A surface loop (25/50-kDa loop) near the
nucleotide pocket of myosin has been proposed to be an important
element in determining the rate of ADP release from myosin, and as a
consequence, the rate of actin-myosin filament sliding (Spudich,
J. A. (1991) Nature 372, 515-518). To test this
hypothesis, loops derived from different myosin II isoforms that
display a range of actin filament sliding velocities were inserted into
a smooth muscle myosin backbone. Chimeric myosins were produced by
baculovirus/Sf9 cell expression. Although the nature of this
loop affected the rate of ADP release (up to 9-fold), in
vitro motility (2.7-fold), and the Vmax
of actin-activated ATPase activity (up to 2-fold), the properties of
each chimera did not correlate with the relative speed of the myosin
from which the loop was derived. Rather, the rate of ADP release was a
function of loop size/flexibility with the larger loops giving faster
rates of ADP release. The rate of actin filament translocation was
altered by the rate of ADP release, but was not solely determined by
it. Through a combination of solute quenching and transient
fluorescence measurements, it is concluded that, as the loop gets
smaller, access to the nucleotide pocket is more restricted, ATP
binding becomes less favored, and ADP binding becomes more favored. In
addition, the rate of ATP hydrolysis is slowed.
 |
INTRODUCTION |
Resolution of the atomic structure of the myosin II motor from
crystallographic x-ray diffraction data has created interest in several
flexible loops on its surface. These include the "HCM" loop (1, 2)
and two loops that were not resolved in the crystal structures and are
the sites of proteolytic cleavage: the loops at the 25/50-kDa junction
and 50/20-kDa junction. These junctional loops also have been referred
to as loop 1 (25/50-kDa loop) and loop 2 (50/20-kDa loop). The HCM loop
is so called because a number of mutations at its base are associated
with the human disease, hypertrophic cardiomyopathy (3). Furthermore,
some members of the myosin family are regulated by phosphorylation of
this loop (4, 5). Loops 1 and 2 have been proposed to be major
determinants of the kinetic properties of myosin (6). Both loops are
highly variable in sequence and length among members of the myosin II
family. If this variability underlies a large degree of the kinetic
diversity among myosins, then it would reveal an evolutionary strategy
for kinetic tuning that would involve regions of the molecule outside
of the core that might not have interactions with the backbone of the
myosin motor. This could allow a rapid evolutionary divergence of motor
properties without altering the core structure or the motor's basic
function.
The suggestion of a fundamental role for the junctional loops of myosin
was based on a study of loop 2, which is located at the actin interface
(7) and on the demonstration of functional differences for naturally
occurring isoforms involving alternations in loop 1 (25/50-kDa loop)
(8). The former study involved the creation of chimeric myosin II
molecules that were the sequence of Dictyostelium myosin II
in all regions other than that of loop 2. The loop 2 sequences were
derived from a number of other myosins. What was observed was an effect
on the steady state ATPase activity. It was shown, at an actin
concentration (20 µM) assumed to be high enough to reveal
the Vmax of the actin-activated ATPase, that
Vmax was correlated with that of the parent
myosin from which the loop 2 sequence was derived. However, a
subsequent study that performed similar experiments in the smooth
muscle myosin II backbone concluded that loop 2 altered the apparent
Km for actin (KATPase), but
not the Vmax of the steady state ATPase (9). Whether loop 2 affects only KATPase or
Vmax or both, it clearly does alter the steady
state ATPase activity of myosin. However, more extensive studies must
be performed to determine whether or not it is the primary determinant
of either the Vmax or Km.
The other junctional loop, loop 1 (25/50-kDa loop), is located near the
catalytic site. Its sequence varies in vertebrate smooth and non-muscle
myosin II isoforms as the result of alternative splicing, and these
splice variants have been shown to lead to alterations in the steady
state ATPase activity and rate of actin filament movement in an
in vitro motility assay (8, 10). This loop has been proposed
to be the primary determinant of the rate of ADP release from myosin,
and as a consequence of this, the determinant of the in
vitro actin filament sliding rate (6).
To examine the role of loop 1, we adopted the same general strategy
that was previously used in evaluating loop 2 (7, 9). We took a common
myosin II backbone (chicken smooth muscle myosin II), and inserted into
it a number of 25/50-kDa loops that were based on the sequences found
in other myosins. We expressed both S11 and HMM-like fragments of
myosin, and assessed their function, using steady state ATPase assays,
stopped flow measurements of the rate of ADP release from myosin bound
to actin, and the rate of actin filament sliding in an in
vitro motility assay. Our findings suggest that this loop can
alter all of these kinetic properties to a degree that suggests that it
has a role in determining isoform diversity. However, it does not
appear to be the major determinant of any of the kinetic properties of
myosin. Thus, the 25/50-kDa loop appears to be a modulator of kinetic
properties, which are primarily determined by other structural elements
within the myosin motor. Insights into the mechanism of this kinetic
modulation were obtained via a combination of solute quenching and
nucleotide binding kinetics. The measurements suggest a model in which
the relative mobility of the "switch" elements of the nucleotide
binding pocket are modulated by the nature of the residues occupying
the 25/50-kDa loop.
 |
EXPERIMENTAL PROCEDURES |
Construction of Chimeras--
The cDNA for chicken gizzard
smooth muscle myosin was truncated at either the codon corresponding to
amino acid 859 to create a S1-like fragment, or at amino acid 1112, to
create a HMM-like fragment. In the case of the S1, a Flag peptide
sequence (11), followed by a stop codon was appended, whereas for the
HMM, a Myc epitope sequence (12), followed by the Flag sequence and a
stop was appended. The Flag peptide allowed for affinity purification, whereas the Myc epitope was for motility studies. Both the S1- and
HMM-like constructs were subcloned into the baculovirus transfer vector, pVL 1393 (Invitrogen). Two silent mutations were introduced into the S1 sequence to create unique restriction sites flanking the
25/50-kDa loop. Alternative loops were then introduced by using these
sites to open the plasmid; a linker corresponding to the new loop
flanked by the silent restriction sites was then ligated in place,
creating the chimeric cDNA. The S1 chimera was then used to
generate the corresponding HMM. Generation of the recombinant
baculoviruses coding for the S1- and HMM-like constructs followed
published techniques (13).
Expression and Purification of Proteins--
Baculovirus
expression was used to produce S1- and HMM-like fragments of chicken
smooth muscle myosin II. This involved infection of an insect cell line
(Sf9) with recombinant baculovirus driving high level expression
of foreign protein under the polyhedron promoter. Sf9 cells were
co-infected with recombinant virus containing chicken smooth ELC
(LC17a) and RLC cDNAs and with recombinant virus coding
for a truncated myosin heavy chain. For a subset of experiments, virus
coding for the alternative ELC (LC17b) was substituted.
Three days after infection, Sf9 cells were lysed in a high ionic
strength buffer containing ATP, DTT, Nonidet P-40, and protease inhibitors. This was followed by low speed centrifugation, ammonium sulfate fractionation, and overnight dialysis at 4 °C in the
presence of F-actin (and hexokinase and glucose to remove ATP).
Dialysis was followed by centrifugation at 500,000 × g
for 20 min, which pelleted the F-actin and the F-actin-myosin
complexes. The pellet was washed, and then resuspended in a buffer
containing 1 mM MgATP to dissociate the actin and myosin.
The F-actin was then pelleted again by centrifugation, and the
supernatant was removed and passed through an anti-Flag epitope
antibody affinity column. (For experiments with HMM, the myosin was
incubated with 2 µM calmodulin and 0.15 µM
skeletal muscle myosin light chain kinase in the presence of 100 µM CaCl2 and 1 mM MgATP for 30 min prior to loading on the affinity column. This achieved high levels
of regulatory light chain (RLC) phosphorylation (>90%).) In this
manner, only myosin that bound and released from actin and that
contained a C-terminal Flag epitope bound to the column. The myosin was
eluted via Flag peptide competition. Microdialysis and concentration
into the appropriate buffer was the final purification step.
Actin was purified from rabbit back and leg skeletal muscles, following
the procedure of Spudich and Watt (14). All protein concentrations were
determined using the Bradford (15) method of assay. Purity of all
myosin and actin preparations was confirmed on 10% discontinuous
SDS-polyacrylamide gels (16). Levels of HMM phosphorylation for each
preparation was determined using glycerol/urea gels (17).
Actin Dissociation Rate and Apparent ADP "Off" Rate--
A
mixture of actin and myosin S1 (or HMM) were mixed with ADP (at a
concentration of 50, 100, or 200 µM) and 10 mM imidazole (pH 7.0), 1 mM DTT, 1 mM EGTA, and 1 mM MgCl2 at
25 °C. For experiments with S1, the actin concentration prior to
mixing was 5 µM and the myosin S1 concentration was 2 µM. For experiments with HMM, the actin concentration was
increased to 10 µM and the HMM concentration was 1 µM. The mixture was rapidly mixed with a 2 mM
MgATP using stopped flow techniques. The rate of actin dissociation was
assumed to be rate-limited by the off rate of ADP (i.e.
dissociation from actin cannot occur until the ADP dissociates from
myosin and ATP binds). Thus, the rate of actin dissociation measured,
as monitored by an exponential drop in turbidity, represents the
apparent ADP off rate. It should be noted that, for these and
subsequent measurements involving ADP or ADP analogs, addition of ADP
to myosin or to actomyosin does not reach the same ADP state that is
achieved immediately following hydrolysis of ATP and release of
inorganic phosphate (18). However, the ADP state achieved is likely the state from which ADP ultimately dissociates. The procedures to obtain
and analyze these measurements follow the published procedures of White
(19).
For these experiments, both S1 and HMM fragments were used, to assess
the possibility of cooperative head interactions in the HMM. However,
both the S1 (phosphorylated or dephosphorylated) and the phosphorylated
HMM gave turbidity drops that were best fit by single exponentials.
Kinetic Methodologies for mant Nucleotides and ATP
Binding--
Transient kinetic measurements of mant nucleotide and ATP
binding kinetics were made on a Hi-Tech Scientific PQ/SF-53 stopped flow spectrometer, as described (20).
In Vitro Motility Assay--
The assay was performed essentially
as described by Sellers et al. (21), with the following
modifications in the manner of attachment of HMM-like fragments to the
surface and in the final buffer conditions. Monoclonal anti-Myc
antibodies at a concentration of 0.2 mg/ml were introduced into the
flow cell containing a nitrocellulose-coated coverslip and allowed to
incubate for 5 min. The unbound antibodies were washed out with buffer
A (80 mM KCl, 20 mM MOPS, pH 7.4, 5 mM MgCl2, 0.1 mM EGTA) containing 1 mg/ml bovine serum albumin. HMM-like fragments were then introduced at
a concentration of 0.2 mg/ml and allowed to incubate for 10-15 min.
Unbound HMM-like fragments were washed out with buffer A, and the
antibody-bound myosin was phosphorylated by the addition of buffer A
containing 5 mg/ml myosin light chain kinase, 10
7
M calmodulin, 0.2 mM CaCl2, and 1 mM ATP. This buffer also contained 10 mM actin
(not rhodamine-phalloidin labeled) to irreversibly bind any non-cycling
myosin heads. After 5 min, the coverslip was washed with buffer A,
followed by buffer A with 20 nM rhodamine-phalloidin. The
assay was initiated by adding buffer A containing 200 nM
tropomyosin, 1 mM ATP, 2.5 mg/ml glucose, 0.1 mg/ml glucose
oxidase, 0.02 mg/ml catalase, and 50 mM DTT. The
temperature of the assay was 30 °C. The equipment used to observe
the myosin-mediated translocation of actin filaments and the method
used for quantification of the motion have been described previously
(22).
ATPase Assays of Phosphorylated HMM-like Fragments--
The
ATPase assays involved the measurement of the rate of radioactive
32P generation from
-phosphate-labeled ATP, by the
method of Pollard and Korn (23). The conditions of the assay were 2 mM MgCl2, 10 mM MOPS (pH 7.0), 0.1 mM EGTA, 1 mM ATP at 25 °C.
Vmax and KATPase were
read from Lineweaver-Burk plots. The actin concentration used was over
the range of 0-150 µM.
Solute Quenching--
Solute accessibility of mant-ADP bound to
S1 constructs was measured using acrylamide, as described previously
(24).
Fluorescence Methodologies--
Fluorescence lifetimes and
anisotropy decays of mant nucleotide-labeled myosin constructs were
performed as described previously (24). Anisotropy decay of mant-ADP
was measured at 20.0 °C on a PRA 3000 single photon-counting system
equipped with a DCM dye laser, the output of which was
frequency-doubled to 325 nm as the excitation source for mant-ADP. The
general procedures used to measure anisotropy decay and analyze the
decay data have been described (25). The decay data from mant
nucleotide were analyzed by a bi-exponential model.
 |
RESULTS |
Based on the structure of chicken skeletal muscle myosin S1
fragment (1) and on the structure of the Dictyostelium
myosin motor domain (26), the boundaries of the 25/50-kDa loop were defined as illustrated by the underlined sequences in Fig.
1. The chimeric loops, shown below the
two chicken smooth muscle wild type loops, are grouped according to
relative myosin speed. The chicken fast skeletal, rat cardiac, and
Dictyostelium myosins are all faster than smooth muscle
myosin, whereas the three non-muscle myosins are all slower. All myosin
S1 and HMM-like constructs containing either wild type or chimeric
loops (Fig. 1) yielded 1-2 mg of active myosin from 1 liter of
Sf9 cells (approximately 3 × 106 cells). For
all S1 and HMM-like constructs, the purified myosin fragments displayed
a heavy chain:ELC:RLC stoichiometry of 1:1:1, as measured by
densitometry (correcting for molecular weight differences; data not
shown). All values given for HMM-like constructs are in the presence of
phosphorylation of the RLC. None of the HMM-like constructs displayed
movement or actin-activated ATPase activity in the absence of RLC
phosphorylation.

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Fig. 1.
25/50-kDa loop substitutions. The
underlined amino acids represent either the wild type or
heterologous loops from the indicated myosin II heavy chains inserted
into the chicken smooth muscle myosin II backbone. The first two
sequences are those of the two chicken smooth muscle wild type loops.
Those are followed by four loops from myosins that are faster than
either of the smooth muscle wild types. These are followed by three
loops from non-muscle myosins that are considerably slower than smooth muscle myosin. The last loop ( 25/50 is a large deletion of the smooth muscle loop.
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Table I presents the values for the rate
of ADP release from either smooth muscle myosin S1 or phosphorylated
HMM bound to actin. The values were not different for S1 compared with
the phosphorylated HMM. Fig. 2 depicts
the rate of ADP release from smooth muscle myosin HMM-like constructs
(phosphorylated) and the corresponding rate of in vitro
translocation of actin filaments by each of the constructs. The fastest
values for both parameters were obtained with the gizzard loop. Loops
from the faster myosins gave a range of ADP release values that were
similar to or slower than that obtained for the chicken aorta smooth
muscle myosin loop. The in vitro motility values for these
loops were intermediate to the gizzard and aorta values. Interestingly,
the loops from the slower non-muscle myosins gave ADP release and
in vitro motility values that were as fast or faster than
the those obtained with the aorta loop. To facilitate comparison of the
in vitro motility measurements of the chimeric myosins (Fig.
2), literature values for the parent myosins are given in Table
II. The comparison reveals that there is
no correlation between the speed of the chimeric myosins and the speed
of the myosin from which the 25/50-kDa loops were derived ("loop
donors").

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Fig. 2.
ADP release rates and in vitro
motility for smooth muscle myosin with 25/50-kDa loop
substitutions. ADP off rates from actin-myosin-ADP using expressed
HMM (phosphorylated) fragments of chicken smooth muscle myosin
incorporating the 25/50-kDa loop substitutions shown in Fig. 1 are
depicted as solid bars with standard deviations indicated.
The gizzard loop was significantly faster than all others
(p < 0.01; Student's t test). The
Xenopus non-muscle loop (Xen NM) was faster than
all but the gizzard loop (p < 0.01). The
Dictyostelium myosin II loop (Dicty) and the
deletion loop were slower than all others (p < 0.01).
The chicken fast skeletal loop (Fast skel), the rat
-cardiac loop (rat -card), the rat -cardiac loop
( -card), and the chicken non-muscle myosin II loop
(NMA) were not statistically different from each other. The
chicken non-muscle myosin II loop gave a value intermediate to the
group that included the fast skeletal loop and the two slowest
(Dictyostelium and deletion) loops (p < 0.01). In vitro motility values for the same loop
substitutions in phosphorylated chicken smooth muscle myosin HMM are
depicted as stippled bars. The gizzard and
Xenopus non-muscle loops gave rise to values that were
significantly faster than all others (p < 0.01),
whereas the aorta loop, the Dictyostelium loop, the NMB
loop, and the deletion loop gave the slowest values, that were not
different from each other, but were significantly lower than those for
all other loops (p < 0.01). There were not
statistically significant in vitro motility differences
among the other chimeric HMM constructs.
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The only obvious correlation between the loop sequences and any of the
measured kinetic parameters was a correlation between the length of the
25/50-kDa loop and the rate of ADP release. This relationship is
depicted in Fig. 3A. Note that
the only loop from Fig. 1 that does not fit this size rule is the
gizzard wild type loop.

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Fig. 3.
A, ADP release rates versus
the size of the 25/50-kDa loop. The rates of ADP release from
actin-myosin-ADP using the chimeric HMM constructs depicted in Fig. 1
are plotted as solid squares, with the exception of the HMM
containing the chicken gizzard loop, which is represented by an
open square. Values for the chimeric HMM constructs
containing the alterations in the seven-amino acid insert in the
chicken gizzard 25/50-kDa loop shown in B are plotted as a
closed circle (rabbit loop) or an open circle
(alanine substitutions). B, alterations in the seven-amino
acid insert in the chicken gizzard 25/50-kDa loop are shown in
italics. The rabbit loop incorporates the sequence from the
inserted form of rabbit smooth muscle myosin, which differs from the
chicken inserted form by two amino acids. The alanine replacement loop
involves substitution of four of the seven insert amino acids with
alanines.
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This was further examined by constructing two additional smooth muscle
myosin chimeras for which the 25/50-kDa loops are shown in Fig.
3B. The first substituted the seven amino acid alternative smooth muscle insertion from rabbit in place of that of chicken. The
second replaced five of the seven gizzard insert residues with
alanines. As shown in Fig. 3A, the alanine replacement
decreased the rate of ADP release to a value that better fits the size
rule whereas the rabbit loop gave a value lower than the chicken
gizzard value, but higher than the loop containing the five alanine
substitutions. The corresponding motility and ATPase data for HMM-like
fragments containing the same altered inserts of Fig. 3 are given in
Table IV.
Table III presents the results of
actin-activated ATPase assays with smooth muscle myosin HMM-like
constructs containing the loops depicted in Fig. 1. All of the chimeric
loop substitutions gave essentially the same
Vmax value, which was intermediate to the values
of the gizzard and aorta wild types. Although the Km values showed considerable variability, the chicken fast skeletal loop
and the deletion (
25/50) loop yielded significantly lower Km values than either wild type or for any of the
other chimeras.
To address whether the correspondence between size of the loop and the
rate of ADP release was related to loop flexibility and size, two
additional smooth muscle myosin HMM chimeras were constructed. Into the
gizzard 25/50-kDa loop, three proline substitutions were made, and into
the deletion (
25/50)loop, one proline substitution was made. These
are shown in Fig. 4. The effect of the
proline insertion was, in both cases, to reduce the speed of ADP
dissociation from the actin-myosin HMM·ADP complex. For the gizzard
loop, the reduction was from 79 ± 5/s to 37 ± 5/s. For the
deletion loop, the reduction was from 12 ± 2/s to 8 ± 2/s.

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Fig. 4.
Proline insertions into the 25/50-kDa
loop. Amino acid sequences illustrating proline (bold)
insertions into either the chicken gizzard smooth muscle 25/50-kDa
loop, or into the 25/50 loop.
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It was noted that all of the loops from myosin II isoforms had
positively charged residues near the N-terminal end of the loop, and
all of the loops had a net positive charge. To examine the possible
contribution of this charge, the three lysines of the gizzard loop were
replaced with either aspartate or alanine residues, as depicted in Fig.
5. Either removal or reversal of this
charge resulted in a decrease in both the Vmax
and the Km of the actin-activated ATPase activity
(Table IV). Charge removal had no effect
on the rate of ADP release, whereas charge reversal slowed the release
(Table IV). The in vitro motility decreased in both cases,
but to a much greater extent in the case of charge reversal (Table
IV).

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Fig. 5.
Charge alterations in the 25/50-kDa
loop. The bold amino acids represent either charge
removal (lysine to alanine substitutions) or charge reversal (lysine to
aspartate substitutions) within the chicken gizzard smooth muscle
myosin 25/50-kDa loop.
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Two ELC isoforms are associated with smooth muscle myosin, which
previous studies have examined for functional significance (8, 10). We
extended these previous studies by substituting LC17b for
LC17a in preparations of HMM with the gizzard, aorta, and
deletion loops. The values for the rate of ADP release, in vitro motility, and actin-activated ATPase activity are given in
Table V.
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Table V
Kinetic parameters for phosphorylated HMM-like constructs with altered
25/50-kDa loops as a function of the ELC isoform
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To provide further insights into the mechanism underlying kinetic
modulation via the 25/50-kDa loop, the kinetics of binding of ATP and
2'-deoxy-mant-ADP to S1(gizzard loop), S1(aorta loop), and S1(loop
deletion;
25/50) were measured in the stopped flow. Binding of ATP
was monitored at 10 °C by the increase in tryptophan fluorescence
emission (32). A plot of apparent rate versus [ATP] yielded data that could be fit to a rectangular hyperbola (Fig. 6) for the constructs, defining maximum
rates (
max) and apparent dissociation constants
(Kd) (Table VI). The
maximum rates for the S1(gizzard) construct was 32% larger than that
for the S1
25/50 construct, and 95% larger than the S1(aorta)
construct. (This maximum rate of tryptophan fluorescence enhancement is
thought to represent the ATP hydrolysis rate (32).) Furthermore, the apparent dissociation constant was approximately 5 times larger for
S1
25/50, whereas both the gizzard and aorta constructs had similar
values for their dissociation constants. This difference was also seen
for HMM(gizzard loop) and HMM(
25/50) at 20 °C using the
fluorescent ATP analogue, mant-ATP (Fig.
7). Binding of mant-ATP produced a
fluorescent enhancement that could be described by a single exponential
term. Although the values of
max were very similar, the
value of Kd for HMM(
25/50) was over 2-fold larger.

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Fig. 6.
The kinetics of enhancement of tryptophan
fluorescence produced by binding of ATP to gizzard S1 (open
squares), 25/50 deletion mutant on a gizzard backbone
(closed circles), and 25/50 aorta loop on a gizzard
backbone (open triangles), 10 °C. A solution of S1
in 25 mM Hepes, 20 mM KCl, 1 mM
MgCl2, 1 mM DTT, pH 7.5, was mixed with an
equal volume of an at least 20-fold molar excess of ATP in the same
solution. The rate of the resulting tryptophan fluorescence enhancement
was fitted to a single exponential process, and the apparent rate
constant was plotted versus final {ATP]. Data was fitted
to a rectangular hyperbola, defining apparent binding constants and
maximum rates summarized in Table VI.
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Table VI
Kinetic parameters for binding of mant ATP and ATP to HMM and
S1-like constructs with altered 25/50-kDa loops
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Fig. 7.
The kinetics of enhancement of mant-ATP
fluorescence emission produced by mant-ATP binding to the catalytic
site gizzard HMM (open squares) and the 25/50 deletion
mutant on a gizzard HMM backbone (open circles),
20 °C. A solution of HMM or deletion mutant in 25 mM Hepes, 20 mM KCl, 1 mM
MgCl2, 1 mM DTT, pH 7.5, was mixed with an
equal volume of an at least 20-fold molar excess of mantATP in the same
solution. The mant fluorophor was excited at 295 nm through energy
transfer from a myosin tryptophan residue. The rate of the resulting
fluorescence enhancement was fitted to a single exponential process,
and the apparent rate constant was plotted versus final
[mantATP]. Data was fitted to a rectangular hyperbola, defining
apparent binding constants and maximum rates summarized in Table
VI.
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By contrast, the situation appears reversed for 2'-deoxy-mant-ADP.
Kinetics of binding of this ligand were monitored by energy transfer
from protein tryptophan to the mant fluorophor. A maximum rate was not
observed over the concentration examined (Fig.
8). The slopes of rate versus
nucleotide concentration define apparent second order rate constants
(ka), back extrapolation of the rate defines
apparent dissociation rates (k
), and the ratio
of ka/k
defines apparent
dissociation constants (Kd) (Table
VII). This reveals that compared with
S1(gizzard), the S1
25/50 construct binds 2'-deoxy-mant-ADP with a
5-fold higher ka, with a 13-fold lower
k
, and with a 70-fold lower
Kd.

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Fig. 8.
The kinetics of enhancement of
2'-deoxy-mant-ADP fluorescence emission produced by mant-ADP binding to
the catalytic site gizzard S1 (open squares) and the
25/50 deletion mutant on a gizzard S1 backbone (open
circles), 20 °C. Conditions are as detailed in Fig. 7.
The rate of 2'-deoxy-mant-ADP fluorescence enhancement was found to
have a linear dependence on nucleotide concentration. The slope of
these curves defines apparent second order rate constants,
ka, and extrapolation to zero ligand concentration
defines apparent dissociation rate constants,
k , which are summarized in Table VII.
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Table VII
Kinetic parameters for binding of 2'-deoxy-mant-ADP to native S1 and to
the 25/50 deletion S1 construct
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These results suggest that deletion of the 25/50 loop shifts the
equilibrium of S1 conformations toward one that favors ADP binding, and
should consequently favor a "strong binding" state. To explicitly
test this, S1(gizzard)·2'-deoxy-mant-ADP and
S1D25/50·2'-deoxy-mant-ADP were mixed in the stopped flow with at
least a 15-fold molar excess of actin + 1 mM ATP. A plot of
rate versus [actin] (Fig. 9)
could be fit to a rectangular hyperbola for S1
25/50, defining values of
max for nucleotide release and Kd
for binding to actin. A maximum rate could not be observed for
S1(gizzard), and a plot of rate versus [actin] was linear.
A comparison of the initial slope of the hyperbolic plot for S1
25/50
with that for S1(gizzard), however, reveals that the apparent second
order rate constants are very similar to each other (Fig.
9). In this experiment, the rates of
release of 2'-deoxy-mant-ADP from S1(gizzard) and S1
25/50 were
measured directly by mixing S1(gizzard)·2'-deoxy-mant-ADP and
S1
25/50·2'-deoxy-mant-ADP with 1 mM ATP. The release
transients fit single exponential decays, with rate constants of 7.8 s
1 and 2.1 s
1 for S1(gizzard) and
S1
25/50, respectively, which are in excellent agreement with values
calculated from Fig. 8.

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Fig. 9.
Dependence of the rate of 2'-deoxy-mant-ADP
release from gizzard S1 (open squares) and the 25/50
deletion mutant on a gizzard S1 backbone (open circles) on
actin concentration. A solution of 0.5-5 µM S1 + 25 µM 2'-deoxy-mant-ADP was mixed in the stopped flow with a
10-fold molar excess of actin + 1 mM ATP. The mant
fluorescence emission was monitored by energy transfer from an S1
tryptophan residue, and the resulting fluorescence intensity decrease
was fitted to a single exponential process. Data were fitted to a
rectangular hyperbola, defining apparent binding constants, and maximum
rates summarized in Table VIII. Conditions are as follows: 25 mM Hepes, 20 mM KCl, 1 mM
MgCl2, 1 mM DTT, pH 7.5, 20 °C.
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Table VIII
Kinetic parameters for actin-activated dissociation of
2'-deoxy-mant-ADP from S1(gizzard wild type) and the S1(loop deletion) constructs
|
|
Additional information about the effect of the loop on the geometry of
the catalytic site can be obtained by examining the spectroscopic
properties of S1 constructs labeled in the catalytic site with
fluorescent nucleotide, such as mant-ADP. In aqueous solution, the
fluorescence decay of free mant-ADP can be described by a single
lifetime of 4.0 ns (33). By contrast, the fluorescence decay of
complexes of mant-ADP with either S1(gizzard) or S1
25/50 could be
described by two lifetimes. For S1(gizzard), the lifetimes had values
of 10.2 ± 0.2 ns and 1.8 ± 0.1 ns and with relative contents of 0.56 and 0.44, respectively. For S1
25/50, the
corresponding values were 7.7 ± 0.2 ns and 2.1 ± 0.1 ns and
with relative contents of 0.70 and 0.30, respectively. Addition of
beryllium sulfate to 0.2 mM and sodium fluoride to 5 mM produces a myosin·ADP·BeF complex that mimics the
pre-hydrolytic state (26). Lifetimes of mant-ADP bound to S1(gizzard)
or S1
25/50 in the presence of beryllium fluoride were very similar
to each other: 9.0 ± 0.1 ns and 3.8 ± 0.1 ns for
S1(gizzard) with relative contents of 0.75 and 0.25, and 9.0 ± 0.1 ns and 3.5 ± 0.1 ns for S1
25/50 with relative contents of
0.78 and 0.22. mant-ADP consists of a mixture of the 2'- and 3'-isomers
(34). To avoid the confounding effects of a stereoisomeric mixture of
fluorophors, fluorescence lifetimes were measured for S1(gizzard) or
S1
25/50 complexed to 2'-deoxy-mant-ADP. This revealed that the
fluorescence decays of both S1(gizzard)·2'-deoxy-mant-ADP and
S1
25/50·2'-deoxy-mant-ADP could still be characterized by two
components. For S1(gizzard), the lifetimes were 8.73 ± 0.2 ns
(70%) and 1.8 ± 0.1 ns (30%), whereas for S1
25/50 they were
8.2 ± 0.5 ns (30%) and 3.8 ± 0.3 ns (70%).
As a fluorescence quencher, acrylamide shortens the fluorescence
lifetimes of bound mant-ADP as described by the Stern-Volmer equation
(35), where
o is the singlet lifetime in the absence of
quencher,
is the lifetime at quencher concentration [Q], and
kq is the bimolecular Stern-Volmer quenching rate constant. The quenching rate constant kq can vary
from the rate of diffusion (totally accessible to solute) to zero
(inaccessible). Thus, the effect of acrylamide on fluorescence lifetime
can be used to measure the effect of the 25/50 loop deletion on the
accessibility of bound nucleotide to solvent. Quenching studies were
performed on S1(gizzard)·mant-ADP, S1
25/50·mantADP,
S1·mant-ADP·BeF, and S1
25/50·mant-ADP·BeF. Results are
summarized in Table IX. As has been
reported previously for smooth muscle myosin (24), beryllium fluoride
reduces the solvent accessibility of the mant fluorophor when bound to
S1(gizzard) by approximately a factor of 2, whereas it has much less of
an effect on S1
25/50·mantADP. Furthermore, the values of
kq for S1(gizzard)·mant-ADP·BeF are very similar
to those for S1
25/50·mantADP (Table IX).
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Table IX
Fluorescence lifetime and quenching parameters for complexes of
mant-ADP with S1(gizzard wild type) and the S1(loop deletion) constructs
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Rotational correlation times were calculated from the anisotropy decays
of S1(gizzard)·mant-ADP·BeF and S1
25/50·mant-ADP·BeF. Both
samples had similar limiting anisotropies (0.21 for
S1
25/50·mant-ADP·BeF and 0.23 for S1·mant-ADP·BeF). The
anisotropy decay for both samples was characterized by a single
rotational correlation time that was 10% smaller for S1
25/50
construct (144.9 ± 4.0 ns for S1 versus 130.1 ± 3.6 ns for S1
25/50).
 |
DISCUSSION |
The data shown in Fig. 2 and Tables I and III demonstrate that the
sequence of the 25/50-kDa loop alters the rate of ADP release from
myosin bound to actin over a 9-fold range; the rate of in vitro motility, as measured by actin sliding velocity, over a 2.7-fold range; and the steady state ATPase activity over a 2-fold range. The magnitude of the alterations are less than an order of
magnitude, yet the in vitro motility rates for the myosins from which the chimeric loops were derived vary by 90-fold (Table II).
This suggests that this loop plays a modulatory role in the generation
of isoform diversity, and is not the primary determinant of any of
these kinetic properties. Furthermore, none of the loops produced any
kinetic changes that were faster than those associated with the gizzard
loop, suggesting that other elements of the myosin molecule ultimately
limit all of the kinetic parameters as compared with those of faster
myosins. Additionally, the data summarized in Table V demonstrate that
none of the kinetic properties measured were altered by the ELC isoform
(LC17a versus LC17b). This confirms and extends published reports (8, 10).
It is noteworthy than none of the loop substitutions resulted in a loss
of RLC phosphorylation-mediated regulation of the HMM-like constructs.
This is despite the fact that several of the loop substitutions
resulted in a marked decrease in the KATPase. In
the study of Rovner et al. (9), chimeric substitutions in the 50/20-kDa loop caused a decrease in the
KATPase and a loss of regulation. Based on the
present study, one must conclude that decreasing
KATPase does not in itself cause a loss of
regulation, and that the loss of regulation seen in that study must be
specific to alterations of the 50/20-kDa loop.
If one focuses on the rate of ADP release and in vitro
motility, it is evident that loops derived from myosin molecules that are faster than smooth muscle myosin all produced values that were
intermediate to the smooth gizzard and aorta wild type myosins. Although there is no correlation between the speed of the chimeric molecule and the speed of the parent molecule from which the loop was
derived, there is a striking correlation between the rate of ADP
release and the length of the 25/50-kDa loop (Fig. 3).
Although the shortest loops were associated with the slowest rates of
actin filament translocation, velocity was not strictly proportional to
the rate of ADP release. For example, the deletion loop and
Dictyostelium loop gave rise to in vitro motility
values that were similar to the longer aorta loop, even though the
corresponding values of ADP release rate were much less for the
deletion and Dictyostelium loops. Both the deletion loop and
Dictyostelium loop give rise to steady state ATPase
Vmax values that are greater than that for the
longer aorta loop. One possible interpretation is that, whereas the
rate of ADP release can limit the shortening Vmax of a muscle (and presumably in
vitro motility), as has been suggested previously (36), it is not
the sole determinant. Earlier steps in the cross-bridge cycle may also
contribute to the rate of unloaded actin-based movement.
The one loop sequence that is much faster in terms of ADP release rate
than would be predicted from its length is the chicken gizzard loop.
The chimeric substitutions shown in Fig. 3 reveal that this deviation
is due to specific amino acids within the seven-amino acid insertion
that distinguishes this loop from that of the aorta smooth muscle
myosin II loop sequence. When the seven-amino acid insert was altered
to that found in rabbit smooth muscle, both the rate of ADP release and
in vitro motility declined. When multiple alanine
substitutions were made (Fig. 3), the loop obeyed the same size rule as
all other loops. This may indicate that the kinetic properties are not
a function of loop size, but rather of loop flexibility, and that the
seven-amino acid insert in the gizzard sequence confers a greater
flexibility to the loop than do the substitutions of Fig. 3. If that is
the case, then insertions that decrease the flexibility of the loop
should slow the ADP release rate. To test this, the substitution of
prolines was performed with two different loops within HMM constructs.
In both cases, slowing of ADP release accompanied the proline
insertions. This result supports the concept that it is loop
flexibility that is the important property governing the release rate
of ADP from myosin bound to actin. The data in Table IV demonstrate
that charge reversal, but not charge removal, also slowed the rate of
ADP release. It is interesting to note that two possible 25/50-kDa loops can be inserted into the Placcopecten myosin II heavy
chain, creating either the striated muscle or the slower, catch muscle myosin (37). The catch loop is one amino acid shorter and substitutes two proline residues for two charged residues.
How can one envision a mechanism whereby a loop that is near the
nucleotide binding pocket controls the rate of ADP release? One
possibility is that the longer loops extend to the nucleotide pocket,
where they interact directly with ADP in a manner that accelerates ADP
release. However, this is not supported by data that examined access of
the nucleotide pocket to solvent for the gizzard loop versus
the loop deletion (Table IX). As the data in Table IX indicate,
deletion of the 25/50 loop reduces solute accessibility of the mant
fluorophor by a factor of 2-3, a reduction in accessibility similar to
that produced by addition of beryllium fluoride. This suggests that
deletion of the loop restricts the "opening" of the nucleotide
pocket when ADP occupies the active site, increasing nucleotide
affinity, and reducing the rate of ADP release, both in the absence and
presence of actin (Fig. 9).
The dissociation constant for 2'-deoxy-mant-ADP for S1 calculated from
the kinetic data, at 188 µM, is over 4-fold larger than
that directly measured for acto-S1 (38). This implies that following
initial binding of ligand, the S1-nucleotide complex undergoes a
conformational change that is not detected by energy transfer-induced
fluorescence from the mant nucleotide. This is consistent with the
lifetime data, which does not detect substantial amounts of free
nucleotide at total S1 and ligand concentrations of 50 µM, but which indicates that S1·2'-deoxy-mant-ADP is a
mixture of two states with an equilibrium constant of approximately 4. Deletion of the 25/50 loop reduces this equilibrium constant to approximately 0.4. However, ADP affinity is still enhanced, due both to
acceleration of the forward rate constant and reduction in the reverse
rate constant for the ligand binding step. Previous work by Marston and
Taylor (39) measured a rate of ADP release of 0.9 s
1 for
proteolytically prepared gizzard S1, which is approximately 9 times
slower than what was measured in this study. This is consistent with
our recent finding that nucleotide release from recombinant S1 is 8-10
times faster than that for proteolytic S1, perhaps because heavy chain
of the former is uncleaved.2
By contrast, ATP binding and hydrolysis, as measured by the enhancement of tryptophan fluorescence (32, 40), is slowed for the deletion mutant
and the ATP affinity is reduced by a factor of 5.
Results of transient fluorescence measurements suggest a basis for
these effects on nucleotide binding. For
S1(gizzard)·2'-deoxy-mant-ADP, the major component in the
fluorescence decay was that with the longer lifetime (8.7 ns), whereas
the situation was reversed for the deletion mutant. The conformation of
S1-nucleotide is an equilibrium mixture of "strong" and "weak"
states (41). Thus, the fluorescence decay results suggest that deletion
of the 25/50 loop shifts the equilibrium to a strong state.
Deletion of the 25/50 loop had a marked effect on the kinetics of
actin-activated mant-ADP release (Fig. 9). Data for both S1(gizzard)
S1
25/50 are consistent with the following model (Scheme 1), where
the asterisk refers to states of enhanced fluorescence.
In this scheme, K1 is a rapid equilibrium
relative to the second step, the apparent second order rate constant,
ka = K1k2, and the maximum
rate for the fluorescence transient
max =
k2, because under the conditions of the experiment,
dissociation of mant nucleotide is irreversible. A plot of rate
versus [actin] for S1(gizzard) is linear; hence,
max(gizzard)
max(
25/50). However, ka(gizzard)
ka(
25/50), which implies that
K1(gizzard)
K1(
25/50). Thus, deletion of the 25/50 loop has two effects; it enhances actin affinity for S1·ADP, as would be
expected if this construct is largely in a strong binding state, and it
markedly slows the rate of actin-accelerated nucleotide release. Taken
together, these results suggest that deletion of the loop locks the
nucleotide pocket into a state that strongly binds ADP, and restricts
solute entry. However, this conformation of the pocket retards ATP
entry into the active site and slows hydrolysis.
A structural basis for this type of mechanism is suggested by a close
examination of the location of this loop. The 25/50-kDa loop connects
two helices that are attached to key elements of the nucleotide binding
pocket. At the N-terminal end of the first helix is the P-loop, and at
the C-terminal end of the second helix begins a sequence that
ultimately leads to the switch I component of the active site (42).
Although there is currently not a structure available for myosin in the
absence of both nucleotide and metal at the active site, there are such
structures for a number of G proteins (43). What these structures
suggest is that the release of MgADP may come about by breaking the
coordination of the P-loop with the magnesium ion. This could be
accomplished in myosin by a movement of the helix that contains amino
acid T186 and K185 (Dictyostelium myosin II amino acid
numbers). One can envision that as the flexibility of the 25/50-kDa
loop increases, the probability that the helix will move via thermal
motion sufficiently to release the MgADP is increased. Interestingly,
the crystal structure of kinesin reveals that its P-loop helix is
interrupted by a loop (44). Perhaps that loop in kinesin plays a
functional role that is analogous to the 25/50-kDa loop of myosin.
In 1967, Barany (45) suggested that the same kinetic step that
determines the Vmax of the solution ATPase
activity of myosin also determines the maximal velocity of shortening.
This was based on correlations between the two parameters in myosins
from a large number of different muscles. The data from this study
demonstrate that the rate of in vitro motility (the in
vitro analog of the shortening Vmax of a
muscle) and the ATPase Vmax can be dissociated from each other. However, if one examines the data from the three smooth muscle wild type loops (chicken gizzard, chicken aorta, and
rabbit; Table IV), the proportionality noted by Barany is maintained.
Thus, although there is no fundamental mechanistic link between the two
parameters, Barany's observed correlation may reflect evolutionary
pressure to maintain proportionality between these parameters.
This may reflect interaction distance and duty cycle constraints on the
myosin motor that must be maintained within a narrow window to achieve
optimal muscle performance.
In summary, the nature of the loop at the 25/50-kDa junction alters the
kinetic properties of myosin in a manner that allows for the generation
of isoform diversity. The greatest effect of this loop is on the rate
of ADP release, which seems to be determined by the flexibility of the
loop; greater flexibility leads to an enhancement in the rate of ADP
release. The loop can also alter the steady state ATPase activity (both
Vmax and the apparent Km) and
the rate of ATP hydrolysis. The speed of actin filament translocation is primarily determined by the rate of ADP release, with contributions from earlier steps. Thus, although this loop is not the primary determinant of shortening speed of myosin, as was hypothesized, it is
an important modulatory element of myosin kinetics. It is likely that
the kinetic properties of myosin will be the sum total of numerous such
modulatory regions that must be altered in concert to achieve the
functional range that the myosin motor manifests.