Báo cáo khoa học: The two IQ-motifs and Ca2+/calmodulin regulate the rat myosin 1d ATPase activity pptx

The two IQ-motifs and Ca
2+
/calmodulin regulate the rat
myosin 1d ATPase activity
Danny Ko
¨
hler*, Sandra Struchholz* and Martin Ba
¨
hler
Institute for General Zoology and Genetics, Westfa
¨
lische Wilhelms University, Mu
¨
nster, Germany
Myosins are actin-based motors that serve a variety of
cellular functions. They generally consist of a head and
a tail. The head represents the motor part and contains
nucleotide and actin binding sites. In addition, it
encompasses at its C-terminus a light chain binding
domain. The tail provides additional activities like, e.g.
cargo binding. The motor activity of various myosins
can be regulated by a number of different mechanisms.
In several myosins the light chain binding domain and
the associated light chains have been demonstrated
to exhibit a regulatory function [1–3]. The light chain
binding domains comprise different numbers of
IQ-motifs [1] that represent binding sites for the Ca
2+
-
sensor protein calmodulin (CaM) and CaM-related
EF-hand proteins. Many unconventional myosins have

between one and six CaM light chain(s) associated
with them. CaM contains four Ca
2+
-binding sites, one
pair of lower affinity sites in the N-terminal lobe and
one pair of higher affinity sites in the C-terminal lobe,
respectively [4,5]. Initially, IQ-motifs were identified as
Ca
2+
-independent CaM-binding sites [6,7]. However,
in some instances IQ-motifs have been shown to bind
CaM in a Ca
2+
-dependent manner [1,8,9].
Based on myosin head sequence comparisons, myo-
sins have been subdivided into 18 classes [10–12]. The
mammalian class I myosins studied so far have all
been shown to contain CaM light chains. Analysis of
the regulation of class I myosins by Ca
2+
⁄ CaM has
provided different results with no evidence for a com-
mon mechanism operating in class I myosins. Myosins
1a (brush border myosin I), 1b (myr 1) and 1c (myosin
Ib, myr 2) contain 3–6 CaM binding sites [13–17]. Ele-
vation of the free Ca
2+
concentration leads to a par-
tial loss of CaM from these myosins in vitro. However,
it is not known whether CaM dissociation occurs also

in vivo. Provided that CaM-binding is saturated by the
addition of exogenous CaM, an increase in free Ca
2+
concentration stimulates the basal (actin-independent)
Keywords
calmodulin; Ca
2+
regulation; IQ-motif;
myosin; Myo1d
Correspondence
M. Ba
¨
hler, Institut fu
¨
r Allgemeine Zoologie
und Genetik, Westfa
¨
lische Wilhelms-
Universita
¨
t, Schlossplatz 5, 48149 Mu
¨
nster,
Germany
Fax: +49 251 8324723
Tel: +49 251 8323874
E-mail:
*These authors contributed equally to this
work
(Received 24 January 2005, revised 25

February 2005, accepted 4 March 2005)
doi:10.1111/j.1742-4658.2005.04642.x
The light chain binding domain of rat myosin 1d consists of two IQ-motifs,
both of which bind the light chain calmodulin (CaM). To analyze the
Myo1d ATPase activity as a function of the IQ-motifs and Ca
2+
⁄ CaM
binding, we expressed and affinity purified the Myo1d constructs Myo1d-
head, Myo1d-IQ1, Myo1d-IQ1.2, Myo1d-IQ2 and Myo1dDLV-IQ2. IQ1
exhibited a high affinity for CaM both in the absence and presence of free
Ca
2+
. IQ2 had a lower affinity for CaM in the absence of Ca
2+
than in
the presence of Ca
2+
. The actin-activated ATPase activity of Myo1d was
75% inhibited by Ca
2+
-binding to CaM. This inhibition was observed
irrespective of whether IQ1, IQ2 or both IQ1 and IQ2 were fused to the
head. Based on the measured Ca
2+
-dependence, we propose that Ca
2+
-
binding to the C-terminal pair of high affinity sites in CaM inhibits the
Myo1d actin-activated ATPase activity. This inhibition was due to a
conformational change of the C-terminal lobe of CaM remaining bound to

the IQ-motif(s). Interestingly, a similar but Ca
2+
-independent inhibition of
Myo1d actin-activated ATPase activity was observed when IQ2, fused
directly to the Myo1d-head, was rotated through 200° by the deletion of
two amino acids in the lever arm a-helix N-terminal to the IQ-motif.
Abbreviation
CaM, calmodulin.
FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS 2189
ATPase activity in Myo1a-c and also the actin-activa-
ted ATPase activity of Myo1b. The translocation of
actin filaments by these class I myosins in the gliding
assay was either abolished, slowed or not affected [18–
20]. For myosin 1c it has been demonstrated that bind-
ing of Ca
2+
to the C-terminal pair of Ca
2+
-binding
sites in CaM inhibits its actin-translocating activity
[19]. Myosin 1e, another class I myosin, contains a
single CaM-binding site and an increase in free Ca
2+
concentration did not cause a release of the CaM
bound to this site, but induced a decrease in basal
ATPase activity [21].
Rat myosin 1d (Myo1d, formerly called myr 4)
exhibits a light chain binding domain with two IQ-
motifs. In gel overlay assays, the IQ-motif 1 (IQ1) has
been found to bind CaM with higher affinity in the

absence of Ca
2+
whereas IQ-motif 2 (IQ2) bound
CaM in a Ca
2+
-dependent manner [8]. Currently, it is
not known whether the light chain binding domain
and the CaM light chains serve a regulatory function
in Myo1d. Single molecule mechanical measurements
demonstrated that the light chain binding domain of
Myo1d functions as a rigid mechanical lever rotating
by 90° during the working stroke [22]. In analogy to
other myosins it is assumed that the IQ-motifs adopt
an a-helical conformation that is stabilized by the
associated light chains [1,23].
In the present study we analyzed the regulation of
Myo1d ATPase activity by Ca
2+
⁄ CaM and the two
IQ-motifs. We affinity purified recombinant Myo1d
constructs from stable transfected HeLa cells that dif-
fered in number and position of IQ-motifs and deter-
mined the binding of CaM to these IQ-motifs. The
effects on Myo1d ATPase activity upon Ca
2+
-binding
to CaM associated with the IQ-motifs were investi-
gated. We now report that Ca
2+
-binding to the C-ter-

minal pair of Ca
2+
-binding sites in CaM bound to the
IQ-motif directly following the converter domain
inhibits Myo1d actin-activated ATPase activity. Dele-
tion of two amino acids at the interface between con-
verter and IQ-motif led to a Ca
2+
-independent
inhibition of the actin-activated ATPase activity.
Results
Binding of Ca
2+
⁄ CaM to the two Myo1d IQ-motifs
The light chain binding domain of rat Myo1d contains
two IQ-motifs that are quite distinct in sequence
(Fig. 1B). To investigate their interaction with the
Ca
2+
-sensor molecule CaM, we expressed different
recombinant Myo1d proteins in HeLa cells (Fig. 1A).
The recombinant Myo1d proteins included the head
domain and either no, one or both IQ-motifs. In addi-
tion, we expressed a construct that lacked the first
IQ-motif and contained only the second IQ-motif
(Myo1d-IQ2). This construct was further modified in
that the two C-terminal amino acids (LV) of the con-
verter domain not present in the Myo1d-head con-
struct were deleted (Myo1dDLV-IQ2, Fig. 1). Because
these two amino acids are part of a a-helix continued

by the IQ-motifs, the IQ-motif is rotated counterclock-
wise by 200° and moved  3A
˚
closer towards the head
domain. Following expression of these Myo1d con-
structs and affinity purification in the absence of free
Ca
2+
, we assessed the stoichiometry of CaM bound to
the two IQ-motifs. The stoichiometries of CaM asso-
ciated with Myo1d-head (0 : 1; Fig. 2B), Myo1d-IQ1
(1 : 1; Figs 2A and 3) and Myo1d-IQ1.2 (1.74 : 2),
respectively, have been described previously [22].
Adjusting the free Ca
2+
concentration to 0.1 mm did
not cause a release of CaM bound to IQ 1 in the
Myo1d-IQ1 heavy chain construct (Fig. 3). This dem-
onstrates that IQ 1 has a high affinity for CaM both
in the absence and presence of free Ca
2+
. In contrast,
the binding of CaM to IQ 2 was clearly distinct. The
deletion constructs Myo1d-IQ2 and Myo1dDLV-IQ2
could be purified under identical conditions using anti-
bodies directed against the C-terminal FLAG epitope
A
B
Fig. 1. Schematic representation of rat Myo1d constructs and
IQ-motif sequences. (A) Domain organization of Myo1d: IQ motifs

(white) are labeled with numbers indicative of their respective posi-
tion in relation to the motor domain (gray) and tail domain (black).
All recombinant constructs contain a C-terminal FLAG-epitope (cir-
cle). Amino acids of rat Myo1d, linker residues (italics) and the
FLAG-epitope sequence (underlined) for each of the five different
constructs are indicated. (B) Aligned sequences of the two
IQ-motifs in Myo1d and the generalized consensus IQ-motif are
shown.
Regulation of Myo1d ATPase activity D. Ko
¨
hler et al.
2190 FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS
albeit with a lower protein yield (Figs 2A and 4). Den-
sitometric analysis of the CaM content in affinity puri-
fied Myo1d-IQ2 and Myo1dDLV-IQ2 preparations
revealed a ratio of only 0.24 ± 0.03 CaM per Myo1d-
IQ2 heavy chain and 0.39 ± 0.14 CaM per
Myo1dDLV-IQ2 heavy chain, respectively (Figs 2A
and 4). However, a higher protein yield and a stoichio-
metric association of CaM with Myo1d-IQ2 were
achieved when this construct was purified in the pres-
ence of free Ca
2+
(data not shown), indicating that
IQ2 binds CaM more tightly in the presence of Ca
2+
.
To test if the IQ2 in Myo1d-IQ2 and Myo1dDLV-
IQ2 could be saturated with CaM in the absence or
presence of free Ca

2+
, we mixed purified Myo1d-IQ2
or Myo1dDLV-IQ2 with 10 lm exogenous CaM (Figs 4
and 5). Free CaM was separated from CaM bound to
Myo1d-IQ2 or Myo1dDLV-IQ2 by cosedimentation of
the myosin with F-actin. In the absence of free Ca
2+
Myo1d-IQ2 and Myo1dDLV-IQ2 had stoichiometric
amounts of CaM bound (molar ratio of 0.92 ± 0.12
CaM ⁄ Myo1d-IQ2 heavy chain and of 1.1 ± 0.1
CaM ⁄ Myo1dDLV-IQ2 heavy chain) (Figs 4 and 5).
The 1 : 1 ratio of CaM binding to Myo1d-IQ2 and
Myo1dDLV-IQ2 did not change upon the addition of
100 lm free Ca
2+
to the assay mixture (Figs 4 and 5).
Therefore, we supplemented purified Myo1d-IQ2
and Myo1dDLV-IQ2 preparations for all further
experiments with 10 lm CaM to guarantee the stoichio-
metric binding of CaM.
Regulation of the actin-activated ATPase of
recombinant Myo1d proteins by Ca
2+
⁄ CaM
In the absence of free Ca
2+
, the basal ATPase activity
of the Myo1d-head without an IQ-motif was 0.01 s
)1
.

The V
max
of the actin-activated ATPase was 2.6 s
)1
and
the K
actin
38 lm [22] with an apparent second-order rate
AB
Fig. 2. Different amounts of calmodulin are copurified with Myo1d-
IQ1, Myo1dDLV-IQ2 and Myo1d-head. Affinity-purified Myo1d con-
structs were analyzed on Coomassie blue stained 7.5–15%
SDS ⁄ polyacrylamide gradient gels for their content of copurified
calmodulin. (A) Myo1dDLV-IQ2 (lane 1), Myo1d-IQ1 (lane 2) and cal-
modulin (lane 3); (B) Myo1d-head (lane 1) and calmodulin (lane 2).
Molecular masses are indicated to the left. The arrowheads mark
the position of the Myo1d heavy chains and the asterisks highlight
the copurified light chain calmodulin.
Fig. 3. CaM binds stoichiometrically to Myo1d-IQ1 irrespective of
the free Ca
2+
-concentration. Myo1d-IQ1 purified in the presence of
EGTA was either left in EGTA (EGTA, lanes 3 and 4) or buffer con-
ditions were adjusted to 100 lM free Ca
2+
(Ca
2+
, lanes 1 and 2) fol-
lowed by the addition of F-actin. Samples were centrifuged and
separated into supernatants (S) and pellets (P). Myo1d-IQ1 and

associated calmodulin was cosedimented with F-actin to monitor a
potential release of calmodulin. Proteins were separated on a 7.5%
- 15% SDS-polyacrylamide gradient gel and stained with Coomassie
blue. Electrophoresis of CaM (lane 5) served as a marker and is
indicated by an asterisk. The arrowhead indicates Myo1d-IQ1.
Fig. 4. Stoichiometric binding of exogenous calmodulin to purified
Myo1d-IQ2 in the absence and presence of free Ca
2+
. SDS ⁄ PAGE
(7.5–15%) analysis revealed that affinity purified Myo1d-IQ2 (lane
1) does not contain stoichiometric amounts of CaM. Purified
Myo1d-IQ2 (0.5 l
M) was incubated with 10 lM CaM either in the
presence of 100 l
M free Ca
2+
(Ca
2+
, lanes 2 and 3) or in the
absence of free Ca
2+
(EGTA, lanes 4 and 5). To determine
the amount of calmodulin bound to Myo1d-IQ2, F-actin was added
to the samples and they were centrifuged. Supernatants (S) and
pellets (P) were analyzed by SDS ⁄ PAGE. As a control, calmodulin
was centrifuged with F-actin alone (EGTA, lanes 6 and 7). Purified
CaM served as a marker (lane 8) and is indicated by an asterisk.
The position of Myo1d-IQ2 heavy chain is indicated by an arrow-
head. The molar ratio of CaM to Myo1d-IQ2 determined by densi-
tometry in the actin pellets was 0.92 ± 0.12 in EGTA and

0.87 ± 0.25 in 100 l
M Ca
2+
, respectively.
D. Ko
¨
hler et al. Regulation of Myo1d ATPase activity
FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS 2191
constant K
app
of 0.68 · 10
5
s
)1
Æm
)1
(Fig. 6). These val-
ues were virtually identical for the Myo1d-IQ1 and
Myo1d-IQ1.2 constructs that contained in addition
either IQ1 or IQ1 and IQ2. The two IQ-motifs were
even exchangeable, as the Myo1d-IQ2 fusion protein
also exhibited identical basal and actin-activated
ATPase activities (Fig. 6A,B). However, deletion of the
two C-terminal amino acids of the converter domain in
the construct Myo1dDLV-IQ2 lead to a pronounced
inhibition of the actin-activated ATPase activity while
the basal ATPase activity was unaltered (Fig. 6B). The
actin-activated ATPase activity increased almost
linearly in the range of the actin concentrations tested.
It exhibited a K

app
of 0.12 · 10
5
s
)1
Æm
)1
that was about
six-fold reduced in comparison with Myo1d-IQ2,
indicating a change in coupling between the actin and
nucleotide binding sites.
To investigate whether the Myo1d ATPase is regula-
ted by Ca
2+
⁄ CaM, we determined the actin-activated
ATPase of Myo1d constructs in the presence of 22 lm-
free Ca
2+
. Interestingly, the V
max
of the actin-activated
ATPase activity of the Myo1d-head was reduced by
Fig. 5. Stoichiometric binding of exogenous CaM to purified
Myo1dDLV-IQ2 in the absence and presence of free Ca
2+
. Purified
Myo1dDLV-IQ2 (0.5 l
M) was incubated with 10 lM CaM either in
the absence of free Ca
2+

(EGTA, lanes 3 and 4) or in the presence
of 100 l
M free Ca
2+
(Ca
2+
, lanes 5 and 6). To determine the
amount of calmodulin bound to Myo1dDLV-IQ2, F-actin was added
to the samples and they were centrifuged. Supernatants (S) and
pellets (P) were analyzed by SDS ⁄ PAGE (7.5–15%). As a control,
calmodulin was centrifuged with F-actin alone (EGTA, lanes 1 and
2). Purified CaM served as a marker (lane 7) and is indicated by an
asterisk. The position of Myo1dDLV-IQ2 heavy chain is indicated by
an arrowhead.
Fig. 6. The actin-activated Mg
2
ATPase activity of different Myo1d-
constructs is inhibited by Ca
2+
. Actin-activated ATPase activity
was determined at 37 °C in a buffer containing 30 m
M KCl, 10 mM
Hepes pH 7.4, 2 mM MgCl
2
,3mM EGTA, 1 mM 2-mercaptoethanol,
2m
M NaN
3
and 2 mM ATP. CaCl
2

(3 mM) was added where indica-
ted to obtain a free Ca
2+
concentration of 22 lM. Samples con-
tained 54–270 n
M of the respective Myo1d constructs, 0–67 lM
F-actin and in the case of IQ2 containing constructs exogenous cal-
modulin. (A) Actin dependence of Myo1d-head ATPase activity (s)
in EGTA (solid line) and 22 l
M free Ca
2+
(dashed line) conditions,
respectively. Also shown is the actin dependence of Myo1d-IQ1.2
ATPase activity (n) in EGTA conditions. (B) and (C) Actin depend-
ence of Myo1d-IQ1 (d), Myo1d-IQ2 (m) and Myo1dDLV-IQ2 (
)
ATPase activities in EGTA conditions (B) and 22 l
M free Ca
2+
conditions (C). Data points were fitted according to Eqn (1) in the
Experimental procedures.
Regulation of Myo1d ATPase activity D. Ko
¨
hler et al.
2192 FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS
roughly 20% whereas the K
actin
was unaltered
(Fig. 6A). This Ca
2+

-dependent inhibition was not
reversible when free Ca
2+
was chelated with EGTA
(data not shown). In the Myo1d-IQ1 construct, free
Ca
2+
inhibited the actin-activated ATPase activity
by  75% (Fig. 6C). The calculated K
app
was
0.12 · 10
5
s
)1
Æm
)1
. As the two Myo1d IQ-motifs have
different CaM-binding properties, we analyzed if IQ1
and IQ2 regulate the Myo1d ATPase activity differ-
ently. However, in the construct Myo1d-IQ2 that has
IQ1 exchanged for IQ2, free Ca
2+
caused a compar-
able inhibition of the ATPase with a V
max
of 0.7 s
)1
(Figs 6C and 7). The actin affinity was not affected
significantly with a determined K

actin
¼ 44 lm. The
K
app
derived from the initial slope of the hyperbola
was 0.15 · 10
5
s
)1
Æm
)1
.
In the construct Myo1dDLV-IQ2 that exhibited
already a reduced ATPase activity in the absence of
free Ca
2+
, the addition of free Ca
2+
reduced its ATP-
ase activity further by about 40% and a K
app
of
0.07 · 10
5
s
)1
Æm
)1
was determined.
Inhibition of the Myo1d actin-activated ATPase

activity as a function of the concentration
of free Ca
2+
Next, we determined the ATPase activities for the dif-
ferent Myo1d constructs as a function of the free
Ca
2+
concentration (0.001–158 lm) (Fig. 7). The slight
reduction of the actin-activated ATPase activity of the
Myo1d-head exhibited an IC
50
of pCa  6 (0.3 lm free
Ca
2+
). All of the Myo1d constructs that contained
either one or two IQ motifs, specifically Myo1d-IQ1,
Myo1d-IQ1.2 and Myo1d-IQ2, were inhibited with an
IC
50
of pCa 7 (0.05–0.08 lm Ca
2+
) (Fig. 7). This
IC
50
value corresponds to the affinity of the pair of
Ca
2+
-binding sites in the C-terminal lobe of CaM.
The Myo1dDLV-IQ2 protein that exhibited already a
reduced ATPase activity in the absence of free Ca

2+
did not show any significant changes in ATPase activ-
ity with increasing free Ca
2+
concentrations.
Discussion
To obtain a complete understanding of the physiologi-
cal functions of a given myosin, it is necessary to
understand the mechanisms that regulate its motor
activities. Here we investigated the regulation of the
Myo1d ATPase activity by its light chain binding
domain and the associated light chain CaM. The light
chain binding domain of Myo1d consists of two
IQ-motifs that were found to bind CaM with different
affinities and calcium-sensitivity. Binding of Ca
2+
to
the CaM bound to the first IQ-motif inhibited the
actin-activated ATPase activity by  75%. When
the first IQ-motif was deleted, binding of Ca
2+
to the
CaM bound to the second IQ-motif inhibited the
actin-activated ATPase activity by the same extent. In
both cases, the inhibition of the ATPase activity was
induced by virtually identical free Ca
2+
concentra-
tions. Deletion of two amino acids N-terminal to the
IQ-motif did not affect CaM-binding, but inhibited the

ATPase activity in a Ca
2+
-independent manner to a
similar extent as observed with Ca
2+
for the other
constructs containing either one or two IQ-motifs.
Ca
2+
-independent and Ca
2+
-dependent binding
of CaM to IQ-motifs
The two IQ motifs in Myo1d are supposed to belong
to different classes of IQ motifs [8,23]. Whereas IQ1 in
Myo1d conforms well to the consensus sequence of
IQ-motifs, IQ2 is less well conserved and possesses
hydrophobic residues at positions 1, 5, 8 and 14 as is
typical for Ca
2+
-dependent CaM-binding motifs [1].
Indeed, we found that IQ1 has a higher affinity for
ApoCaM than IQ2. Based on structural studies of
ELC binding to IQ1 of a myosin II [24,25] and Mlcp1
binding to the IQ-motifs in Myo2p [23], we presume
that the C-terminal lobe of ApoCaM binds to the
N-terminal parts of IQ1 (LQKVWR) and IQ2
Fig. 7. Inhibition of the actin-dependent ATPase activities of differ-
ent Myo1d-constructs as a function of free Ca
2+

concentrations.
Calcium dependence of ATPase activity of Myo1d-head (s),
Myo1d-IQ1.2 (n), Myo1d-IQ1 (d), Myo1d-IQ2 (m) and Myo1dDLV-
IQ2 (
). ATPase activities were measured at 37 °C in a solution
containing 24 l
M F-actin, 2 mM ATP, 30 mM KCl, 10 mM Hepes
pH 7.4, 2 m
M MgCl
2
,3mM EGTA, 1 mM 2-mercaptoethanol and
2m
M NaN
3
. To adjust free Ca
2+
concentrations (0–158 lM), corres-
ponding amounts of CaCl
2
were added.
D. Ko
¨
hler et al. Regulation of Myo1d ATPase activity
FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS 2193
(IIRYYR), respectively, in a semi-open conformation.
We expect that the N-terminal lobe of ApoCaM binds
in a closed conformation to the C-terminal part of IQ1
(GTLAR). The reduced affinity of IQ2 for ApoCaM
as compared to IQ1 is probably due to a lack of inter-
action between the N-terminal lobe of CaM and the

C-terminal part of IQ2 (RYKVK). The exchange of
Gly at position 7 for an Arg with a bulky side chain in
IQ2 is likely to interfere sterically with the binding of
the N-lobe of ApoCaM as has been demonstrated for
Mlc1p binding to IQ4 of Myo2p [23]. ApoCaM has
also been reported to bind weakly to the single IQ
motif present in Myo VI [26]. This IQ motif deviates
from the consensus sequence (IQXXXRGXXXR ⁄ K)
in that the glycine consensus residue at position 7 is
changed to a methionine. Charge repulsion due to a
stretch of positively charged amino acids in the C-ter-
minal part of IQ2 (Arg733, Lys735, Lys737) may addi-
tionally repel the N-lobe farther away from IQ2. The
N-lobe of CaM might thus be free to interact with
sequences in the Myo1d tail or with other proteins.
The affinity of IQ2 for ApoCaM was not affected
when the two C-terminal amino acids (LV) of the con-
verter domain a-helix were deleted. This deletion is
predicted to introduce a counterclockwise rotation by
200° and a shift by  3A
˚
towards the head domain of
the CaM bound to the IQ-motif. We conclude that no
steric hindrance for CaM binding was introduced by
this deletion.
We have shown previously that 1.74 CaM molecules
were associated with affinity purified Myo1d-IQ1.2.
This stoichiometry of bound CaM is higher than the
sum of CaM molecules bound to Myo1d-IQ1 (1.1) and
Myo1d-IQ2 (0.24). This result indicates that CaM

binds in a cooperative manner to the light chain bind-
ing domain of Myo1d. The binding of CaM to IQ1
may induce a stabilization of the IQ2 a-helical struc-
ture and thereby facilitate binding of the C-terminal
lobe of ApoCaM to the N-terminal half of IQ2.
We found that purified Myo1d-IQ2 and Myo1dDLV-
IQ2 could be fully saturated by the addition of exogen-
ous ApoCaM or Ca
2+
⁄ CaM. This finding allowed
us to investigate the effects of Ca
2+
-binding to
ApoCaM associated with the IQ2. Myo1d-IQ2 affinity
purified under Ca
2+
conditions contained stoichiomet-
ric amounts of copurified CaM demonstrating that IQ2
actually has a higher affinity for Ca
2+
⁄ CaM than for
ApoCaM. This result is in accordance with previous
observations in a gel overlay assay [8] and the above
mentioned sequence similarity of IQ2 with Ca
2+
-
dependent CaM-binding motifs. The two lobes of
Ca
2+
⁄ CaM may both bind in an open conformation to

IQ2, explaining the increased affinity.
Mechanism of inhibition of the Myo1d ATPase
activity by Ca
2+
⁄ CaM
As reported previously, in the absence of free Ca
2+
Myo1d-head, Myo1d-IQ1 and Myo1d-IQ1.2 exhibited
very similar basal and actin-activated ATPase activities.
In the presence of actin, the ATPase activities reached
V
max
values of 2.6–3.1 s
)1
[22]. The addition of one or
two IQ motifs to the head domain did not affect ATPase
rates and actin affinities. In the present study, we show
that IQ1 can even be replaced by IQ2 without that the
ATPase rates and actin affinities get significantly
altered. However, binding of Ca
2+
to CaM associated
with the IQ motif directly following the head (converter)
domain induced a significant inhibition of the Myo1d
actin-activated ATPase activity. This inhibition was
independent of whether one or two IQ motifs were pre-
sent or IQ1 or IQ2 were directly fused to the head
region. The free Ca
2+
concentrations that were neces-

sary to induce the inhibition of the actin-activated
ATPase activity correlated well with the reported affin-
ity of the C-terminal lobe of CaM for Ca
2+
. The N-ter-
minal lobe of CaM exhibits a 10-fold lower affinity for
Ca
2+
[19]. These results support the notion that the
C-terminal lobe of CaM is bound to both IQ1 and IQ2
in a semi-open conformation and switches upon Ca
2+
-
binding to an open conformation. The open conforma-
tion inhibits the actin-activated ATPase activity of
Myo1d. The C-terminal lobe of CaM bound in an open
conformation to the IQ motif directly following the
head region might inhibit the actin-activated ATPase
activity by specific interactions with the head region.
The ELC bound to IQ1 of smooth muscle myosin II has
been observed to contact in the prepower stroke state a
loop in the head domain that modulates nucleotide
affinity [27]. Therefore, a change in nucleotide affinity
might be the reason for the reduced ATPase activity.
Changes in nucleotide affinity have also been reported
for Dictyostelium discoideum myosin II head constructs
with varying lengths of the C-terminal a-helix of the
converter domain that is continuous with the IQ-motifs
[28]. Therefore, Ca
2+

⁄ CaM may modulate the actin-
activated ATPase activity by affecting the stability or
flexibility of the a-helix N-terminal to the IQ-motif(s).
Fusion of the IQ2 directly to the Myo1d head con-
struct led to the deletion of the two amino acids immedi-
ately N-terminal to the IQ-motif. This deletion caused a
similar but Ca
2+
-independent inhibition of the Myo1d
actin-activated ATPase activity. This deletion is predic-
ted to rotate the CaM associated with the IQ-motif by
200° on the a-helix emanating from the converter
domain and to shorten this a-helix by roughly 3 A
˚
. The
fact that this construct demonstrates a similar reduction
Regulation of Myo1d ATPase activity D. Ko
¨
hler et al.
2194 FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS
in ATPase activity might either be explained by coinci-
dence or by the assumption that it mimics the Ca
2+
-
dependent changes induced in the other IQ-motif
constructs. However, the latter possibility seems only
feasible when the inhibitory mechanism does not involve
a stereospecific interaction of CaM with the head region.
A common mode of regulation may include effects on
conformation and ⁄ or flexibility of the light chain bind-

ing domain and N-terminal a-helix that are transduced
to the head region. Such effects may become more pro-
nounced when the head is bearing strain.
As reported here for Myo1d, the first of several IQ-
motifs was demonstrated to control the Ca
2+
-sensitiv-
ity of the kinetics of Myo1b (MI
130
; myr 1) [29].
Myo1e (myr 3; Myosin IC) has only a single IQ-motif
and Ca
2+
-binding to the CaM associated with this IQ-
motif negatively regulates the basal ATPase activity
[21]. Therefore, the first IQ-motif in class I myosins
might serve generally as a Ca
2+
-regulatory element.
On the other hand, conversion of the CaM C-lobe
upon Ca
2+
-binding from a semi-open to open IQ-
motif binding configuration is unlikely to provide a
general inhibitory mechanism for class I myosin
ATPase activities. Although binding of Ca
2+
to the
C-lobe of CaM has been reported to inhibit actin gli-
ding powered by Myo1c (myosin Ib, myr 2), Myo1c

ATPase activity was not affected at this Ca
2+
concen-
tration and was enhanced at 10-fold higher Ca
2+
con-
centrations simultaneously with the dissociation of one
CaM [19]. The actin-activated ATPase activities of
Myo1a (brush border myosin I) and Myo1b (MI
130
;
myr 1) were actually increased in the presence of
Ca
2+
⁄ CaM [20,30]. In Myo1e the basal ATPase activ-
ity was reduced with a Ca
2+
-sensitivity suggestive of a
contribution by both the C- and N-terminal lobes of
CaM [21]. In conclusion, the regulatory functions of
Ca
2+
⁄ CaM in different class I myosin molecules
appears to be quite diverse and no common mecha-
nisms have emerged yet. The molecular basis for these
differences remains to be elucidated. The detailed char-
acterization of Myo1d regulation by Ca
2+
⁄ CaM provi-
ded here represents a necessary step towards this goal.

Experimental procedures
Plasmid construction
Construction of the expression plasmids Myo1d-head-
FLAG, Myo1d-IQ1-FLAG and Myo1d-IQ1.2-FLAG
which encode amino acids 1–697, 1–721 and 1–743 of rat
Myo1d, respectively, has been described previously [22].
Plasmids Myo1d-IQ2-FLAG and Myo1dDLV-IQ2-FLAG
have the first of the two IQ-motifs deleted, so that the
second IQ-motif is fused to the head domain directly.
Myo1dDLV-IQ2-FLAG is further missing the last two
codons for amino acids 698–699 (LV) of the head. For the
generation of these two deletion constructs, a two step
PCR strategy was employed. At first, two overlapping frag-
ments were amplified by PCR. The sequences flanking the
deleted region were fused in the reverse primer used for
amplification of the 5¢-fragment. To construct Myo1d-IQ2-
FLAG, the two overlapping fragments were amplified with
the two primer pairs 5¢-GGCAAACTTGATGATGAGCG
CTGC-3¢ (forward 1) ⁄ 5¢-CAGAGCTGCCTTGACGA-
GCATCTG-3¢ (reverse 1) and 5¢-CAGATGCTCGTCAA
GGCAGCTCTG-3¢ (forward 2) ⁄ 5¢-ATTCCAGCACACT
GGTCACTT-3¢ (reverse 2). To obtain Myo1dDLV-IQ2-
FLAG, the two primer pairs forward 1 ⁄ 5¢-CAGAGC
TGCCTTCATCTGGGCGCG-3¢ (reverse 1¢) and 5 ¢-ATT
CCAGCACACTGGTCACTT-3¢ (forward 2¢) ⁄ reverse 2
were used, respectively. After annealing and extension of
the two overlapping fragments, the resultant fragment
served as the template in a final PCR using the primer pair
forward 1 ⁄ reverse 2. The resulting products were cloned
into pIRES Myo1d-head-FLAG using the unique BstXI

and Eco47III restriction sites. PCR derived fragments were
verified by sequencing.
Cell culture
HtTA-1 HeLa cells [31] were cultured at 37 °C and 5%
(v ⁄ v) CO
2
in Dulbecco’s modified Eagle’s medium supple-
mented with 10% (v ⁄ v) fetal bovine serum, 100 UÆmL
)1
penicillin and 100 lgÆmL
)1
streptomycin. Cells were trans-
fected by the addition of plasmid DNA precipitated with
calcium phosphate. Single colonies were isolated after
selection in 200 lgÆmL
)1
hygromycin for 2 weeks. Cells
were expanded and analyzed for expression of Myo1d
constructs by immunoblotting with the rat Myo1d anti-
body SA 522. Selection by hygromycin was maintained
continuously.
Protein expression and purification
Recombinant Myo1d proteins were purified as described in
detail previously [22]. Briefly, cells were grown in 20–30 cul-
ture dishes (diameter 150 mm) to 80% confluence, washed
with NaCl ⁄ P
i
, collected by scraping and permeabilized with
lysis buffer [150 mm NaCl, 20 mm Hepes pH 7.4, 2 mm
MgCl

2
,1mm EGTA, 0.5% (v ⁄ v) Triton X-100, 2 mm
ATP, 0.1 mgÆmL
)1
Pefabloc, 0.01 mgÆmL
)1
leupeptin,
0.02 UÆmL
)1
aprotinin] for 1 h on ice. After clearing the
lysate by two subsequent centrifugation steps, the super-
natant was mixed with 1 mL FLAG-antibody agarose
(Sigma-Aldrich) and incubated for 2 h in the cold. The
beads were washed twice with buffer WP (50 mm KCl,
10 mm Hepes pH 7.4, 2 mm MgCl
2
,1mm EGTA, 1 mm
D. Ko
¨
hler et al. Regulation of Myo1d ATPase activity
FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS 2195
2-mercaptoethanol and 2 mm NaN
3
) and finally, bound pro-
tein was eluted by buffer WP supplemented with
125 lgÆmL
)1
soluble FLAG peptide (Sigma-Aldrich). In
some cases, Myo1d-IQ2 was purified in the presence of
50–100 lm free Ca

2+
concentrations. Occasionally, 5 lm
calmodulin was added during the elution of Myo1d-IQ2 to
enhance protein solubility. Eluted proteins were dialyzed
against buffer WP and concentrated using microcon filters
(cut-off 10 kDa) if necessary. All recombinant Myo1d pro-
teins were cleared by ultracentrifugation (150 000 g for
20 min) immediately before use. To saturate all light chain
binding sites, cleared Myo1d-IQ2 and Myo1dDLV-IQ2
preparations were preincubated with 10 lm calmodulin for
20 min on ice. Densitometric analysis of Coomassie-stained
protein bands on SDS gels was performed with an ultrascan
laser densitometer. Values represent the mean of at least
three different preparations. Actin was purified from rabbit
skeletal muscle as described by Pardee and Spudich [32].
Purified calmodulin was purchased from Sigma-Aldrich.
ATPase assays
Steady-state ATPase activities were determined at 37 °Cas
described in detail previously [21]. All assay mixtures con-
tained 30 mm KCl, 10 mm Hepes pH 7.4, 2 mm MgCl
2
,
2mm ATP, 3 mm EGTA, 1 mm 2-mercaptoethanol, 2 mm
NaN
3
and various actin concentrations. To measure Ca
2+
-
dependent activities, a constant actin concentration of
24 lm was used. Free Ca

2+
concentrations between 0.001
and 158 lm were adjusted by adding the appropriate
amounts of CaCl
2
to obtain the desired value in the pres-
ence of 3 mm EGTA. Actin-dependent ATPase activities
were measured in the absence or presence of 22 lm free
Ca
2+
in a concentration range between 0 and 75 lm actin.
Purified Myo1d constructs were used in a range between 54
and 270 nm. V
max
and K
m
values were determined by fitting
the measured ATPase rates (v) to the Michaelis–Menten
equation
v ¼ V
max
½actin=ðK
actin
þ½actinÞ ð1Þ
with the program kaleidograph.
Sedimentation assays
To separate myosin-associated CaM from soluble CaM,
myosin was incubated with 2 lm F-actin in a buffer con-
taining 30 mm KCl, 10 mm Hepes pH 7.4, 2 mm MgCl
2

,
3mm EGTA, 1 mm 2-mercaptoethanol and 2 mm NaN
3
for 15 min on ice. Where indicated, the free Ca
2+
concen-
trations were adjusted accordingly. Assay mixtures with
Myo1d-IQ2 purified under calcium conditions contained
50–100 lm free Ca
2+
. Free Ca
2+
was chelated in these
preparations by the addition of appropriate amounts of
EGTA. After high speed centrifugation at 150 000 g for
20 min, supernatants with soluble CaM were separated
from pellets containing acto-myosin complexes with tightly
bound CaM and analyzed by SDS ⁄ PAGE and densito-
metry.
Acknowledgements
We thank Margrit Mu
¨
ller and Edith Bru
¨
ne for techni-
cal assistance. We acknowledge the financial support
of the DFG (Ba 1354 ⁄ 6–1).
References
1Ba
¨

hler M & Rhoads A (2002) Calmodulin signaling via
the IQ motif. FEBS 513, 107–113.
2 Barylko B, Binns DD & Albanesi JJ (2000) Regulation
of the enzymatic and motor activities of myosin I. Bio-
chim Biophys Acta 1496, 23–35.
3 Wolenski JS (1995) Regulation of calmodulin-binding
myosins. Trends Cell Biol 5, 310–316.
4 Linse S, Helmersson A & Forsen S (1991) Calcium
binding to calmodulin and its globular domains. J Biol
Chem 266, 8050–8059.
5 Maune JF, Klee CB & Beckingham K (1992) Ca
2+
binding and conformational change in two series of
point mutations to the individual Ca
2+
-binding sites of
calmodulin. J Biol Chem 267, 5286–5295.
6 Alexander KA, Wakim BT, Doyle GS, Walsh KA &
Storm DR (1988) Identification and characterization of
the calmodulin-binding domain of neuromodulin, a neu-
rospecific calmodulin-binding protein. J Biol Chem 263,
7544–7549.
7 Baudier J, Deloulme JC, Dorsselaer AV, Black D &
Matthes HWD (1991) Purification and characterization
of a brain-specific protein kinase C substrate, neurogra-
nin (p17): identification of a consensus amino acid
sequence between neurogranin and neuromodulin
(GAP43) that corresponds to the protein kinase C phos-
phorylation site and the calmodulin-binding domain.
J Biol Chem 266, 229–237.

8Ba
¨
hler M, Kroschewski R, Sto
¨
ffler H & Behrmann T
(1994) Rat myr 4 defines a novel subclass of myosin I:
identification, distribution, localization, and mapping of
calmodulin-binding sites with differential calcium sensi-
tivity. J Cell Biol 126, 375–389.
9 Farnsworth CL, Freshney NW, Rosen LB, Ghosh A,
Greenberg ME & Feig LA (1995) Calcium activation of
Ras mediated by neuronal exchange factor Ras-GRF.
Nature 376, 524–527.
10 Mermall V, Post PL & Mooseker MS (1998) Unconven-
tional myosins in cell movement, membrane traffic, and
signal transduction. Science 279, 527–533.
11 Sellers JR (2000) Myosins: a diverse superfamily. Bio-
chim Biophys Acta 1496, 3–22.
Regulation of Myo1d ATPase activity D. Ko
¨
hler et al.
2196 FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS
12 Berg JS, Powell BC & and. Cheney RE (2001) A millen-
nial myosin census. Mol Biol Cell 12, 780–794.
13 Swanljung-Collins H & Collins JH (1991) Ca
2+
stimu-
lates the Mg
2(+)
-ATPase activity of brush border myo-

sin I with three or four calmodulin light chains but
inhibits with less than two bound. J Biol Chem 266,
1312–1319.
14 Ruppert C, Kroschewski R & Ba
¨
hler M (1993) Identifi-
cation, characterization and cloning of myr 1, a mam-
malian myosin-I. J Cell Biol 120, 1393–1403.
15 Sherr EH, Joyce MP & Greene LA (1993) Mammalian
myosin I alpha, I beta, and I gamma: new widely
expressed genes of the myosin I family. J Cell Biol 120 ,
1405–1416.
16 Ruppert C, Godel J, Mu
¨
ller RT, Kroschewski R, Rein-
hard J & Ba
¨
hler M (1995) Localization of the rat myo-
sin I molecules myr 1 and myr 2 and in vivo targeting of
their tail domains. J Cell Sci 108, 3775–3786.
17 Reizes O, Barylko B, Li C, Su
¨
dhof TC & Albanesi JP
(1994) Domain structure of a mammalian myosin I
beta. Proc Natl Acad Sci USA 91, 6349–6353.
18 Wolenski JS, Hayden SM, Forscher P & Mooseker MS
(1993) Calcium-calmodulin and regulation of brush bor-
der myosin-I MgATPase and mechanochemistry. J Cell
Biol 122, 613–621.
19 Zhu T, Beckingham K & Ikebe M (1998) High affinity

Ca
2+
binding sites of calmodulin are critical for the
regulation of myosin Ibeta motor function. J Biol Chem
273, 20481–20486.
20 Perreault-Micale C, Shushan AD & Coluccio LM
(2000) Truncation of a mammalian myosin I results in
loss of Ca
2+
-sensitive motility. J Biol Chem 275, 21618–
21623.
21 Sto
¨
ffler HE & Ba
¨
hler M (1998) The ATPase activity of
Myr3, a rat myosin I, is allosterically inhibited by its
own tail domain and by Ca
2+
binding to its light chain
calmodulin. J Biol Chem 273, 14605–14611.
22 Ko
¨
hler D, Ruff C, Meyho
¨
fer E & Ba
¨
hler M (2003) Dif-
ferent degrees of lever arm rotation control myosin step
size. J Cell Biol 161, 237–241.

23 Terrak M, Wu G, Stafford WF, Lu RC & Domin-
guez R (2003) Two distinct myosin light chain struc-
tures are induced by specific variations within the
bound IQ motifs-functional implications. EMBO J 22,
362–371.
24 Houdusse C & Cohen A (1995) Target sequence recog-
nition by the calmodulin superfamily: implications from
light chain binding to the regulatory domain of scallop
myosin. Proc Natl Acad Sci USA 92, 10644–10647.
25 Houdusse A, Silver M & Cohen C (1996) Structure
of the regulatory domain of scallop myosin at 2 A
˚
resolution: implications for regulation. Structure 4,
1475–1490.
26 Bahloul A, Chevreux G, Wells AL, Martin D, Nolt J,
Yang Z, Chen LQ, Potier N, Van Dorsselaer A, Rosen-
feld S, Houdusse A & Sweeney HL (2004) The unique
insert in myosin VI is a structural calcium-calmodulin
binding site. Proc Natl Acad Sci USA 101, 4787–4792.
27 Dominguez R, Freyzon Y, Trybus KM & Cohen C
(1998) Crystal structure of a vertebrate smooth muscle
myosin motor domain and its complex with the essential
light chain: visualization of the pre-power stroke state.
Cell 94, 559–571.
28 Kurzawa SE, Manstein DJ & Geeves MA (1997) Dic-
tyostelium discoideum myosin II: characterization of
functional myosin motor fragments. Biochemistry 36,
317–323.
29 Geeves MA, Perreault-Micale C & Coluccio LM (2000)
Kinetic analyses of a truncated mammalian myosin I

suggest a novel isomerization event preceding nucleotide
binding. J Biol Chem 275, 21624–21630.
30 Collins K, Sellers JR & Matsudaira P (1990) Calmodu-
lin dissociation regulates brush border myosin I (110-
kD-calmodulin) mechanochemical activity in vitro .
J Cell Biol 110, 1137–1147.
31 Gossen M & Bujard H (1992) Tight control of gene
expression in mammalian cells by tetracycline-responsive
promoters. Proc Natl Acad Sci USA 89, 5547–5551.
32 Pardee JD & Spudich JA (1982) Purification of muscle
actin. Methods Cell Biol 24, 271–289.
D. Ko
¨
hler et al. Regulation of Myo1d ATPase activity
FEBS Journal 272 (2005) 2189–2197 ª 2005 FEBS 2197