From the Department of Biochemistry, University of
Nevada, Reno, Nevada 89557 and the ¶ Department of Chemistry,
Washington State University, Pullman, Washington 99164
Received for publication, July 11, 2002, and in revised form, November 13, 2002
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ABSTRACT |
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The goal of this study was to provide structural
information about the regulatory domains of double-headed smooth muscle
heavy meromyosin, including the N terminus of the regulatory light
chain, in both the phosphorylated and unphosphorylated states. We
extended our previous photo-cross-linking studies (Wu, X., Clack,
B. A., Zhi, G., Stull, J. T., and Cremo, C. R. (1999)
J. Biol. Chem. 274, 20328-20335) to determine regions
of the regulatory light chain that are cross-linked by a cross-linker
attached to Cys108 on the partner regulatory light
chain. For this purpose, we have synthesized two new biotinylated
sulfhydryl reactive photo-cross-linking reagents, benzophenone,
4-(N-iodoacetamido)-4'-(N-biotinylamido) and benzophenone,
4-(N-maleimido)-4'-(N-biotinylamido).
Cross-linked peptides were purified by avidin affinity chromatography
and characterized by Edman sequencing and mass spectrometry. Labeled
Cys108 from one regulatory light chain cross-linked to
71GMMSEAPGPIN81, a loop in the
N-terminal half of the regulatory light chain, and to
4RAKAKTTKKRPQR16, a region for which there is
no atomic resolution data. Both cross-links were to the partner
regulatory light chain and occurred in unphosphorylated but not
phosphorylated heavy meromyosin. Using these data, data from our
previous study, and atomic coordinates from various myosin isoforms, we
have constructed a structural model of the regulatory domain in an
unphosphorylated double-headed molecule that predicts the general
location of the N terminus. The implications for the structural basis
of the phosphorylation-mediated regulatory mechanism are discussed.
The actin-activated ATPase activity and motor properties of smooth
muscle and nonmuscle myosins are regulated by phosphorylation of the N
terminus of the RLC1 (1-4). The
RLC is a subunit of the two head domains
(S1) with each S1 containing one motor domain, ELC and RLC. The two S1
domains are attached to a long Domain requirements for regulation have been elucidated through studies
of various proteolytic and expressed subfragments of SMM. HMM, which
lacks the C-terminal two-thirds of the tail, is double-headed and
regulated (5), but expressed HMMs with truncated tails failed to form
double-headed structures and were found to be unregulated (6-8) as was
S1 (9-11) and single-headed myosin (12, 13). Therefore, two heads are
critical for regulation. Two motor domains were found to be required
for regulation of a nonmuscle myosin (14). Most mutant constructs that
have altered regulation, appear to have not only an increased ATPase
activity in the unphosphorylated state but a decreased ATPase activity in the phosphorylated state. This suggests that the phosphorylated state does not reflect a simple case in which the inhibitory mechanism inherent to the native unphosphorylated state has been removed.
The structural basis of the regulatory mechanism is unknown. There are
no crystal structures of double-headed, and therefore, regulated
constructs. There are no atomic resolution data for the N terminus of
the RLC (residues 1-24), which includes the phosphorylated serine at
position 19 (smooth muscle isoform). The N terminus of the RLC is
highly conserved in myosins that are regulated by phosphorylation and
has been shown to be critical to the regulatory properties of the
molecule (15).
It is likely that the mechanism whereby phosphorylation controls the
motor ATPase activity is common and important to other isoforms that
are only modulated by phosphorylation or that accomplish regulation
through Ca2+ binding. For example, a class of mutations
found in the The goal of this study was to provide structural information about the
regulatory domains of double-headed smooth muscle HMM, including the N
terminus of the RLC. In previous work (22) we used a
photo-cross-linker, BPIA, to label cysteines in a set of single-cysteine mutants of the RLC, exchange the labeled RLC mutants onto native HMM, and compare the ability of the protein to be photo-cross-linked in two states, unphosphorylated, and
thiophosphorylated. For A23C, Q15C and Cys108 we found
cross-linking between the two RLC but only in the unphosphorylated state. This result means that the cysteine sulfur atoms can approach within ~8-9 Å of the RLC of the other head, suggesting that they are oriented toward the partner head. For the other mutants, S59C and
T134C, no cross-linking was observed for either state suggesting that
these residues are probably not oriented toward the surface of the
partner head and cannot approach the other head within 8-9 Å at any time.
Here we have extended our previous photo-cross-linking studies (22) to
determine sites on one RLC that can be cross-linked by a cross-linker
attached to Cys108 of the other RLC. Previously, we used
the photo-cross-linker BPIA, but for the present work we have
synthesized two new photo-cross-linking reagents, BBPIA and its
respective maleimide derivative, which use the same photochemical
mechanism as BPIA, but contain a biotin affinity tag to facilitate
purification of cross-linked peptides. BBPIA-cross-linked peptides were
purified by avidin affinity chromatography and characterized by Edman
sequencing and MALDI-MS. BBPIA-labeled Cys108 was found to
cross-link to both 71GMMSEAPGPIN81 and
to 4RAKAKTTKKRPQR16 of the RLC in smooth u-HMM.
Likely targeted residues within these sequences were identified. Both
of these cross-links were between the two RLC. Using these data, data
from our previous study, and atomic coordinates from various myosin
isoforms (21, 23, 24), we have constructed a model of a regulatory
domain in a double-headed molecule. The implications for the structural
basis of the phosphorylation-mediated regulatory mechanism are discussed.
IAA (Sigma) was recrystallized from hexanes before use.
Reactions did not proceed smoothly without this precaution. Flash chromatography was performed with silica gel 60 (EM Reagents; 230-400
mesh). TLC was performed with aluminum-backed TLC plates (5 × 10 cm; 0.2 mm) coated with silica gel 60 F254 (E. Merck, EM Separations). All reactions were protected from room light.
Synthesis of Compound 1 (see Fig. 1)--
The
following is a modification of the method of Gilbert and Rando (25). To
a solution of biotin (Sigma, free acid, 142 mg, 0.58 mmol),
4,4'-diaminobenzophenone (161 mg, 0.76 mmol), diisopropylethylamine
(106 mg, 0.82 mmol), and 1-hydroxy-7-azabenzotriazole (120 mg, 0.88 mmol) in DMF (5 ml) were added to DCC (134 mg, 0.70 mmol) and stirred
for 12 h. The reaction was quenched with H2O (50 ml),
and the solution was extracted with n-butanol (3 × 25 ml). The combined organic layers were washed with brine (2 × 20 ml), dried using MgSO4, filtered, and concentrated to give
a tan solid. The material was purified by flash chromatography
(CH2Cl2/MeOH (80:20)) to give
1 (381 mg, 76%) as a tan solid: mp 269-270 °C; IR
(NaBr) Synthesis of Compound 2--
Syntheses were performed in a
standard 2.0-ml plastic microcentrifuge tube. DCC (12.3 mg, 58 µmol)
was added to a solution of recrystallized IAA (Sigma, 16.2 mg, 86 µM) in THF (200 µl), and the mixture was stirred for 30 min at 0 °C. To this solution was added 1 (10.1 mg, 23 µmol) in DMF (400 µl) and stirred for 30 min. The progress of the
reaction was monitored by analytical TLC (MeOH/CCl3
(20:80)). The mixture was centrifuged in a microcentrifuge, the
supernatant was removed, and the product was precipitated by addition
of cold H2O (1.5 ml). The precipitated product was dissolved in DMF (400 µl) and precipitated again by adding cold H2O (1.5 ml). The supernatant was removed to give
2 (11.8 mg, 86%) as a tan solid. The solid was dissolved in
DMF (20-30 mM) and stored at Synthesis of the Tritiated Form of Compound 2--
All steps
were performed in a hood. IAA (17.6 mg, freshly recrystallized) was
placed into a tared 3-ml flat-base conical vial equipped with a stir
bar. A positive pressure micropipette (Rainin Microman) was used to
transfer [3H]IAA (3 mCi (PerkinElmer Life
Sciences; ~100-200 mCi mmol Synthesis of Compound 3--
To a solution of 1 (151 mg, 0.34 mmol) in DMF (1.5 ml) was added maleic anhydride (Sigma, 67.2 mg, 0.68 mmol) and stirred for 30 min at room temperature. Sodium
acetate (30.0 mg, 23 µmol) and acetic anhydride (1 ml) were added
with stirring for 1 h at 100 °C. The mixture was poured into
H2O (50 ml), and the resulting precipitate purified using
flash chromatography (CHCl3/MeOH (80:20)) to give
3 (105 mg, 59%) as a tan solid; 1H NMR
(CD3OD) Protein Preparations--
SMM (26) from frozen chicken gizzards
was used to prepare regulated HMM (5) as described (9) except that all
buffers (except digest) contained fresh
diisopropylfluorophosphate (100 µM) to prevent
post-quench digestion. DIFP converts in time to an unknown compound
that causes HMM to lose regulation, giving a fast FTP turnover rate.
Ammonium sulfate was added to 60% saturation; higher concentrations
precipitated the protease. Native RLC was isolated from SMM (27).
Extinction coefficients were: SMM,
Thiophosphorylation by SMM light chain kinase (5, 22) was verified
using 10% Tris-glycine gels. Samples (25-40 µg) were precipitated
with 3 volumes of cold acetone before. Sufficient sample buffer (8 M urea, 33 mM Tris-glycine (pH 8.6), 0.17 mM EDTA, 10 mM fresh DTT, bromphenol blue) was
added to give 6-7 mg/ml protein. These gels gave superior results to
urea and/or urea-glycerol gels (27, 28).
Photo-cross-linking and Purification of Cross-linked RLC-RLC
Dimer--
Cys108 of native RLC was 75-95% labeled with
[3H]BBPIA (23,500 cpm/nmol; 60% counting efficiency) as
described (22) and dialyzed (Pierce, snakeskin) overnight to remove
excess [3H]BBPIA. Native RLC of u-HMM was replaced with
[3H]BBPIA-labeled RLC by exchange (5, 12, 22).
Unexchanged RLC was removed by gel filtration (5).
[3H]BBPIA-labeled u-HMM was dialyzed to 10 mM
MOPS (pH 7.0), 0.1 mM EGTA, 0.05 mM DTT,
centrifuged (350,000 × g) at 4 °C for 10 min, and
filtered through a 0.45-micron filter prior to irradiation. Ten percent
of the sample was retained, and 90% was irradiated as described (29).
Samples were lyophilized, dissolved in 6 M GndHCl, 10 mM MOPS (pH 6.5), 1 mM DTT, and 1 mM EDTA, heated to 50 °C for 30 min, and filtered.
RLC-RLC dimers (40 kDa) were separated from uncross-linked RLC and
heavy chains by gel filtration in the above buffer (two TSK SW4000
columns (Tosohaas) in series at 0.2 ml/min at 25 °C). The RLC-RLC
dimer was identified on SDS gels and by scintillation counting.
Four Independent Experiments to Purify and Characterize
Photo-cross-linked Peptides--
In experiment 1, RLC-RLC dimer (11.4 nmol) was digested with 6 µg of endoproteinase Asp-N (Roche Molecular
Biochemicals sequencing grade) for 48 h in 0.1 M
GndHCl, 50 mM Tris (pH 8.5) at 34 °C, after which
another 6 µg of endoproteinase Asp-N was added and allowed to react
at 4 °C for another 48 h. NaCl (0.5 M) was added to
the sample prior to loading onto a 1-ml neutravidin column (Pierce)
equilibrated in 0.1 M GndHCl, 100 mM Tris (pH
8.5), 0.5 M NaCl, 1 mM DTT, and 1 mM EDTA. The column was washed with the above buffer and 25 mM ammonium bicarbonate, 1 mM DTT. During this
wash, 40-45% of the loaded tritium eluted from the column, as was
found for all experiments. Further binding could not be achieved with
fresh avidin. Biotinylated peptides were eluted with 70% formic acid
as buffers recommended by the manufacturer were ineffective. The sample
(2.2 nmol) was lyophilized and applied to a C8 reversed-phase column
(Brownlee Aquapore, narrowbore) and eluted with a linear gradient of
0.1% trifluoroacetic acid/H2O versus 0.1%
trifluoroacetic acid/80% ACN. Radiolabeled peptides (2.4 nmol) eluted
over one-third of the gradient. Fractions were lyophilized to near
dryness and analyzed by MALDI-MS (Table I).
In experiment 2, an Asp-N digest of RLC-RLC dimers (8.6 nmol) was
prepared as in experiment 1. An unirradiated u-HMM (0.86 nmol; not gel
filtered) was also prepared. Immobilized neutravidin (Pierce; in a
syringe column attached to a pump) was equilibrated with 10 volumes of
50 mM Tris-Cl (pH 8.25 at 25 °C), 0.3 M
GndHCl, 0.5 M NaCl, 0.5 mM DTT, and 0.5 mM EDTA (loading buffer). The sample (adjusted to 0.5 M NaCl) was loaded, and the column was washed with 10 column volumes of each of the following; 1) loading buffer, 2) loading
buffer at 2 M GndHCl, 3) loading buffer at 2 M
NaCl, 4) 50 mM ammonium bicarbonate, 0.5 mM
EDTA, 5) buffer 4 with 0.1% Triton X-100 added, 6) 5 mM
ammonium bicarbonate, 7) buffer 6 with 10% ACN, 8) 50 mM
sodium acetate pH 4.0, 10% ACN, and 9) H2O. The
biotinylated peptides were eluted with 88% formic acid and immediately
applied to a Superdex Peptide column (Amersham Biosciences; 24 ml, 0.5 ml/min) equilibrated in 0.1% trifluoroacetic acid, 30% ACN (see Fig.
4). Fractions (0.3 ml) were lyophilized prior to sequencing (see Fig.
5) and MALDI-MS (see Fig. 6).
In experiment 3, RLC-RLC dimer (5 nmol) treated with Asp-N as for
experiments 1 and 2. EDTA (1 mM) was added to inhibit
Asp-N; Glu-C (Roche Molecular Biochemicals sequencing grade; 1/100 w/w) was added and allowed to digest at 25 °C for 3 days. The digest was
filtered and loaded onto a 0.5-ml monomeric avidin column (Pierce,
Ultralink), and the column was washed as described in experiment 2. The
biotinylated peptides were eluted with 70% trifluoroacetic acid. After
lyophilization, the sample was dissolved in 200 mM ammonium
bicarbonate and clarified by centrifugation. Acylaminoacyl-peptidase (30 µg; Roche Molecular Biochemicals sequencing grade; E.C.
3.4.19.1), EDTA (1 mM), and Mass Spectral Analysis--
Mass spectra were obtained on an
Applied Biosystems Voyager DE/RP MALDI instrument in linear positive
ion mode. Laser power was 15% above threshold. The matrix was
For experiment 1, three spectra from three different fractions from the
C8 reversed-phase column were analyzed on at least two different days.
Values reported are the average ± standard deviations (Table I).
An external calibration was performed with standard peptides
(Sequezyme Calmix 2 from Applied Biosystems). For experiment 3, masses [M + H]+ were detected in at least 3 of 5 different measurements made on different days on the same sample.
Values reported are the average ± standard deviations (Table II).
Calibration with internal standards resulted in two problems that
prevented simultaneous observation of both unknown masses and
standards. First, the sample peaks were suppressed to below detection
levels, and second, a sample milieu effect caused the measured masses
of peptide standards to shift to higher masses ( Synthesis of Photo-cross-linkers--
We have developed two new
trifunctional photo-cross-linkers (Fig.
1, compounds 2 and
3) to facilitate purification of photo-cross-linked
peptides. They are derivatives of the widely used benzophenone
chromophore that forms C-C bonds with polypeptides upon irradiation
with UV light (31). We have previously described the synthesis of the
sulfhydryl-reactive photo-cross-linker, BPIA, including a tritiated
form to facilitate isolation of cross-linked peptides (29). The two new
probes contain three chemical functionalities; an iodoacetyl
(2, BBPIA) or a maleimide (3, BB-maleimide) to
selectively react with the cysteine thiolate anion, the photoreactive benzophenone, and the biotin affinity tag to facilitate purification of
parent-target covalently cross-linked peptides. We describe easy,
economical syntheses for which training in synthetic organic chemistry
is not required. The synthesis of 2 provides for safe
handling of tritiated compounds with the tritium incorporated on the
last step.
Regulation of Labeled HMM--
Fig.
2 shows that the BBPIA-labeled HMM
(sample D) was not as perfectly regulated as untreated HMM
(sample A), HMM with RLC labeled with the less bulky BPIA
(sample B), or HMM exchanged with unlabeled RLC
(sample C). The BBPIA-labeled tp-HMM had a normal activity,
but the unphosphorylated form was more active than normal. This
suggested that the presence of BBPIA at Cys108 may alter
the functional properties of u-HMM to some extent. However, in this
case there remains a sufficient level of regulation to justify
continuing with the study. A more complete assessment of regulation of
this and other similarly labeled HMMs using the more sensitive
single-turnover approach (5, 14) is in progress.
Cross-linking Occurs between the Two RLC--
Since the regulatory
properties of the BBPIA-labeled u-HMM remained largely functional, we
expected the photo-cross-linking pattern seen for BBPIA-HMM to be
similar to that previously observed for BPIA-HMM (shown to be
regulated; (22)). As expected, irradiation caused the formation of
RLC-RLC cross-linked dimers but only in the unphosphorylated state
(Fig. 3). To determine the site(s) on the
RLC into which the BBPIA was photo-inserted (target), the irradiated
sample was denatured and passed over an analytical gel filtration
column to isolate the RLC-RLC dimers.
Identification of the Target Peptides--
We performed four
independent experiments to purify and characterize parent-target
cross-linked peptides. In experiment 1, an Asp-N digest of RLC-RLC
dimers was applied to an avidin column, and the eluate was separated on
a reversed-phase column. Table I shows
the peptides in the column fractions identified as targets by MALDI-MS.
Peptides 1-4 matched the N terminus. To verify this match, a sample
enriched in the N-terminal peptide was treated with acetic anhydride
(32) to acetylate lysines. A new set of peaks emerged (data not shown)
consistent with 5-6 acetylations (addition of 210-252 mass units), as
predicted for the N terminus. Peptides 5-14 were members of a family
of peptides spanning residues 70-98, and peptides 15-21 spanned
residues 49-51.
In experiment 2, avidin-purified biotinylated peptides were
gel-filtered (Fig. 4). For the
unirradiated sample, most of the radioactivity eluted in fractions
35-40 that contained parent peptides. For the irradiated sample, the
major portion of the radioactivity eluted earlier in the profile, as
would be expected for the larger parent-target cross-linked peptides.
Edman sequencing was performed for 15 cycles on fractions 6-8 from
Fig. 4 (irradiated sample), and two major peptide sequences were
observed (Fig. 5); the parent peptide
that was also detected in the MALDI-MS spectra from experiment 1 ((M + H)+ = 1634.92), and the target sequence
66DEYLEGMMSEAPGPI80 ... were found in
approximately equal quantities, strongly suggesting that the peptides
were cross-linked together. The acetylated N terminus did not sequence.
The sequence 49DKE51 was not detected in Fig.
5, but it was observed in the MALDI-MS (peptides 16-21 from Table I).
Together these data suggest that, while present, the sequence
49DKE51 was in minor amounts relative to the
target sequence
66DEYLEGMMSEAPGPI80. . . . .
Experiment 3 was performed to further define the cross-linked regions
identified in experiments 1 and 2. An avidin-purified Asp-N/Glu-C/acetyl aminopeptidase digest of RLC-RLC dimers was treated
with Zip-Tips without further purification. Two samples were obtained.
Edman sequence analysis of the first sample (Fig. 6A) identified the target
sequence starting with Gly71 that is contained within the
sequences identified from experiments 1 (Table I) and 2 (Fig. 5). Table
II shows the MALDI-MS data, which is
consistent with the sequencing data. Edman sequence analysis for the
second sample (Fig. 6B) shows that the major target peptide was the deacetylated N terminus as the sequences starting with Gly71 was not detected even though they were detected in
the MALDI-MS spectra (Table II). Data from experiment 3 are consistent
with data from Table I and Fig. 5 and suggest that
71GMMS74, 78GPIN81, and
the first 25 residues from the N terminus are targeted. From Fig. 5 it
appears that Ser74 is a cross-linked amino acid, but it is
not definitive because the yield for serine is often low. Methionine is
often targeted by activated benzophenone, presumably at the methylene
carbon adjacent to the sulfur (31). In this case the sequencing data suggest strongly that methionine is not targeted. The targeted residue
within the 78GPIN81 was not identified, but the
sequencing data suggest that it is Asn81.
In experiment 4, a tryptic digest was performed to identify targeted
residues from the N terminus by MALDI-MS (Fig.
7). Other previously identified targeted
regions were not observed in this experiment because the peptides were
too large. Five different peptides were matched to a parent mass plus
an arginine residue. Arg4 is the only single arginine
predicted from a tryptic digest and is therefore a targeted residue. A
lysine residue was also targeted and is most likely Lys12,
but Lys150 was also possible. The dipeptide AK could be
residues 5-6 and/or 7-8. By combining the information from Figs.
6B and 7, we can be reasonably sure that Ser1,
Ser2, Ala5, Ala7, Thr9,
and Gln15 are not targeted. Lys3,
Lys8, and Thr10 are potentially targeted, and
Arg4, Lys6, Lys11,
Lys12, Arg13, Pro14, and
Arg16 are probably targeted. Therefore, the region
4RAKAKTTKKRPQR16 can approach within
cross-linking distance of benzophenone.
Summary of Target Peptides and Residues--
By analysis of
MALDI-MS and Edman sequencing data from four independent
photo-cross-linking experiments, we have shown that Cys108
of an RLC must be nearby two general regions of the partner RLC in
u-HMM; the region including 71GMMS74 and
78GPIN81 with Ser74 and
Asn81 as likely targeted residues, and the region
4RAKAKTTKKRPQR16 with Arg4,
Arg6, Lys11, Lys12,
Arg13, Pro14, and Arg16 as likely
targeted residues. These regions are not cross-linked in tp-HMM.
Products of Protein Photo-cross-linking with Benzophenone
Derivatives--
Our study has revealed new mechanistic information
about benzophenone photochemistry. It is known that the product of
benzophenone cross-linked to the Effect of BBPIA upon Regulatory Properties of HMM--
Labeling of
Cys108 with the bulky BBPIA, unlike the smaller BPIA (22),
minimally altered regulation (Fig. 2). In contrast, labeling of Q15C
and A23C with BBPIA significantly disrupted regulation (data not
shown), whereas regulation was intact with BPIA (22). This suggests
that these residues are positioned in critical locations important for regulation.
Motor Domains Are Not Required for the
Phosphorylation-dependent Structural Changes--
We have
previously shown (22) that the HMM cross-linking pattern was not
altered by ADP or ATP. And we showed that RLC-RLC cross-linking
occurred in an unphosphorylated construct lacking motor domains and
that phosphorylation abolished the cross-linking. Therefore, it appears
that our experiments are sensing a phosphorylation-induced conformational change in the RLC that does not require motor domains. Similarly, Rosenfeld et al. (33) showed that RLC rotational motion in a construct lacking the motor domains is increased by RLC
phosphorylation. None of the interactions in Table
III were observed in tp-HMM in any
nucleotide state.
Structural Model of u-HMM Regulatory Domain Structure--
Table
III summarizes data we considered to develop a computational model of
the u-HMM regulatory domain. The general features of the model are
shown in Fig. 8, the details of which
will be published elsewhere. It describes the relative orientation of the regulatory domains during the cross-linking event. To build this
symmetrical model both benzophenone moieties (one from each RLC
Cys108) were locked within 1.4 Å of Gly78 of
the RLC from the other head, and the structure was adjusted to avoid
Van der Waal's overlap and to agree with Table III. No other
symmetrical models were consistent with the data in Table III. The two
RLC are side by side in an antiparallel manner. Fig. 8 is not meant to
indicate specific RLC interactions, but merely suggests their relative
orientation and separation during the cross-linking event. The two
Phe25 (the most N-terminal residues for which we have
atomic resolution data) are close together at the interface between the
two RLCs in the center of Fig. 8A and in the lower portion
of the RLCs in Fig. 8C.
To test our model, we performed an additional cross-linking experiment.
Irradiation of BPIA-labeled T83C HMM formed RLC-RLC dimers, only in the
unphosphorylated state, and nucleotide did not appear to affect the
result (data not shown). This result is consistent (Fig. 8) as
Thr83 is positioned at the top of the groove between the
two RLC within cross-linking distance to the partner RLC.
Model Predicts Location of RLC N Terminus--
Our model allows us
to predict the position of the first 24 RLC residues, a region for
which there is no atomic resolution data. This area is of particular
interest because it contains the critical regulatory phosphorylation
site at Ser19. First, we generated an independent model of
these first 24 residues by using secondary structure and disorder
prediction tools and analysis of kinase structures with bound substrate
peptides. Our model predicts that Ser1 to Thr10
or Lys11 forms a helix followed by a disordered region that
cannot be assigned secondary structure. This latter portion could
maximally extend ~32 Å from Lys11 to Met24.
Fig. 8B shows the independently modeled N-terminal 24 residues (gray) from the lower RLC (blue) placed
onto the regulatory domain model in a manner consistent with our data.
We have shown that residues within
4RAKAKTTKKRPQR16 can approach within 8-9 Å of
the Cys108 sulfur of the partner RLC (Fig. 6B,
Tables I and II, and Fig. 7). All the probable and potentially targeted
residues found within residues 1-11 (Fig. 7), a region that we have
predicted to be a helix, are located on one face of such a helix. This
suggests that residues 1-11 may be folded into a helix. However
because so many residues within this region were targeted, the helix
and the benzophenone are not highly restricted in space relative to one
another. Much of the N terminus including Ser19
(red) lies in a groove between the two RLC. This placement
may explain the fact that we observed altered regulation in A23C and Q15C mutants labeled with a bulky group. Previous experiments with the
less bulky BPIA-labeled Q15C and A23C on u-HMM (22) showed that RLC-RLC
dimers were formed upon irradiation, but the site of labeling was not
identified. This model places these two residues, flanking the
Ser19 (pink), within cross-linking distance of
the partner RLC.
As seen in Fig. 8B, the N terminus interacts with the
C-terminal domain of the partner RLC. Several studies have shown that elements of the C-terminal domain are crucial to proper regulatory properties (34-36). It is tantalizing to suggest that the N terminus is positioned to control the conformation of the heavy chain helix to
which the two RLC bind. It has been previously suggested that Ca2+ binding in up-regulated scallop myosin may play a role
in stabilizing the regulatory domain through tightened interactions
between the RLC, ELC, and heavy chain (21). The N terminus may also be
strategically placed to control the interface between the two RLC and
the attitude and flexibility of the linker connecting the two domains
of the RLC. This latter region is known to be important to the
regulatory mechanism (37) and has been noted to be different between
the skeletal and scallop structures (21).
Model Predicts Phosphorylation-induced Motion of RLC N
Terminus--
Phosphorylation must result in movement of
Cys108 out of cross-linking distance to the
71GMMSEAPGPIN81 and
4RAKAKTTKKRPQR16 targets of the partner RLC as
photo-cross-linked RLC-RLC dimers were not observed in tp-HMM. It may
be that movements of the two targets are consequences of one another.
Our model shows that cross-linking could occur from Gln15
and Ala23 to the partner RLC, in agreement with Table III.
However, upon phosphorylation, neither Gln15 or
Ala23 can cross-link to the partner RLC suggesting a
phosphorylation-induced movement of a significant portion of the N
terminus. This could occur if the phospho-serine folded back the N
terminus upon itself through coordination with its or other basic
residues. In our model, the phosphorylated serine is in a strategic
position to dramatically alter the interactions of the N terminus with
such a coordination. It is interesting that phosphorylation sites are often in regions of known disorder (38), and we predict that the
region around Ser19 is disordered. Disorder is also common
in protein-protein interactions that are part of regulatory switches
(38). The role of phosphorylation may be to transition this region of
the N terminus from an extended (shown in our model) to a folded structure.
Does Phosphorylation Dissociate Regulatory Domains?--
Our model
is consistent with previous work showing that isolated u-SMM regulatory
domains dimerize (33) and further suggests that interactions between
the two RLC provide the stabilizing forces. However, phosphorylation
has little effect upon the stability of the dimers (33). Our results
using zero-length cross-linking were consistent with these
findings.2 Rosenfeld et
al. (33) showed that the rotational motion of a fluorophore on
Cys108 in a construct lacking motor domains is increased by
RLC phosphorylation. However, the rotational motion was slower than
expected for mobile regulatory domains moving independently of the
rods. In light of these findings, the transition we observe from
cross-linking in the u-HMM to no cross-linking in the tp-HMM may be the
result of local motions that do not destabilize RLC interactions. Our data, which shows that the N terminus moves out of cross-linking distance to Cys108, may be sufficient to explain the
changes in rotational motion observed by Rosenfeld et al.
(33).
Implications of the Model for S2 Structure--
The distance
between the C termini of the heavy chains at the head-rod junction is
47 Å in our model, suggesting that the rod cannot adopt a coiled-coil
up to the head-rod junction. We were unable to find any satisfactory
models with such a coiled rod conformation. Our model is consistent
with the idea that optimal mechanical performance of SMM may require
the rod to uncoil near the heads (39).
Comparison to Other Models--
The data from Table III do not fit
a model developed from a three-dimensional reconstruction of frozen
hydrated expressed smooth u-HMM on a lipid bilayer (40). Neither can
RLC Cys108 reach RLC residues 71-81 from either head, nor
are inter-head Cys108 to N termini interactions likely
without large rearrangements. Since the rod interacts with the lipid
bilayer and the heads are arranged on top of the rod, it may be such an
interaction stabilizes a structure not found in solution.
We previously reported (22) that our cross-linking data did not fit a
computed model (24) of scallop myosin regulatory domains (atomic
resolution data from Xie et al. (20) in the presence of
Ca2+) attached to a model of the C-terminal portion of the
SMM 10 S Studies Support the Model--
The 10 S or folded
conformation of u-SMM, like u-HMM, is kinetically inactive. Several
studies have shown that the N terminus of the RLC is required to form a
10 S SMM structure (41, 42) with the tail folded onto the heads.
However, myosin containing an N-smooth/C-skeletal RLC chimera, which
contains a complete N terminus, also fails to fold to 10 S (34). This
suggests that an interaction of the N terminus with the C-terminal
domain may be critical to generate a binding site on the heads for the
tail in 10 S. Since single-headed myosin fails to form the 10 S (29, 41) it appears that elements from both heads are required to generate
this binding site. We have previously shown (29) that BPIA-labeled
Cys108 photo-cross-links to the tail in the 10 S
conformation. All these data point to a structure where the RLC N
terminus interacts with the RLC C-terminal domain on the partner head
and that this interaction is required for tail interaction near
Cys108. These data taken together are highly consistent
with our model, which places Cys108 in the RLC C-terminal
domain close to the N terminus of the partner RLC. We propose that an
interaction similar to that in Fig. 8 is important for down-regulation
of SMM.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical coiled-coil domain (tail or
rod). The regulatory domain is defined as an RLC and ELC attached to the portion of the heavy chain to which they bind. The unphosphorylated forms of these regulated myosins have low ATPase activity and are
unable to move actin filaments, whereas the phosphorylated forms are
activated in both respects.
-myosin isoform from cardiomyopathy patients are found
clustered near the N terminus of the RLC (16-18) and myosin RLC
phosphorylation is a key determinant of the stretch activation response
in Drosophila muscles (19). Ca2+ binding to the
ELC turns on molluscan myosins. The Ca2+ ion mediates
interactions between the RLC and the ELC in addition to the heavy chain
(20, 21). Molluscan myosins, like the smooth and nonmuscle isoforms,
require two heads for regulation. It is likely that the
Ca2+-mediated regulatory mechanism and the
phosphorylation-mediated regulatory mechanism will have many structural parallels.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3442, 3352, 3292, 1703, 1619, 1594, 1521, 1314, 1169 cm
1; 1H NMR (CD3OD)
7.71-7.57 (m, 6H, phenyl), 6.65 (d, J = 8.8 Hz, 2H,
phenyl), 4.45 (dd, J = 3.0, 7.2 Hz, 1H, H-4), 4.28 (dd,
J = 4.1, 7.5 Hz, 1H, H-3), 3.29 (ddd, J = 4.7, 6.3, 9.2 Hz, 1H, H-2), 2.89 (m, 1H, H-5A), 2.68 (d,
J = 12.7 Hz, 1H, H-5B), 2.40 (t, J = 7.2 Hz, 2H, H-
), 1.75-1.49 (m, 6H, H-
A, H-
B, H-
, H-
); 13C NMR (DMF-d7)
192.8 (PhCOPh), 172.1 (C-10), 163.3 (C-2'), 153.9 (phenyl), 142.9 (phenyl), 133.6 (phenyl),
132.5 (phenyl), 130.4 (phenyl), 125.1 (phenyl), 118.3 (phenyl), 112.8 (phenyl), 61.9 (C-3), 60.2 (C-4), 56.1 (C-2), 40.4 (C-5), 36.8 (C-
),
28.9 (C-
), 28.7 (C-5), 25.7 (C-
); high resolution MS (M + 1)+ m/z calculated 439.1726, found
439.1729.
80 °C; 1H
NMR (CD3OD)
7.76 (m, 8H, phenyl), 4.49 (dd,
J = 4.1, 7.9 Hz, 1H, H-4), 4.32 (dd, J = 4.6, 7.9 Hz, 1H, H-3), 3.91 (s, 2H, -CH2I), 3.23 (ddd,
J = 4.9, 6.4, 9.0 Hz, 1H, H-2), 2.94 (dd,
J = 5.0, 12.7 Hz, 1H, H-5A), 2.71 (d, J = 12.9 Hz, 1H, H-5B), 2.45 (t, J = 7.4 Hz, 2H, H-
),
1.62 -1.40 (m, 6H, H-
A, H-
B, H-
, H-
); 13C NMR
(DMF-d7)
192.6 (PhCOPh), 171.4 (C-10), 163.6 (C-2'),
153.5 (phenyl), 143.2 (phenyl), 133.9 (phenyl), 132.8 (phenyl), 130.9 (phenyl), 125.7 (phenyl), 119.0 (phenyl), 113.0 (phenyl), 74.3 (-CH2I), 62.3 (C-3), 60.6 (C-4), 56.4 (C-2), 40.9 (C-5),
36.8 (C-
), 29.3 (C-
), 29.1 (C-
), 26.1 (C-
); MALDI-MS (M + 1)+ calculated 607.088, found 607.093;
E
1 cm
1 in MeOH. The
Rf was 0.65 on TLC in MeOH/CCl3
(20:80). The product reacted with cysteine (pH 8), and the cysteine
adduct did not migrate on TLC.
1) from the original
vessel to the conical vial with dry THF. THF was removed with a stream
of argon. After adding hexanes (250 µl), the vial was warmed to
60 °C with an aluminum block hot plate until the IAA just dissolved.
The vial was cooled to room temperature and then placed on ice for 30 min. The mother liquor was removed with a drawn glass pipette, and the
crystals were washed with ice-cold hexanes. Crystals were dried
overnight under a stream of argon and quantitated by weight. The
coupling reaction was carried out as described above.
7.91-7.76 (m, 2H, phenyl), 7.02 (s, 2H,
maleimide), 4.50 (dd, J = 4.5, 7.8 Hz, 1H, H-4), 4.32 (dd, J = 4.2, 6.9 Hz, 1H, H-3), 3.22 (ddd,
J = 4.9, 6.3, 9.0 Hz, 1H, H-2), 2.95 (dd, J = 4.5, 12.2 Hz, 1H, H-5A), 2.71 (d, J = 12.6 Hz, 1H, H-5B), 2.45 (t, J = 7.2 Hz, 2H, H-
),
1.84 -1.49 (m, 6H, H-
A, H-
B, H-
, H-
); 13C NMR
(DMF-d7)
194.5 (PhCOPh), 172.8 (C-10), 170.4 (maleimide-CO), 163.8 (C-2'), 144.8 (phenyl), 137.5 (phenyl), 136.1 (phenyl), 135.7 (alkenyl-H), 132.2 (phenyl), 132.0 (phenyl), 130.9 (phenyl), 126.9 (phenyl), 119.1 (phenyl), 62.5 (C-3), 60.8 (C-4), 56.7 (C-2), 41.1 (C-5), 37.6 (C-
), 29.5 (C-
), 29.4 (C-
), 26.3 (C-
); MALDI-MS (M + 1)+ calculated 519.170, found
519.164. The Rf was 0.60 on TLC in MeOH/CCl3 (20:80). The product of the reaction with
cysteine (pH 8) did not migrate from the baseline.
-mercaptoethanol (1 mM) were added, and the digest was allowed to proceed for
24 h at 37 °C. The sample was reapplied to a fresh monomeric
avidin column and eluted as previously described. After repeated
lyophilization the sample was resuspended in 0.1% trifluoroacetic
acid/65% ACN to give a precipitate too large to be due to
labeled peptides. Therefore the sample was repeatedly centrifuged, and
the pellets were washed with 0.1% trifluoroacetic acid/65% ACN. The
combined supernatants were lyophilized and treated with Zip-Tips
(Millipore; C8) in 0.1% trifluoroacetic acid/10% ACN. Complete
Zip-Tip binding required an overnight incubation. Peptides were
released from 5 Zip-Tips with 65% ACN (see Fig. 7A). An
second sample was prepared by treating the residual from the previous
sample with more Zip-Tips in 0.1% trifluoroacetic acid and eluting
with 65% ACN (see Fig. 7B). In experiment 4, samples were
prepared as in experiment 1, except that proteolysis was with trypsin
(1/100 w/w) overnight at 25 °C.
-cyano-4-hydroxy-cinnamic acid prepared as a saturated solution in
50%/50% (v/v) ACN/water with 0.25% trifluoroacetic acid. Data were
analyzed using Kaleidograph and MS programs from the Expasy website,
FindPept Tool and PeptideMass. Observed masses were identified by
comparison to calculated masses generated by summing the masses of
photo-cross-linker, parent peptides, and predicted target peptides. We
subtracted 18 mass units, since 18 mass units can be lost from
benzophenone cross-linked peptides treated under similar conditions
(30). Most observed masses matched to this in silico data set.
~4-5 Da) than
was found for standards in the absence of sample. Therefore an external
calibration was performed with standard added to the sample. Then, data
were acquired on the sample, alone and the external calibration was applied.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
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Fig. 1.
Compounds synthesized. Compound
1 is the starting material for 2 (BBPIA) and
3 (BB-maleimide). The iodoacetyl functionality is sulfhydryl
reactive, and the benzophenone contains a photoreactive carbonyl.
Biotin is an affinity tag.
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Fig. 2.
Actin-activated steady-state MgATPase
activities at 25 µM actin of HMM
samples. Conditions were 25 °C in 10 mM MOPS (pH
7.0), 0.1 mM EGTA, 2 mM MgCl2, 1 mM DTT, and 1 mM ATP. The rate of hydrolysis of
[ -32P]ATP (DuPont) was measured (43). Dark
bars, unphosphorylated; light bars, phosphorylated. All
HMM samples, except in A, were exchanged with the indicated
RLC. A, untreated, not exchanged (data from Ref. 22).
B, Wild type construct containing four non-native amino
acids at the N terminus, BPIA-labeled, unirradiated (data from Ref.
22). C, Wild type, unlabeled, unirradiated (data from
Ellison et al. (5). D, native BBPIA-labeled
unirradiated. Data were calculated based upon the HMM mass of 375 kDa.
Rates in the absence of actin ranged from 0.01-0.04 s
1
head
1. Values are the average of two measurements; ranges
were not more than 0.01 s
1.
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[in a new window]
Fig. 3.
Effect of irradiation upon
u-HMM containing BBPIA-labeled RLC. Gradient gel
(Tris-glycine, 4-20% acrylamide, Invitrogen) was stained with
Coomassie Blue (lane 1), and sister gel was transferred for
Western blot with anti-RLC antibodies (lanes 2-6).
Lanes 1 and 2, HMM standard; lane 3,
unphosphorylated unirradiated; lane 4,
unphosphorylated irradiated 20 min; lane 5,
thiophosphorylated unirradiated; and lane 6,
thiophosphorylated irradiated for 20 min. UnP,
unphosphorylated; P, thiophosphorylated.
Analysis of MALDI-MS data from experiment #1
View larger version (26K):
[in a new window]
Fig. 4.
Gel filtration of avidin affinity-purified
peptides (experiment 2). Peptides eluted from the avidin column
were applied to a Superdex Peptide column (24 ml; Amersham Biosciences)
equilibrated in 0.1% trifluoroacetic acid/30% ACN and eluted at 0.5 ml/min. Fractions (0.3 ml) were collected, and 20 µl was assayed for
radioactivity. Solid bars, unirradiated; open
bars, irradiated.
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[in a new window]
Fig. 5.
Results of Edman sequencing of irradiated
fractions 6-8 (experiment 2). The amount of aspartic acid (33.1 pmol) in cycle 1 was arbitrarily distributed evenly as both the parent
and the target sequences began with aspartic acid. X
indicates the BBPIA-labeled cysteine residue. Single letter amino acid
without and with parentheses indicates unambiguous or
ambiguous assignment, respectively.
View larger version (63K):
[in a new window]
Fig. 6.
Edman sequencing of Asp-N, Glu-C,
aminopeptidase digest (experiment 3). Single letter amino acid
without parentheses indicates unambiguous assignment.
Parenthesis indicates ambiguous assignment because the same
amino acid from a coeluting avidin fragment
((S)25ARKCS ... shown at the right) was in
that cycle or in the cycle before or after. If an amino acid was
present in both parent and target sequences the total pmol was evenly
attributed. X = BBPIA-labeled Cys108.
A, the sample from Fig. 6A. G*, not determined as
Gly is a common contaminant in the first cycle. B,
residual sample from Fig. 6B. The amount of Asp in
cycle 1 was 96 pmol.
Analysis of MALDI-MS data from experiment #3
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Fig. 7.
Peptides in N-terminal region identified by
MALDI-MS (experiment 4). The first 16 residues of the RLC sequence
are shown. Each line spanning the residues represents an independent
measurement of a mass matching that region. Darker lines represent more
than one observation. Dotted lines mean that the match could be in more
than one place; either of two AK positions, respectively. The position
of the lines above the sequence is not important.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-carbon of glycine can dehydrate
(31) and that peptides containing a targeted methionine exist in a form that is 18 mass units lighter than expected (30). Most of our cross-linked peptides were 18 mass units lighter than predicted (31).
For example, peptide 1-25 was found in the dehydrated form (Table I).
This peptide does not contain glycine and we have no evidence for
cross-linking of methionine. We identified Lys and Arg as targeted
amino acids in this region. This suggests that these amino acids are
also prone to dehydration as may be Ser and Asn (Tables I and II). We
do not know if this dehydration occurs during the photolysis or in the
mass spectrometer. Most likely, a proton on the methylene carbon
adjacent to the heteroatom of the side chain leaves as water along with
the OH of the biphenyl alcohol of benzophenone after C-C bond
formation, thus forming a double bond. This new information should
alert investigators to analyze MS data considering both hydrated and
dehydrated forms of cross-linked peptides, as we have here.
Data used to develop structural model of the regulatory domain of u-HMM
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Fig. 8.
Model of unphosphorylated regulatory
domains. The red RLC and orange ELC are
attached to the olive HC; this is one regulatory domain. The
motor domain (not shown) would extend to the right. The
blue RLC and purple ELC are attached to the
light blue HC; this is another regulatory domain.
A, the C termini of the heavy chain helices (residue
Leu837) are extending out of the page toward you. The
distance between these two terminal residues is 47 Å. Benzophenone is
shown in yellow space-filling attached to
Cys108. The benzophenone on the right is
attached to the red RLC. The RLC sequence
71GMMSEAPGPIN81 is shown in green
ribbon. The -carbon of Gly78 (space-filling) is <1 Å from the carbonyl carbon of the benzophenone. Ser59 is
shown in white ribbon. T134C is also in white
ribbon, but is mostly obscured. Phe25 of each RLC is
found in the center of the structure, near the linker (residues
Gly95-Pro98) between the RLC N-terminal and the
C-terminal domains. B, same orientation as A and
illustrates the predicted orientation of the blue RLC N
terminus (residues 1-24; shown in gray). The N terminus has
been positioned manually. The equivalent region of the red
RLC is not shown for clarity. Ser19 is pink and
10TKKRP14 is shown in cyan.
Cys108 is shown in yellow ribbon. C,
B rotated 90 ° toward you about the x-axis.
Heavy chains now extend downward. Thr134 is visible as
white ribbon in the upper portion of both RLC.
Ser59 is most clearly seen in the red RLC. All
modeling was performed on a Silicon Graphics, Inc. (Mountain View, CA)
Octane work station using Insight II (Version 2000; Accelrys).
-helical coiled-coil S2 (tail) domain. The data presented here
reinforce that conclusion. To generate our model from the scallop
model, the coiled-coil must be unwound to allow the opposite faces of the RLC to interact. We are unaware of any structural data in the
scallop system that supports the computed model.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Rob Ronald, Dr. Yin Luo, and Dr. Thomas Bell for helpful discussions and Geetha Ramaprian for technical assistance. We thank Derek Pouchnik for zero-length cross-linking results. We are grateful for RLC antibody from Dr. James Stull and Dr. Kristine Kamm.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: ArQule, Inc., Woburn, MA 01801-5140
Current address: Concurrent Pharmaceuticals, Cambridge,
MA 02142
** Current address: Dept. of Engineering Science, University of Oxford, Oxford OX1 3PJ, UK
To whom correspondence should be addressed: University of
Nevada Central Receiving, Dept. of Biochemistry/330, 1664 N. Virginia St., University of Nevada, Reno, NV 89557. Tel.: 775-784-7033; Fax: 775-784-1419; E-mail: cremo@unr.edu.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M206963200
2 D. Pouchnick and C. R. Cremo, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RLC, regulatory light chain; ELC, essential light chain; SMM, smooth muscle myosin; HMM, heavy meromyosin; BPIA, 4-iodoacetamido-benzophenone; BBPIA, biotin affinity-tagged BPIA; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; u-HMM, unphosphorylated HMM; TLC, thin layer chromatography; DMF, N,N-dimethylformamide; DCC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; IAA, iodoacetic acid; THF, tetrahydrofuran; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid; GndHCl, guanidine HCl; ACN, acetonitrile; tp-HMM, thiophosphorylated HMM; S1, subfragment 1; Asp-N, Glu-C, Arg-C proteolytic cleavage, cleavage on the N-terminal side of Asp, C-terminal side of Glu, and C-terminal side of Arg, respectively.
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