calmodulin. Moreover,
essentially no binding of calmodulin was detected to IQGAP1 constructs
with point mutations in the IQ motifs, despite the presence of an
intact CHD.
X-ray and NMR structures of calmodulin bound to "classic" target
peptides reveal that calmodulin is compact, with the globular domains
close together, joined by a loop of the extended flexible central helix
(reviewed in Ref. 6). Whereas the classic calmodulin target
peptides adopt an amphiphilic
-helical conformation (6, 28), recent
evidence reveals that calmodulin (and the target peptides) can
adopt a variety of conformations when interacting with different
targets. For example, Ca2+/calmodulin induced dimerization
of the gating domain of a Ca2+-activated K+
channel (4). In this structure, the calmodulin-binding domain forms an elongated dimer with a calmodulin molecule bound at each end.
In addition, when bound to anthrax adenylyl cyclase, calmodulin adopts
an extended conformation (5), rather than the compact conformation seen
with other targets. These recent surprising findings accentuate the
diversity and variability in the interaction of calmodulin with its
target molecules.
Although IQ motifs were described 10 years ago (9), a detailed
structure of the calmodulin-IQ complex has not yet been published. A
model of calmodulin bound to an IQ motif has been constructed, using
the crystal structure of the regulatory domain of scallop myosin (11,
29). The highly conserved portion of the IQ motif (IQXXXR;
the first residue may be Ile, Leu, or Val (10)) is the most critical
region and determines both the conformation and positioning of the
C-terminal lobe of calmodulin (29). The second part of the IQ motif
core (GXXXR) is not well conserved and has a minor role in
fixing the position of the calmodulin N-terminal lobe. IQ motifs having
both parts are termed "complete," whereas those lacking the second
part are termed "incomplete." This distinction is important because
binding of calmodulin to complete IQ motifs does not require
Ca2+ (29). The C-terminal lobe of calmodulin is expected to
be semi-open and the N-terminal lobe closed when bound to a complete IQ
motif. By contrast, when bound to an incomplete IQ motif, the
N-terminal lobe would adopt an open conformation provided that
Ca2+ is present (29). It is thought that apocalmodulin and
Ca2+/calmodulin are likely to bind different sites in the
IQ motif (11).
The results presented here complement the model. Some of our data
support the scheme, whereas other observations differ from those
anticipated from the model. For example, consistent with the
predictions of Houdusse and Cohen (11), both hydrophobic and
electrostatic interactions appear to contribute to the binding of
apocalmodulin and both seem necessary; disruption of either mode of
association by mutation of critical residues reduces binding. The
expectation from the model was that apocalmodulin would bind all the
complete IQ motifs. However, our data revealed that apocalmodulin did
not bind to IQ1, although it fulfills the criteria of a complete IQ
motif. As indicated by our results, caution should be exercised in
extrapolating data obtained with model IQ peptides because these do not
always mimic the behavior of the intact protein. For example,
neuromodulin and neurogranin bind calmodulin only in the absence of
Ca2+, but isolated peptides of the calmodulin-binding
domains bind Ca2+/calmodulin (30-32). Similarly, the
interaction of calmodulin with the complete catalytic domain of edema
factor of adenylyl cyclase was different from its interaction with the
peptide of the calmodulin-binding domain; the peptide induced a
conformation of calmodulin opposite to that caused by the whole
catalytic domain (5). These observations emphasize the importance of
studies with intact proteins as conducted here.
It is generally believed that calmodulin targets that contain IQ motifs
have a higher affinity for the Ca2+-free form of calmodulin
(8, 12, 31, 33, 34). Moreover, for some proteins such as brush border
myosin I (35), Ca2+ induces the dissociation of bound
calmodulin. Our mutagenesis data reveal that the interaction between
calmodulin and IQ motifs is substantially more complex than current
thinking and selected IQ motifs may have higher affinity for
Ca2+/calmodulin. This observation is supported by the
reports from both our laboratory and others that the IQ-containing
proteins IRS-1 (36) and NinaC myosin (37) bind
Ca2+/calmodulin with higher affinity than
apocalmodulin. The results presented here extend previous
findings, indicating that the binding of Ca2+/calmodulin to
IQ motifs exhibits several features different from those of
apocalmodulin. First, Ca2+/calmodulin binds to all four IQ
motifs of IQGAP1, whereas apocalmodulin does not interact appreciably
with the first two (IQ1 or IQ2) IQ motifs. These findings provide the
molecular mechanism for our initial observation that 2-fold more IQGAP1
bound calmodulin in the presence of Ca2+ (24). Second,
electrostatic interactions appear less important for
Ca2+/calmodulin binding than hydrophobic interactions.
Substitution of the polar but uncharged Gln for critical basic Arg
residues in all four IQ motifs did not attenuate the interaction of
Ca2+/calmodulin, but was sufficient to eliminate
apocalmodulin binding. Third, individual hydrophobic residues have
critical roles that differ between apocalmodulin and
Ca2+/calmodulin. The I of the IQ motif appears to be
essential for binding Ca2+/calmodulin, but has a less
significant role in the interaction with apocalmodulin. Binding to
apocalmodulin, but not Ca2+/calmodulin, could be eliminated
without altering this Ile.
This study employed direct experimentation to test models of the
interaction of calmodulin with IQ motifs. Nevertheless, some caveats of
our work should be borne in mind. Fourteen amino acids had to be
substituted in the four IQ motifs of IQGAP1 to abrogate binding of
Ca2+/calmodulin. It is conceivable that these substitutions
could modify secondary or tertiary structure, thereby altering the
orientation of the IQ motifs to one another or to other regions of
IQGAP1. The observation that IQGAP1·apoCaM(
) and
IQ1,2,3,4R IQGAP1 (which have 7 and 10 substitutions, respectively) bind to Ca2+/calmodulin with the same affinity as to wild
type IQGAP1 renders this possibility less likely. It remains possible,
however, that the mutant IQGAP1 could bind to calmodulin by an
interaction different from that in the native protein. This premise can
be eliminated only by solving the structures of wild type and mutant IQGAP1.
The functional sequelae of calmodulin binding to IQ motifs remain
incompletely understood. For the unconventional myosins, IQ motifs are
thought to influence the chemomechanical properties of the myosins
(10). Binding of calmodulin has a substantial effect on IQGAP1,
modulating its interaction with other targets (16, 17, 24). Previous
analysis documented that Ca2+ is required for calmodulin to
block the binding of Cdc42 to IQGAP1 (24), implying that
Ca2+/calmodulin induces a conformation in IQGAP1 different
from that produced by apocalmodulin. The findings presented here
contribute to our understanding of the molecular mechanism underlying
these functional effects. IQ3 and IQ4 of IQGAP1 bind both apocalmodulin and Ca2+/calmodulin, whereas IQ1 and IQ2 bind only
Ca2+/calmodulin. Therefore, the conformation adopted by
IQGAP1 bound to Ca2+/calmodulin is likely to
result from the interaction of Ca2+/calmodulin with IQ1
and/or IQ2. A second possibility is that simultaneous occupation of all
four IQ motifs is necessary to eliminate Cdc42 binding. Third,
Ca2+/calmodulin may interact with amino acid residues in
the IQ motifs different from those recognized by apocalmodulin,
inducing a different shape in IQGAP1. These mechanisms are not mutually
exclusive and more than one may be operative. Together, the findings
obtained by mutating individual residues in sequential IQ motifs in an intact protein enhance our comprehension of the molecular interactions between calmodulin and IQ motifs, as well as elucidating the modulatory role of Ca2+. We look forward to the crystal structures of
IQ motifs bound to Ca2+/calmodulin and apocalmodulin to
yield further insight into these important protein regulatory motifs.