Identification of the Single Specific IQ Motif of Myosin V from Which Calmodulin
Dissociates in the Presence of Ca2+ †Hiroshi Koide,‡ Tatsuya Kinoshita,‡ Yusuke Tanaka,‡ Shin’ichiro Tanaka,‡ Naoki Nagura,‡
Gabriele Meyer zu Hörste,‡ Atsushi Miyagi,‡ and Toshio Ando*,‡,§
Department of Physics, Kanazawa UniVersity, Kakuma-machi, Kanazawa 920-1192, Japan, andCREST, Japan Science and Technology Agency, Saitama, Japan
ReceiVed July 10, 2006; ReVised Manuscript ReceiVed July 21, 2006

ABSTRACT: Each heavy chain of dimeric chick brain myosin V (BMV) has a neck domain consisting of
six IQ motifs with different amino acid sequences. The six IQ motifs form binding sites for five calmodulin(CaM) molecules and one essential light chain (either 17 or 23 kDa). When the calcium concentration ishigh, a small fraction of the 10 total CaM molecules dissociates from one molecule of BMV, resulting inloss of actin-based motor activity. At low Ca2+ concentrations, two molecules of exogenous CaM associatewith one molecule of CaM-released BMV. This suggests that there is a single specific IQ motif responsible
for the calcium-induced dissociation of CaM. In this study, we identify the specific IQ motif to be IQ2,
the second IQ motif when counted from the N-terminal end of the neck domain. In addition, we showedthat the essential light chains do not reside on IQ1 and IQ2. These findings were derived from proteolysis
of BMV at high Ca2+ concentrations specifically at the neck region and SDS-PAGE analyses of thedigests.

Myosin V is a double-headed molecular motor thatfunctions as an organelle transporter in various cells (1-3)
and moves processively along actin tracks (4). Myosin V’s
ATPase kinetics effectively account for the processivity (57). The movement proceeds in 36 nm steps (8-10), in a
hand-over-hand fashion (11, 12), with the two heads alternating between the leading and trailing positions.
Myosin V consists of two heavy chains and several lightchains. The constituent light chains differ between chickbrain myosin V (BMV)1 and murine myosin V. In BMV,
each heavy chain is associated with five calmodulin (CaM)
light chains, one essential light chain (ELC, either 23 or 17kDa), and one 8 kDa light chain (identical to the 10 kDa
dynein light chain) (13, 14). In murine myosin V, there is a
CaM molecule in place of the ELC (7). The heavy chain
has an 88 kDa head domain at the N-terminus, adjacent toa neck domain consisting of six IQ motifs of ∼24 aminoacids each (15), which are the binding sites for CaM and
ELC. Next to the neck domain is a coiled coil region that is
responsible for dimerization which forms a 30 nm stalk; aglobular tail domain lies at the end of the heavy chain.
†This study was supported by CREST/JST and a Grant-in-Aid for
Basic Research (S) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
* To whom correspondence should be addressed. E-mail:tando@kenroku.kanazawa-u.ac.jp. Phone: +81 76-264-5663. Fax: +81
76-264-5739.
‡
Kanazawa University.
§
CREST, Japan Science and Technology Agency.
1
Abbreviations: CaM, calmodulin; ELC, essential light chain; RLC,
regulatory light chain; BMV, chick brain myosin V; PK, proteinase K;
BSA, bovine serum albumin; PMSF, phenylmethanesulfonyl fluoride;
EGTA, ethylene bis(oxyethylenenitrilo)tetraacetic acid; HMM, heavy
meromyosin; S-1, subfragment 1; SDS, sodium dodecyl sulfate; PAGE,
polyacrylamide gel electrophoresis.

The motor activity of myosin V is regulated by Ca2+ (13,16). At low Ca2+ concentrations (pCa > 7.0), myosin V has
full motility, while at high Ca2+ concentrations (pCa < 6.0),
it has no motility. At high Ca2+ concentrations, CaM partiallydissociates from the myosin V neck domains (4, 17, 18).
Initially, there was disagreement with regard to the potential
relationship between CaM dissociation and loss of motor
activity (16, 19), but it is now clear that dissociation of CaM
greatly weakens the affinity of myosin V for actin, which
results in a loss of motor activity (20).
Part of the myosin V neck region that is exposed following
CaM dissociation can be refilled with exogenous CaM at
low Ca2+ concentrations (4). Using exogenous Cy3-labeled
CaM, we previously observed that endogenous CaM that had
partially dissociated in a relatively high Ca2+ concentration
solution (pCa ∼ 5.0) was replaced with only 2.1 Cy3-labeled
CaMs per BMV molecule (4). This was recently confirmedby another group (20). They estimated the number of Cy3labeled CaM molecules associated with individual myosin
V molecules whose endogenous CaM had been substituted
with Cy3-labeled CaM at various Ca2+ concentrations, using
a fluorescence microscope to quantify the number of photobleaching steps. When pCa < 5.6, almost all BMV
molecules had two associated Cy3-labeled CaMs, while avery small fraction (<5%) had three associated Cy3-labeled
CaMs. These observations suggest that there is a single
specific IQ motif responsible for dissociation of CaM frommyosin V.
We sought to identify the specific IQ motif. In this study,
we demonstrate the following novel findings. (1) At highCa2+ concentrations, proteinase K (PK) specifically cleaves
BMV at the neck domain, producing single-headed BMV(S-1) with an exposed IQ motif. (2) The exposed IQ motif

10.1021/bi0613877 CCC: $33.50 © xxxx American Chemical Society
Published on Web 08/26/2006
PAGE EST: 6.4

B

Koide et al.

is refilled with exogenous CaM at low Ca2+ concentrations,and the resulting reconstituted S-1 (reS-1) has motor activity.
Then, we present analyses of the quantities of CaM in S-1
and reS-1 and successfully identify the single specific IQ
motif as IQ2. This conclusion agrees with the previous
finding by Trybus et al. that IQ2 is at least one of the sites
from which CaM dissociates in Ca2+ (16).
MATERIALS AND METHODS
Proteins. BMV was prepared as previously described (21),stored at 0 °C, and used within 10 days. Actin was preparedfrom rabbit skeletal muscles and purified as previously
described (22). Bovine brain CaM, bovine serum albumin(BSA, fraction V), and PK were purchased from SigmaAldrich. Streptavidin and casein were obtained from Life
Technologies and Nacalai Tesque, respectively.
Ca2+-Induced Dissociation of CaM from BMV. A BMV
sample (ca. 100 nM) in a Ca2+-free solution [buffer A
containing 100 mM KCl, 20 mM imidazole (pH 7.6), 2 mM
MgCl2, 1 mM EGTA, and 2 mM 2-mercaptoethanol] wasdivided into two aliquots. One was cosedimented with F-actin(2 µM), and its pellet was subjected to SDS-PAGE withfive different loads to obtain a standard density-load curve.
The other was first incubated for 5 min at 25 °C in buffer A
and then incubated for 5 min at 25 °C in a Ca2+-containing
solution (buffer A containing 1.01 mM CaCl2). Bound anddissociated CaM were separated by cosedimentation (163000g
for 40 min) of BMV with F-actin (2 µM). The pellet andsupernatant were subjected to SDS-PAGE, and the ratio of
dissociated to bound CaM was estimated from the standard
density-load curve.
Digestion of BMV by Proteinase K. BMV was digested
with PK using PK:BMV ratios from 1:200 to 1:25 (w/w).
BMV (ca. 100 nM) was mixed with PK in buffer A with orwithout 1.01 mM CaCl2 and incubated for 10 min at 25 °C.
The reaction was quenched by the addition of 100 µM
phenylmethanesulfonyl fluoride (PMSF). The digests were
subsequently analyzed by SDS-PAGE. Actin-binding components in the samples were separated by cosedimentation
with F-actin (163000g for 40 min) and analyzed by SDSPAGE.
Preparation of BMV S-1, reS-1, and HeaVy Meromyosin.
BMV (ca. 100 nM) was digested with PK using a 1:40 ratio(w/w) of PK to BMV for 10 min at 25 °C in buffer A with
1.01 mM CaCl2. After the reaction had been quenched withthe addition of 100 µM PMSF, the sample was placed onice, mixed with 5 µM F-actin and 2 mM EGTA, andcentrifuged at 263000g for 40 min. The pellet was dissolvedin a high-ionic strength solution containing 20 mM imidazole(pH 7.6), 600 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and2 mM ATP, and the solution was centrifuged again. S-1 wascollected in the supernatant. In the preparation of reS-1, 1µM CaM or labeled CaM was added to the digested BMV
samples at the same time as F-actin and 2 mM EGTA. For
measurement of the motor activity of reS-1, the supernatantcollected as reS-1 (biotinylated CaM was used) was further
purified using a Q-Sepharose column (Amersham Pharmacia
Biotech, 5 mm in diameter and 20 mm in length); thesupernatant was diluted three times with a solution containing
20 mM imidazole (pH 7.6), 2 mM MgCl2, and 1 mM EGTA
and loaded on a column equilibrated with buffer B [20 mM

Biochemistry
imidazole (pH 7.6), 200 mM NaCl, 2 mM MgCl2, 1 mM
EGTA, 1 mM 2-mercaptoethanol, and 20 µM PMSF] and 2
mM ATP. After the column was run with buffer B containing
ATP and then with buffer B without ATP, reS-1 was eluted
with a high-ionic strength solution (200 mM NaCl in buffer
B was replaced with 600 mM NaCl). The collected sample
was dialyzed against a low-ionic strength solution (200 mM
NaCl in buffer B was replaced with 25 mM KCl). BMV
heavy meromyosin (HMM; the C-terminal tail domain of
BMV is truncated) was prepared in the same way as S-1,
except that BMV was digested by PK in the absence of Ca2+.
HMM was purified by Q-Sepharose column chromatography
in the same way as reS-1.
CaM Labeling. CaM (35 µM) was mixed with 70 µM Cy3
monofunctional N-hydroxysuccinimide ester (Amersham
Bioscience) or 150 µM biotin-(AC5)2 sulfo-OSu (Dojindo
Laboratories) in a solution containing 0.3 M KCl, 20 mM
boric acid (pH 8.5), and 0.1 mM CaCl2 and incubated for 1
h at 25 °C. Following the addition of 5 mM dithiothreitol,
the samples were exhaustively dialyzed against buffer A toremove unreacted Cy3 or biotin. The amount of incorporated
Cy3 was two molecules per CaM, with the amount of Cy3
determined from its absorbance at 550 nm (550 ) 1.5 ×
105 M-1 cm-1) and CaM concentration determined by the
Lowry method. The amount of incorporated biotin was twomolecules per CaM, with a biotin concentration determined
as described previously (22).
SDS-PAGE. Slab-gel SDS-PAGE was performed using
3% stacking gels and 8 to 16% linear-gradient separationgels. Each sample (50 µL) to be examined was mixed with
25 µL of a SDS-containing sample buffer [0.31 M Tris-HCl(pH 6.8), 30% glycerol, 9.4% SDS, 31% 2-mercaptoethanol,and 0.003% bromophenol blue]. After samples had been run,the gels were stained with a silver staining solution and/or
Quick-CBB (Wako Chemicals). For analysis of band intensity, the gels were stained with Quick-CBB alone. Biotinylated CaM in the samples was detected using a property of
streptavidin, its persistence to SDS at even a high temperature(25, 26). Each sample was mixed with SDS sample buffer
and divided into two aliquots. One (30 µL) was mixed withstreptavidin (7 µM, 5 µL) and incubated for 10 min at 35°C, and the other (30 µL) was mixed with 5 µL of waterand incubated for 10 min in boiling water. In the streptavidinmixed samples, biotin-CaM species migrated in gels as acomplex with streptavidin. Each lane in the stained gels wasanalyzed using analysis software developed in our lab.
In Vitro Actin Filament Sliding Assay. The assay with
intact BMV was performed as previously described (4). The
assay with reS-1 was performed as follows. A biotin-BSA
sample (1 mg/mL, 50 µL) in buffer A was applied to a flow
cell (10-20 µL) made from two nitrocellulose-coated coverslips and incubated for 3 min. After the sample had been
washed with buffer A, 50 µL of casein (1 mg/mL in bufferA) was placed in the cell, incubated for 3 min, and washedwith buffer A. Streptavidin (0.2 mg/mL, 30 µL) in buffer A
was applied to the cell, incubated for 3 min, and washedwith buffer A. ReS-1 reconstituted using biotinylated CaM(ca. 10 nM, 40 µL) was applied to the cell, incubated for 6
min, and washed with buffer A. Rhodamine phalloidinelabeled F-actin (15 nM, 100 µL) was applied to the cell andincubated for 2 min. Last, the buffer solution was replacedwith motility buffer containing 2 mM ATP and an oxygen-

Biochemistry

Identification of Specific IQ in Myosin VC

FIGURE 1: SDS-PAGE analysis of the light chain components
dissociated or undissociated from BMV at high Ca2+ concentrations.
BMV incubated at high Ca2+ concentrations was cosedimented with
F-actin, and its supernatant (S) and pellet (P) were subjected to
SDS-PAGE. As a reference for band intensity, myosin V mixed
with F-actin at low Ca2+ concentrations was also subjected to SDSPAGE with different loads (from 1/5 to 1). The maximum load (1)is the same as the total load of the pellet and supernatant. HC,
myosin V heavy chain; DLC, 10 kDa dynein light chain; MWM,
molecular mass markers.

scavenging system (0.2 mg/mL glucose oxidase, 0.36 mg/
mL catalase, and 4.4 mg/mL glucose) in buffer A. The sliding
movement of actin filaments was observed at 25 °C using
an SIT video camera (C2741-08, Hamamatsu Photonics)
coupled with an image intensifier (VSA-1845, Video Scope).
The image intensifier was connected to a microscope (IX71,
Olympus) equipped with an oil-immersion objective (PlanApo 100×, NA 1.40, Olympus) and an objective lens-typeevanescent illumination with a diode green laser (GCL-050L, 532 nm, Crystal Laser) and a filter set (U-MWG2,
Olympus). The image recording and analysis were performed
as previously described (4).
Atomic Force Microscopic Imaging. An atomic force
microscope (AFM) developed in the lab and nearly the sameas that described previously (23, 24) was used. One drop(ca. 1.5 µL) of the purified BMV S-1, reS-1, or HMM (ca.
2 nM) was placed on freshly cleaved mica (1 mm indiameter) for 3 min, rinsed with buffer A, and imaged in asolution containing 20 mM KCl, 25 mM imidazole (pH 7.6),
2 mM MgCl2, and 1 mM EGTA.
RESULTS
Quantity of Dissociated CaM. It was previouosly reported
that approximately three CaM molecules (ca. 30% of thetotal of 10 CaMs) dissociated from one molecule of BMV
in Ca2+ (17). The number was reported to be approximately
two with mouse myosin V constructs having two or six IQ
motifs (19). On the other hand, the number of exogenous
CaMs (Cy3-labeled CaMs) incorporated into partially CaM
dissociated BMV was reported to be two per BMV (4); thisnumber was recently confirmed by the fluorescence microscopic observation of individual BMV molecules with Cy3labeled CaM (20). Although the number of released CaMs
need not necessarily equal the number of incorporated
exogenous CaMs, it is more likely that this is so. Here we
re-examine the number of dissociated CaMs by separatingbound and free CaMs using an actin cosedimentation method.
As shown in Figure 1, addition of Ca2+ resulted in partial
dissociation of CaM from the heavy chain. The densities of
CaM in the supernatant and pellet were the same (within

FIGURE 2: SDS-PAGE analysis of cleavage of myosin V by PK
in the presence or absence of Ca2+. (A) Cleavage patterns as afunction of the weight ratio of PK to BMV and the presence ofCa2+. The weight ratio is indicated at the top. The left lane (marked
with BMV) contained intact BMV. The marks + and - at the top
indicate the presence and absence, respectively, of Ca2+ during
proteolysis. The right lane contained molecular mass markers(MWM). (B) Separation by cosedimentation with F-actin of actinbinding and nonbonding components of the digests obtained in theabsence (left) or presence (right) of Ca2+. S denotes supernatantand P pellet. (C) Stick diagram of the myosin V heavy chain andsketch of myosin V. In the stick diagram, the dotted line represents
a junction corresponding to the 50 kDa/20 kDa junction of
conventional myosin II. The shaded portion and gray bar represent
the IQ motifs and PEST site, respectively. The arrowheads denotethe PK cleavage sites. (D) AFM images of actin-binding fractions.
The top image (scan range, 250 nm) is a sample obtained withcleavage of BMV by PK in the absence of Ca2+ (i.e., HMM); theimage library in the middle line (scale bar, 10 nm) was obtainedwith S-1 (a sample obtained with cleavage of BMV by PK in thepresence of Ca2+), and the image library in the bottom line (scalebar, 10 nm) was obtained with reS-1. (E) Light chain components
contained in BMV and S-1 obtained by cleavage of BMV by PK
in the presence of Ca2+. The faint band just above the 17 kDa ELC(left lane) is not identified. DLC is the dynein 10 kDa light chain.
The amounts of CaM contained in the loaded BMV and S-1 areapproximately the same (i.e., the number of moles of S-1 loaded is∼5 times more than that of BMV).

(10%) as that of the BMV samples whose loaded amounts
were one-fifth or four-fifth, respectively, of the total load(pellet and supernatant) of the Ca2+-treated sample. Thus,
these analyses indicate that approximately two CaMs per
BMV were released. Therefore, we can conclude that the
number of released endogenous CaMs is approximately equal
to the number of incorporated exogenous CaMs. Figure 1
additionally showed that there were two bands around 17
kDa, and the top band (fainter band) appeared in the
supernatant and did not appear almost completely in thepellet. Although at present we cannot identify this Ca2+sensitive light chain, there is a possibility that it is aregulatory light chain (RLC).
PK Digestion of BMV. Digestion of BMV with PK in theabsence of Ca2+ yielded primarily a 130 kDa fragment(Figure 2A). The amounts of the other fragments diminishedwith an increasing amount of PK, with the exception of theD

Koide et al.

65 kDa fragment, the amount of which slightly increased.
The 130 and 65 kDa domains demonstrated F-actin binding(Figure 2B, left panel). These fragments’ molecular masses
and F-actin binding properties are very similar to those
produced by calpain digestion of BMV in the presence ofCa2+ (17). On the basis of the previously reported analysis(17) of calpain-digested BMV fragments, we understand the
130 kDa fragment to be the N-terminal half of myosin V
digested at (or near) the PEST site and the 65 kDa fragment
to be the N-terminal head domain fragment (see Figure 2C).
The cleavage site that produces the 65 kDa fragment
corresponds to the 50 kDa/20 kDa junction of conventional
myosin II, and the 65 kDa fragment is associated with afragment containing a neck domain (17).
On the other hand, PK digestion in the presence of Ca2+
yielded primarily a 90 kDa fragment (Figure 2A). Thisdomain also demonstrated F-actin binding (Figure 2B, rightpanel). On the basis of these results and the fact that Ca2+
induces dissociation of two CaMs from one BMV molecule,
we take the 90 kDa fragment to be the N-terminal head
domain with a neck domain fragment (i.e., a heavy chain of
BMV S-1) that is produced by cleavage of the neck domain,
likely at the exposed IQ motif. The AFM images (Figure2D) of the actin-binding fractions produced by digestion at
low or high Ca2+ concentrations seem to support thisinterpretation. The fraction obtained at low Ca2+ concentrations revealed a two-headed structure with a short tail (Figure
2D, top image), while the fraction obtained at high Ca2+
concentrations revealed a single-headed structure with a
globular head domain associated with a short neck (Figure
2D, middle line). SDS-PAGE of the S-1 fraction purified
by cosedimentation with F-actin indicated that S-1 did notcontain ELC at all but instead CaM (Figure 2E). The apparent
molecular mass of the S-1 heavy chain, 90 kDa, suggeststhat the neck domain may contain only IQ1 [on the basis of
the amino acid sequence (15), the molecular mass of suchan N-terminal heavy chain is calculated to be 91.5 kDa].
Although the validity of this inference is uncertain, it is verycertain that the light chain associated with IQ1 is CaM andnot ELC.
Reassociation of CaM and S-1. After partial dissociationof CaM at pCa < 5.6, BMV can reassociate with two
exogenous CaM molecules at low Ca2+ concentrations (4,20). We examined whether this property persists with BMV
S-1. Cy3-labeled CaM and EGTA were added to BMV
digested with PK in the presence of Ca2+, and the sample
was copelleted with F-actin. The composition of the pellet
was analyzed by SDS-PAGE, and surprisingly, Cy3-labeled
CaM was found in the pellet (Figure 3). This finding indicatesthat S-1 possesses an exposed IQ motif fragment that stillretains an (almost) complete sequence of the original IQ
motif and therefore the ability to bind CaM. This means that
PK digests the heavy chain at (or near) the junction between
the exposed IQ motif and the next IQ motif on the C-terminal
side. The AFM images of reS-1 (Figure 2D, bottom line)
revealed a structure different from S-1 (Figure 2D, middleline); the globular head domain looked similar, but the neckregion was approximately 2 times longer than that of S-1.
To examine if reS-1 possesses motor function, an in vitro
actin filament gliding assay was performed, using exogenous
biotinylated CaM, and the resulting reS-1 was being adhered
to a streptavidin-coated glass surface. The reS-1 retained

Biochemistry

FIGURE 3: SDS-PAGE analysis of incorporation of Cy3-labeled
CaM into BMV S-1. Cys3-labeled CaM, F-actin, and EGTA wereadded to BMV digests obtained in the presence of Ca2+ and thencentrifuged. The pellets were analyzed by SDS-PAGE. The leftlane is a Quick CBB-stained gel and the center lane a fluorescence
image of the unstained gel, and the right lane contained molecularmass markers.

FIGURE 4: Histogram of the speed of actin filament sliding propelled
by reS-1 reconstructed using biotinylated CaM.

motor function, and the sliding speed of actin filaments was
140 nm/s (Figure 4), one-fourth of that with BMV (560 nm/s) under similar conditions.
Identification of a Single, Specific IQ Motif. It was difficult
to accurately determine the concentrations of the purified
S-1 or reS-1, because they were quite low (<100 nM).
Therefore, we could not directly determine the number of
CaMs contained in S-1 or reS-1. However, only one
endogenous CaM can dissociate, and only one exogenous
CaM can reassociate with one heavy chain. On the basis of
this fact, from the ratio (r) of the amount of CaM containedin S-1 to that in reS-1, we could determine the number (N)of CaMs contained in one molecule of reS-1 [N ) 1/(1 r)] and use it to determine the single, specific IQ motif fromwhich CaM dissociates at high Ca2+ concentrations. First,
we analyzed SDS-PAGE of S-1 and reS-1 to compare theband densities of the 90 kDa heavy chain fragment and CaM(Figure 5A). Although the concentration difference between
the two samples was very small, it was compensated using
the 90 kDa band densities to determine the ratio r. Analysisof SDS-PAGE with three separate sample preparations
resulted in an average r of 0.43, which gave an N of 1.8.
Next, we analyzed SDS-PAGE of only reS-1 (reconstitutedusing biotinylated CaM) mixed or unmixed with streptavidin(Figure 5B). In this case, the concentrations of the two
samples were nearly identical. Prior to the performance of
SDS-PAGE, the sample mixed with streptavidin wasBiochemistry

Identification of Specific IQ in Myosin V

Er of 0.47 results in an N of 1.9. On the basis of these analyses,
we conclude that the single, specific IQ motif is IQ2.
DISCUSSIONFIGURE 5: SDS-PAGE analysis of the amounts of CaM that arecontained in S-1 and reS-1. (A) The sample is a suspension of thepellet obtained by centrifuging a mixture of F-actin and S-1 or reS1. The left lane contained reS-1 reconstituted using nonlabeled CaM
and right lane S-1. (B) The sample was reconstituted using
biotinylated CaM. For the left lane, the sample in SDS sample buffer
was heated at 100 °C for 10 min, and for the right lane, the sample
mixed with streptavidin (SA) in SDS sample buffer was incubatedat 35 °C for 10 min.

FIGURE 6: (A) Difference in migration on the gel between
nonlabeled CaM (left five lanes) and biotinylated CaM (right fivelanes). These samples in SDS sample buffer were heated in boilingwater for 10 min. (B) The integrated band densities as a functionof load for nonlabeled CaM (O) and biotinylated CaM (0). This
analysis was performed for the gel shown in panel A.

incubated in SDS sample buffer for 10 min at 35 °C.
Streptavidin incubated in SDS at even 70 °C retains its
tetrameric structure and an ability to bind to biotin (25, 26).
CaM unfolded in SDS at a temperature as low as 35 °C andmigrated on SDS-PAGE at the same rate as CaM heated at
100 °C (the sample not mixed with streptavidin was
incubated in boiling water). The reS-1 not mixed withstretpavidin showed a sharp CaM band, with a blurred band
situated just above (Figure 5B, left lane). This blurred band
was identified as biotinylated CaM (Figure 6A). Even withsuch blurred bands, the integrated band densities were
quantitatively similar to those of sharp bands of nonlabeled
CaM (Figure 6B). Therefore, the region surrounded by arectangle shown in the left lane of Figure 5B was analyzed
to derive the total amount of CaM contained in the reS-1
sample. On the other hand, the reS-1 mixed with streptavidin
did not exhibit such a blurred band just above a sharp CaM
band (Figure 5B, right lane), indicative of binding of
biotinylated CaM to streptavidin in the gel. The mean ratio(over five separate sample preparations), r, was 0.47 ( 0.03,
similar to the result from the previous analysis (0.43). A ratio

In the presence of 2 mM CaCl2, digestion of BMV witha calcium-dependent cystein peptidase, calpain, yields actin
binding fragments (130 and 65 kDa) and an 80 kDa tailfragment (17). The cleavage sites of the heavy chain arelocated one amino acid from the PEST site and within thehead domain (equivalent to the 50 kDa/20 kDa junction inconventional myosins). Judging from their molecular masses,
these cleavage sites are very similar to those cleaved by aserine endopeptidase (PK) in the absence of Ca2+ (pCa >7). From the above result with calpain digestion of BMV in
Ca2+, the neck region exposed due to CaM dissociation does
not seem to have such a flexible structure that is susceptible
to proteolytic cleavage. However, in this study, we foundthat digestion of BMV with PK yielded an actin-binding 90
kDa fragment only in the presence of Ca2+. The resulting
S-1 still retained the ability to bind to exogenous CaM,
suggesting that the exposed, specific IQ motif is intact
following proteolysis and that the cleavage site must be
situated very near the junction between the specific IQ motif(IQN) and the next IQ motif (i.e., IQN+1). Dissociation of
CaM from the specific IQ motif may affect the neighboring
CaM or ELC in a way that the junction region may be forced
to assume a flexible structure susceptible to cleavage by PK.
Alternatively, binding of Ca2+ to CaM that has attached to
IQN+1 may move the CaM toward IQN+2, thus further
exposing the junction region, which is then attacked by PK.
It has recently been suggested by Martin and Bayley (27,28) that such movement of CaM likely takes place in
response to the binding of a Ca2+-saturated CaM to two
adjacent IQ motifs (i.e., bridge formation). PK has a verybroad cleavage specificity. It cleaves peptide bonds adjacentto the carboxylic group of aliphatic and aromatic amino acids.
Because of its very broad specificity, we cannot precisely
localize the cleavage site that produced the 90 kDa fragment.
We here identify the single, specific IQ motif from which
CaM dissociates at high Ca2+ concentrations as IQ2. Thisfinding seems consistent with the AFM images of S-1 andreS-1 (Figure D) and with previously reported results (16,28, 29). The affinity of CaM for six separate peptides (IQ1IQ6 sequences from murine myosin V) varies and depends
on the presence of Ca2+ (28). At low Ca2+ concentrations,the IQ2 peptide has the lowest affinity for CaM (dissociation
constant of ∼1.6 µM), while the dissociation constants withthe other IQ peptides are 1-2 orders of magnitude smaller.
An increasing Ca2+ concentration enhances the affinity of
CaM for IQ3-IQ6, while the affinity for IQ1 slightly
decreases. In contrast to these changes, the affinity of CaM
for IQ2 significantly decreases (dissociation constant of ∼13µM). In addition, a monomeric murine myosin V constructcomposed of the motor domain and two IQ motifs (IQ1 andIQ2) partially releases CaM in the presence of Ca2+, whilea construct with one IQ motif (IQ1) retains CaM both in thepresence and in the absence of Ca2+ (16, 29). However, there
are reports that do not seem to agree with our view of
dissociation of CaM from only IQ2 (30-32). Sakamoto etal. (30, 31) were able to exchange CaM into the 2IQ HMM
construct from murine myosin V even though it contains IQ1and IQ6 in this order. Since CaM does not dissociate from

F

Koide et al.

IQ1 (16), the exchange must have occurred at IQ6. However,
the affinity of Ca2+-saturated CaM for each IQ motif depends
on the neighboring IQ motifs, because one CaM moleculecan bind to double-length IQ sequences (i.e., bridging) (27,28). Therefore, it is plausible that the affinity of CaM for
IQ6 in IQ1-IQ6 would be much lower than that for IQ6 innatural IQ5 and IQ6. Yildiz et al. (32) observed stepwise
movement of myosin V along actin tracks, in which theendogenous CaM was partially replaced with fluorescently
labeled CaM so that fewer than one fluorophore could be
introduced into each myosin V molecule. A constant step
size of 74 nm was mainly observed. In some cases,
alternative steps of 52 and 23 nm, or 42 and 33 nm, wereobserved, indicating that several IQ motifs contain exchangeable CaM. However, the maximum number of exchangeable
CaMs is reported to be three per myosin V, although the
maximum exchange occurs with a small probability (<5%)(20). Therefore, the alternative steps with different sizes musthave been observed with such a small probability and do
not contradict our conclusion.
In this study, we did not seek to identify the light chainthat appeared just above the 17 kDa ELC on SDS-PAGE
and dissociated almost completely from myosin V in Ca2+.
In previous studies, its existence was not recognized, becauseof its very small quantity. On the basis of its Ca2+ sensitivity
and molecular mass (∼18 kDa), the unidentified light chain
may be a regulatory light chain (RLC). Its quantity is muchsmaller than the amount of ELCs, and therefore, only a small
fraction of BMV contains the light chain. We showed that
ELCs do not reside on either IQ1 or IQ2. However, ELCs
as well as CaM can bind to IQ1 when the 1IQ S-1 construct(containing IQ1) from BMV was coexpressed with one of
the non-muscle myosin ELC isoforms (either LC-1sa,
truncated LC-1sa, or LC-17b) or CaM (33). From thecoexpression with two ELC isoforms or with one ELC
isoform and CaM, the relative order of affinity for the IQ1
is suggested to be as follows: LC-1sa > LC-17b > CaM.
However, differences in the expressed amounts of these lightchains are not reported. Kinetic parameters of acto-myosin
V ATPase reaction are not strongly influenced by the lightchain isoforms, except for the Km for actin (i.e., the actin
concentration at the half-maximal ATPase rate). Because ofthis small influence on the kinetics, the crystal structure of
the myosin V head determined using the 1IQ S-1 constructwith LC-1sa (34) may not be so different from the structureof S-1 with the native complement of CaM.
It is intriguing that IQ2 mediates Ca2+ regulation of myosin
V motor function. Release of CaM from IQ1 might significantly affect the structure of the motor domain, which would
lead to irreversible change. If Ca2+ induced dissociation of
CaM from IQ3 or one of the more distant IQ motifs, there
might be little effect on the motor domain structure or motor
function. Thus, it appears that IQ2 has necessarily beennaturally selected. A situation similar to this occurs in ascallop myosin that shows myosin-linked Ca2+ regulation(35, 36); the ELC associates with the first IQ motif and isin contact with the motor domain, and the regulatory lightchain adjoins the ELC and does not directly interact withthe motor domain.
In conclusion, PK specifically cleaves myosin V at theneck region in the presence of Ca2+, which produces BMV
S-1 having intact IQ1 and IQ2. The IQ1 and IQ2 motifs

Biochemistry
associate with CaM (not ELC), and high Ca2+ concentrations
induce dissociation of CaM specifically from IQ2, which
results in a loss of BMV motor function.
ACKNOWLEDGMENT
We thank Noriyuki Kodera and Yuta Saito for technicalassistance.
REFERENCES
1. Prekeris, R., and Terrian, D. M. (1997) Brain myosin V is asynaptic vesicle-associated motor protein: Evidence for a Ca2dependent interaction with the synaptobrevin-synaptophysin complex, J. Cell Biol. 137, 1589-1601.
2. Evans, L. L., Lee, A. J., Bridgman, P. C., and Mooseker, M. S.
(1998) Vesicle-associated brain myosin-V can be activated tocatalyze actin-based transport, J. Cell Sci. 111, 2055-2066.
3. Tabb, S., Molyneaux, B. J., Cohen, D. L., Kuznetsov, S. A., andLangford, G. M. (1998) Transport of ER vesicles on actin
filaments in neurons by myosin V, J. Cell Sci. 111, 3221-3234.
4. Sakamoto, T., Amitani, I., Yokota, E., and Ando, T. (2000) Direct
observation of processive movement by individual myosin V
molecules, Biochem. Biophys. Res. Commun. 272, 586-590.
5. De La Crutz, E. M., Wells, A. L., Rosenfeld, S. S., Ostap, E. M.,
and Sweeney, H. L. (1999) The kinetic mechanism of myosin V,
Proc. Natl. Acad. Sci. U.S.A. 96, 13726-13731.
6. Rosenfeldt, S. S., and Sweeney, H. L. (2004) A model of myosin
V processivity, J. Biol. Chem. 279, 40100-40111.
7. Wang, F., Chen, L., Arcucci, O., Harvey, E. V., Bowers, B., Xu,
Y., Hammer, J. A., and Sellers, J. R. (2000) Effect of ADP andionic strength on the kinetic and motile properties of recombinantmouse myosin V, J. Biol. Chem. 275, 4329-4335.
8. Mehta, A. D., Rock, R. S., Rief, M., Spudich, J. A., Mooseker,
M. S., and Cheney, R. E. (1999) Myosin-V is a processive actinbased motor, Nature 400, 590-593.
9. Rief, M., Rock, R. S., Mehta, A. D., Mooseker, M. S., Cheney,
R. E., and Spudich, J. A. (2000) Myosin-V stepping kinetics: A
molecular model for processivity, Proc. Natl. Acad. Sci. U.S.A.
97, 9482-9486.
10. Mehta, A. D. (2001) Myosin learns to walk, J. Cell Sci. 114,
1981-1998.
11. Yildiz, A., Forkey, J. N., McKinney, S. A., Ha, T., Goldman, Y.
E., and Selvin, P. R. (2003) Myosin V walks hand-over-hand:Single fluorophore imaging with 1.5-nm localization, Science 300,
2061-2065.
12. Forkey, J. N., Quinlan, M. E., Shaw, M. A., Corrie, J. E. T., andGoldman, Y. E. (2003) Three-dimensional structural dynamics
of myosin V by single-molecule fluorescence polarization, Nature
422, 399-404.
13. Cheney, R. E., O’ Shea, M. K., Heuser, J. E., Coelho, M. V.,
Wolenski, J. S., Espreafico, E. M., Forscher, P., Larson, R. E.,
and Mooseker, M. S. (1993) Brain myosin-V is a two-headed
unconventional myosin with motor activity, Cell 75, 13-23.
14. Espindola, F. S., Suter, D. M., Partata, L. B., Cao, T., Wolenski,
J. S., Cheney, R. E., King, S. M., and Mooseker, M. S. (2000)
The light chain composition of chicken brain myosin-Va: Calmodulin, myosin-II essential light chains, and 8-kDa dynein lightchain/PIN, Cell Motil. Cytoskeleton 47, 269-281.
15. Espreafico, E. M., Cheney, R. E., Matteoli, M., Nascimento, A.
A., De Camilli, P. V., Larson, R. E., and Mooseker, M. S. (1992)
Primary structure and cellular localization of chicken brain
myosin-V (p190), an unconventional myosin with calmodulin lightchains, J. Cell Biol. 119, 1541-1557.
16. Trybus, K. M., Kremensova, E., and Freyzon, Y. (1999) Kinetic
characterization of a monomeric unconventional myosin V
construct, J. Biol. Chem. 275, 27448-27456.
17. Nascimento, A. A. C., Cheney, R. E., Tauhata, S. B. F., Larson,
R. E., and Mooseker, M. S. (1996) Enzymatic characterizationand functional domain mapping of brain myosin-V, J. Biol. Chem.
271, 17561-17569.
18. Cameron, L. C., Carvalho, R. N., Araujo, J. R. V., Santos, A. C.,
Tauhata, S. B., Larson, R. E., and Sorenson, M. M. (1998)
Calcium-induced quenching of intrinsic fluorescencein brain
myosin V is linked to dissociation of calmodulin light chains, Arch.
Biochem. Biophys. 355, 35-42.

Biochemistry

PAGE EST: 6.4

19. Homma, K., Saito, J., Ikebe, R., and Ikebe, M. (2000) Ca2+dependent regulation of the motor activity of myosin V, J. Biol.
Chem. 275, 34766-34771.
20. Nguyen, H., and Higuchi, H. (2005) Motility of myosin V regulatedby the dissociation of single calmodulin, Nat. Struct. Mol. Biol.
12, 127-132.
21. Cheney, R. E. (1998) Purification and assay of myosin V, Methods
Enzymol. 298, 3-18.
22. Kunioka, Y., and Ando, T. (1996) Innocuous labeling of thesubfragment-2 region of skeletal muscle heavy meromyosin witha fluorescent polyacrylamide nanobead and visualization ofindividual heavy meromyosin molecules, J. Biochem. 119, 10241032.
23. Ando, T., Kodera, N., Takai, E., Maruyama, D., Saito, K., andToda, A. (2001) A high-speed atomic force microscope forstudying biological macromolecules, Proc. Natl. Acad. Sci. U.S.A.
98, 12468-12472.
24. Ando, T., Kodera, N., Naito, Y., Kinoshita, T., Furuta, K., andToyoshima, Y. Y. (2003) A high-speed atomic force microscopefor studying biological macromolecules in action, Chem. Phys.
Chem. 4, 1196-1202.
25. Bayer, E. A., Ehrlich-Rogzinski, S., and Wilchek, M. (1996)
Sodium dodecyl sulfate-polyacrylamide gel electrophoretic method
for assessing the quaternary state and comparative thermostabilityof avidin and streptavidin, Electrophoresis 17, 1319-1324.
26. Manning, M., and Colon, W. (2004) Structural basis of protein
kinetic stability: Resistance to sodium dodecyl sulfate suggests
a central role for rigidity and a bias toward β-sheet structure,
Biochemistry 43, 11248-11254.
27. Martin, S. R., and Bayley, P. M. (2002) Regulatory implicationsof a novel mode of interaction of calmodulin with a double IQmotif target sequence from murine dilute myosin V, Protein Sci.
11, 2909-2923.

Identification of Specific IQ in Myosin V

G

28. Martin, S. R., and Bayley, P. M. (2004) Calmodulin bridging of
IQ motifs in myosin-V, FEBS Lett. 567, 166-170.
29. Krementsov, D. N., Krementsova, E. B., and Trybus, K. M. (2004)
Myosin V: Regulation by calcium, calmodulin, and the taildomain, J. Cell Biol. 164, 877-886.
30. Sakamoto, T., Wang, F., Schmitz, S., Xu, Y., Xu, Q., Molloy, J.
E., Veigel, C., and Sellers, J. R. (2003) Neck length andprocessivity of myosin V, J. Biol. Chem. 278, 29201-29207.
31. Sakamoto, T., Yildiz, A., Selvin, P. R., and Sellers, J. R. (2005)
Step-size is determined by neck length in myosin V, Biochemistry44, 16203-16210.
32. Yildiz, A., Forkey, J. N., McKinney, S. A., Ha, T., Goldman, Y.
E., and Selvin, P. R. (2003) Myosin V walks hand-over-hand:Single fluorophore imaging with 1.5-nm localization, Science 300,
2061-2065.
33. De La Cruz, E. M., Wells, A. L., Sweeney, H. L., and Ostap, E.
M. (2000) Actin and light chain isoform dependence of myosin
V kinetics, Biochemistry 39, 14196-14202.
34. Coureux, P.-D., Wells, A. L., Menetrey, J., Yengo, C. M., Morris,
C. A., Sweeney, H. L., and Houdusse, A. (2003) Nature 425, 419423.
35. Xie, X., Harrison, D. H., Schlichting, I., Sweet, R. M., Kalabokis,
V. N., Szent-Györgyi, A. G., and Cohen, C. (1994) Structure of
the regulatory domain of scallop myosin at 2.8 Å resolution,
Nature 368, 306-312.
36. Houdusse, A., and Cohen, C. (1995) Target sequence recognitionby the calmodulin superfamily: Implications from light chain
binding to the regulatory domain of scallop myosin, Proc. Natl.
Acad. Sci. U.S.A. 92, 10644-10647.
BI0613877