To analyze the Myo1d ATPase activity as a function of the IQ-motifs and Ca2+⁄ CaM binding, we expressed and affinity purified the Myo1d constructs Myo1d-head, Myo1d-IQ1, Myo1d-IQ1.2, Myo1d
Trang 1myosin 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 Ca2+
-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 Ca2+-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
Ca2+-independent CaM-binding sites [6,7] However,
in some instances IQ-motifs have been shown to bind CaM in a Ca2+-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 Ca2+⁄ 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 Ca2+ 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 Ca2+ concentration stimulates the basal (actin-independent)
Keywords
calmodulin; Ca2+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: baehler@nwz.uni-muenster.de
*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 Ca2+⁄ 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
Ca2+ IQ2 had a lower affinity for CaM in the absence of Ca2+ than in the presence of Ca2+ The actin-activated ATPase activity of Myo1d was
75% inhibited by Ca2+-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 Ca2+-dependence, we propose that Ca2+ -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 Ca2+-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.
Trang 2ATPase 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 Ca2+ to the C-terminal pair of Ca2+-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 Ca2+
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 Ca2+ whereas IQ-motif 2 (IQ2) bound
CaM in a Ca2+-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
by90 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 Ca2+⁄ 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 Ca2+-binding
to CaM associated with the IQ-motifs were
investi-gated We now report that Ca2+-binding to the
C-ter-minal pair of Ca2+-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 Ca2+-independent
inhibition of the actin-activated ATPase activity
Results
Binding of Ca2+⁄ 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
Ca2+-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 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 3 A˚ closer towards the head domain Following expression of these Myo1d con-structs and affinity purification in the absence of free
Ca2+, 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 Ca2+ 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 Ca2+ 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 relaposi-tion 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.
Trang 3albeit 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 Ca2+ (data not shown), indicating that
IQ2 binds CaM more tightly in the presence of Ca2+
To test if the IQ2 in Myo1d-IQ2 and Myo1dDLV-IQ2 could be saturated with CaM in the absence or presence of free Ca2+, 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 Ca2+ 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 Ca2+ 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 Ca2+⁄ CaM
In the absence of free Ca2+, the basal ATPase activity
of the Myo1d-head without an IQ-motif was 0.01 s)1 The Vmaxof the actin-activated ATPase was 2.6 s)1and the Kactin38 lm [22] with an apparent second-order rate
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 calcal-modulin (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 l M CaM either in the presence of 100 l M free Ca2+ (Ca2+, 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 Ca2+, respectively.
Trang 4constant Kapp of 0.68· 105s)1Æm)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 Kappof 0.12· 105s)1Æm)1that 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 Ca2+⁄ CaM, we determined the actin-activated ATPase of Myo1d constructs in the presence of 22 lm-free Ca2+ Interestingly, the Vmaxof 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 l M CaM either in
the absence of free Ca2+(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 m M
Hepes pH 7.4, 2 m M MgCl2, 3 m M EGTA, 1 m M 2-mercaptoethanol,
2 m M NaN3and 2 m M ATP CaCl2(3 m M ) was added where
indica-ted to obtain a free Ca2+ concentration of 22 l M Samples
con-tained 54–270 n M of the respective Myo1d constructs, 0–67 l M
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.
Trang 5roughly 20% whereas the Kactin was unaltered
(Fig 6A) This Ca2+-dependent inhibition was not
reversible when free Ca2+ was chelated with EGTA
(data not shown) In the Myo1d-IQ1 construct, free
Ca2+ inhibited the actin-activated ATPase activity
by 75% (Fig 6C) The calculated Kapp was
0.12· 105s)1Æm)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 Ca2+ caused a
compar-able inhibition of the ATPase with a Vmax of 0.7 s)1
(Figs 6C and 7) The actin affinity was not affected
significantly with a determined Kactin¼ 44 lm The
Kapp derived from the initial slope of the hyperbola
was 0.15· 105s)1Æm)1
In the construct Myo1dDLV-IQ2 that exhibited
already a reduced ATPase activity in the absence of
free Ca2+, the addition of free Ca2+reduced its
ATP-ase activity further by about 40% and a Kapp of
0.07· 105s)1Æm)1was determined
Inhibition of the Myo1d actin-activated ATPase
activity as a function of the concentration
of free Ca2+
Next, we determined the ATPase activities for the
dif-ferent Myo1d constructs as a function of the free
Ca2+concentration (0.001–158 lm) (Fig 7) The slight reduction of the actin-activated ATPase activity of the Myo1d-head exhibited an IC50of pCa 6 (0.3 lm free
Ca2+) 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
IC50 of pCa 7 (0.05–0.08 lm Ca2+) (Fig 7) This
IC50 value corresponds to the affinity of the pair of
Ca2+-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 Ca2+ did not show any significant changes in ATPase activ-ity with increasing free Ca2+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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+-independent manner to a similar extent as observed with Ca2+ for the other constructs containing either one or two IQ-motifs
Ca2+-independent and Ca2+-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 Ca2+-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 m M ATP, 30 m M KCl, 10 m M Hepes
pH 7.4, 2 m M MgCl 2 , 3 m M EGTA, 1 m M 2-mercaptoethanol and
2 m M NaN3 To adjust free Ca 2+ concentrations (0–158 l M ),
corres-ponding amounts of CaCl 2 were added.
Trang 6(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 3 A˚ 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 bindbind-ing 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 Ca2+⁄ CaM This finding allowed
us to investigate the effects of Ca2+-binding to
ApoCaM associated with the IQ2 Myo1d-IQ2 affinity
purified under Ca2+ conditions contained
stoichiomet-ric amounts of copurified CaM demonstrating that IQ2
actually has a higher affinity for Ca2+⁄ 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 Ca2+
-dependent CaM-binding motifs The two lobes of
Ca2+⁄ CaM may both bind in an open conformation to
IQ2, explaining the increased affinity
Mechanism of inhibition of the Myo1d ATPase activity by Ca2+⁄ CaM
As reported previously, in the absence of free Ca2+ 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
Vmaxvalues 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 Ca2+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 Ca2+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 Ca2+ The N-ter-minal lobe of CaM exhibits a 10-fold lower affinity for
Ca2+ [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 Ca2+ -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, Ca2+⁄ 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 Ca2+-independent inhibition of the Myo1d actin-activated ATPase activity This deletion is predic-ted to rotate the CaM associapredic-ted 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
Trang 7in ATPase activity might either be explained by
coinci-dence or by the assumption that it mimics the Ca2+
-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 Ca2+
-sensitiv-ity of the kinetics of Myo1b (MI130; myr 1) [29]
Myo1e (myr 3; Myosin IC) has only a single IQ-motif
and Ca2+-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 Ca2+-regulatory element
On the other hand, conversion of the CaM C-lobe
upon Ca2+-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 Ca2+ 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 Ca2+
concen-tration and was enhanced at 10-fold higher Ca2+
con-centrations simultaneously with the dissociation of one
CaM [19] The actin-activated ATPase activities of
Myo1a (brush border myosin I) and Myo1b (MI130;
myr 1) were actually increased in the presence of
Ca2+⁄ CaM [20,30] In Myo1e the basal ATPase
activ-ity was reduced with a Ca2+-sensitivity suggestive of a
contribution by both the C- and N-terminal lobes of
CaM [21] In conclusion, the regulatory functions of
Ca2+⁄ 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 Ca2+⁄ 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 37C and 5% (v⁄ v) CO2 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⁄ Pi, collected by scraping and permeabilized with lysis buffer [150 mm NaCl, 20 mm Hepes pH 7.4, 2 mm MgCl2, 1 mm 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 MgCl2, 1 mm EGTA, 1 mm
Trang 82-mercaptoethanol and 2 mm NaN3) 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 Ca2+ 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 37C as
described in detail previously [21] All assay mixtures
con-tained 30 mm KCl, 10 mm Hepes pH 7.4, 2 mm MgCl2,
2 mm ATP, 3 mm EGTA, 1 mm 2-mercaptoethanol, 2 mm
NaN3and various actin concentrations To measure Ca2+
-dependent activities, a constant actin concentration of
24 lm was used Free Ca2+ concentrations between 0.001
and 158 lm were adjusted by adding the appropriate
amounts of CaCl2 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
Ca2+in a concentration range between 0 and 75 lm actin
Purified Myo1d constructs were used in a range between 54
and 270 nm Vmaxand Kmvalues were determined by fitting
the measured ATPase rates (v) to the Michaelis–Menten
equation
v¼ Vmax½actin=ðKactinþ ½actinÞ 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 MgCl2,
3 mm EGTA, 1 mm 2-mercaptoethanol and 2 mm NaN3
for 15 min on ice Where indicated, the free Ca2+
concen-trations were adjusted accordingly Assay mixtures with
Myo1d-IQ2 purified under calcium conditions contained
50–100 lm free Ca2+ Free Ca2+ 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)
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