From the Bioscience Division, Los Alamos National
Laboratory, Los Alamos, New Mexico 87545; the ¶ Department of
Physiology, University of Texas Southwestern Medical Center at Dallas,
Texas 75390; and the
Division of Molecular Biology and
Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri
64110
Received for publication, December 4, 2000
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ABSTRACT |
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A novel translocation step is inferred
from structural studies of the interactions between the intracellular
calcium receptor protein calmodulin (CaM) and one of its regulatory
targets. A mutant of CaM missing residues 2-8 ( A wide variety of Ca2+-dependent cellular
processes are regulated by the ubiquitous Ca2+-binding
protein CaM,1 which interacts
with a diverse array of target proteins, including a number of protein
kinases (1). Ca2+/CaM-dependent activation of
MLCK has provided a number of important insights into the molecular
mechanisms underlying CaM-dependent kinase activation. All
isoforms of MLCK include a conserved protein kinase catalytic core (2)
that is followed immediately by a regulatory segment consisting of
autoinhibitory and CaM-binding sequences. The catalytic core is
believed to have the distinctive bilobal structure characterized first
by the crystal structure of the cAMP-dependent protein
kinase (3). A cleft between the lobes defines the catalytic site for
substrate phosphorylation. In its inhibited state, the regulatory
segment of MLCK maintains numerous contacts with the larger lobe of the
catalytic core, including a few sites of contact in the catalytic cleft
itself, thus preventing myosin light chain binding and catalysis
(4-6). Our previous scattering studies of a catalytically competent
truncation mutant of skMLCK (7) support the idea of the autoinhibitory release mechanism (6) for kinase activation in which CaM binding induces a significant movement of the regulatory segment away from the
surface of the catalytic core necessary for binding and phosphorylating
myosin regulatory light chain. More recently, we showed that substrate
binding induces a closure of the catalytic cleft and a reorientation of
CaM that allows for more extensive surface contact between the
catalytic core and the N-terminal sequence of CaM (8).
CaM has two globular Ca2+-binding lobes connected by a
central helix (9) that functions as a "flexible tether" (10, 11). The concept of the flexible tether function obtained its strongest support from structural studies of CaM complexed with isolated peptides
containing the core CaM-binding sequences from kinases that show CaM
undergoes a dramatic conformational collapse (11-15). Flexibility in
the central helix allows the two lobes of CaM to come together,
enfolding the helical-configured target peptides. A similar
conformational collapse of CaM was observed in scattering studies of
its complex with a catalytically competent skMLCK (7, 8). The known
structures of all the CaM·peptide complexes show little or no
interaction between the N-terminal leader sequence of CaM (residues
1-8, ADQLTEEQ) and the bound peptides. Nevertheless, deletion of
residues 2-8 results in a CaM mutant ( We present here the results of scattering experiments on Sample Preparation--
The Scattering Data Acquisition--
Scattering data were acquired
using the x-ray instruments at Los Alamos (18, 19) and reduced to
I(Q) versus Q as described in (18). Q is
(4 Modeling the Scattering Data--
Model I(Q) and
P(r) profiles for the two ellipsoid models were calculated
using SASMODEL (21), which uses a rapid Monte Carlo integration routine
to generate model P(r) functions (18). The P(r)
and I(Q) profiles based on models using crystal structure coordinates were calculated using PRPDB (19). Both SASMODEL and PRPDB
calculate I(Q) as the Fourier transformation of
P(r). P(r) and I(Q) functions are then
compared with the experimental data by calculating Scattering Data--
The scattering data (Fig.
1A) for free Two-Ellipsoid Model of the Complexes Reveals Sizes and Dispositions
of Components--
To gain further insights to the relative sizes and
dispositions of CaM or Higher Resolution Structural Model for the Complex Reveals Position
of the Catalytic Cleft with Respect to the CaM-binding Site--
To
evaluate how sensitive the scattering data are to the position of CaM
or
Simulated scattering data for the
The best-fits to the scattering data obtained using the higher
resolution structural models predict a specific orientation of the
kinase catalytic cleft with respect to bound CaM or
Because of the potential importance of the changes in CaM residues 1-8
in the activation mechanism (16), three peptides were synthesized for
competitive binding studies; the native sequence, the native sequence
with Glu residues substituted for Ala, and a scrambled native sequence.
None of these peptides affected the catalysis or CaM activation
properties at 1 mM (data not shown). This result suggests
that the N-terminal sequence is not simply functioning as a latch to
hold bound CaM in the requisite position away from the catalytic site.
Our results indicate that removal of the N-terminal leader
sequence from CaM results in a complex with skMLCK in which the mutant
protein blocks the catalytic cleft. This result is in stark contrast
with the complex with native CaM, in which bound CaM appears to
translocate so that the catalytic cleft is exposed, and the N-terminal
helix of CaM interacts with the C-terminal portion of the catalytic
core. The higher resolution modeling of the native CaM·skMLCK complex
also resolves the ambiguity that remained in the interpretation of the
original two ellipsoid modeling of the neutron data regarding which
side of the kinase, with respect to the catalytic cleft, is the
CaM-binding site (8). In both native and mutant complexes CaM adopts a
collapsed structure in which both lobes likely interact with the core
CaM-binding sequence in the kinase. These results are consistent with
the fact that although native CaM and NCaM) binds skeletal
muscle myosin light chain kinase with high affinity but fails to
activate catalysis. Small angle x-ray scattering data reveal that
NCaM occupies a position near the catalytic cleft in its complex
with the kinase, whereas the native protein translocates to a position near the C-terminal end of the catalytic core. Thus, CaM
residues 2-8 appear to facilitate movement of bound CaM away from the
vicinity of the catalytic cleft.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
NCaM) that binds with high
affinity but completely fails to activate skMLCK (16). The mutant
protein also has a reduced capacity to activate the smooth muscle form
of the kinase but can fully activate neuronal nitric-oxide synthase
activity, suggesting that the N-terminal leader sequence plays a
specialized role in a subset of CaM-target complexes.
NCaM and
native CaM in their respective complexes with a catalytically competent
skMLCK (17) that reveal the dramatic structural basis for the inability
of
NCaM to activate skMLCK catalysis.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
NCaM was expressed in
Escherichia coli and purified as described in (16). CaM,
skMLCK, and the complexes were prepared as described in (8). All
samples contained excess Ca2+ so that the CaMs were
Ca2+ saturated. Buffer conditions were 25 mM
MOPS, pH 8.0, 25 mM KCl, 5 mM magnesium
acetate, 1 mM dithiothreitol, and 2.5% glycerol.
sin
)/
where
is half the scattering angle and
is the
wavelength of the scattered radiation. Structural information is
derived by calculating the inverse Fourier transform of
I(Q), which yields P(r), the probable frequency
distribution of interatomic vectors, r, within the scattering molecule
(calculated using the program GNOM (20)). P(r) goes to zero
at the maximum linear dimension, dmax, of the
scattering particle and its zeroth and second moments give forward
scatter, I0 and radius of gyration, Rg, respectively. The forward scatter,
I0, is directly proportional to the product of
the protein concentration, c, (in mg/ml) and the molecular
weight of the scattering molecule, M. I0 is thus very sensitive to changes in the size
of the scattering particle because of complex formation or aggregation.
2 for
the experimental and model I(Q) profiles,
where the summation is over N, the number of Q values
for which I(Q) is measured, Iobs is
the observed intensity at each Q value,
(Eq. 1)
is the S.D. for
each Iobs, and Icalc is
the calculated intensity at each Q value for the model being tested. A
value of 1.0 is a perfect fit with the observed data within the errors.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
NCaM indicate
it has a slightly more compact structure than native CaM (Table
I). Fig. 1C compares the
vector length distribution functions, P(r), for the two
proteins. Based on calculations of model P(r) functions using the crystal structure for CaM (not shown), this difference can be
accounted for solely by the deletion of the N-terminal residues in the
mutant protein. The scattering data thus indicate that
NCaM has a
two lobed structure, similar to native CaM. In contrast, the solution
structures of the
NCaM and CaM complexes with skMLCK differ
dramatically. The experiments on the complexes were performed with and
without substrates; a peptide containing the phosphorylation sequence
from myosin regulatory light chain (pRLC, sequence KKRAARATSNVFS) and a
nonhydrolyzable analog of adenosine-triphosphate (AMPPNP). The same
differences were observed between the native CaM and
NCaM complexes
in the presence or absence of substrates. We used data collected in the
presence of the substrates for our analyses, because the native
CaM·skMLCK complex is less prone to aggregation under these
conditions. The I0 values indicate that the
protein solutions contain monodisperse particles with the molecular
weights expected for 1:1 complexes (Table I). As we have reported
previously (7, 8), the P(r) function and
Rg value for the CaM·skMLCK complex (Fig.
1D) indicates an elongated structure (Table I). In contrast,
data for the
NCaM·skMLCK complex indicate a more symmetrical
structure, evident in its significantly smaller
dmax and Rg values (Table
I).
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Fig. 1.
Scattering data and P(r)
functions for CaM, NCaM, and their complexes
with skMLCK. A, I(Q) versus Q for
CaM (
) and
NCaM (
). B, the CaM·skMLCK (
) and
NCaM·skMLCK (
) complexes with substrates AMPPNP and myosin
light chain peptide are presented. Data are binned and shown offset on
an arbitrary log scale. Solid and dotted lines
overlaying each data set in B represent I(Q)
versus Q calculated for the best-fit uniform density and
higher resolution models, respectively (see Fig. 2). Data were fit from
Q = 0.02 to 0.20 Å
1. C, P(r)
functions scaled by their respective molecular weights for CaM (
)
and
NCaM (
). D, the CaM·skMLCK (
) and
NCaM·skMLCK (
) complexes with the solid lines
showing the P(r) calculated for the best-fit high resolution
model shown in Fig. 2.
Structural parameters for NCaM, CaM, and their respective complexes
with skMLCK in the presence of substrates (AMPPNP and myosin light
chain peptide)
NCaM and skMLCK, we simulated x-ray
scattering data using SASMODEL (see "Materials and Methods"). Each
complex was modeled as two uniform density ellipsoids, representing CaM or
NCaM and skMLCK. During this procedure the ellipsoids were required to be in contact, and their dimensions were loosely
constrained to be in a range consistent with the dimensions for CaM and
skMLCK determined in previous scattering studies (8). Specifically, the
ellipsoid semi-axis lengths were constrained to be in the ranges 38-48
Å, 23-35 Å, and 16-22 Å for skMLCK and 22-28 Å, 18-24 Å, and
14-19 Å for CaM. For the
NCaM complex, we also allowed the bound
NCaM to assume the more elongated shape associated with its
uncomplexed state, to test the possibility that the
NCaM might not
collapse upon binding. In each calculation, ~104
different models were tested, with randomly chosen ellipsoid dimensions, relative orientations, and contact points. The best-fit models are depicted in Fig. 2
(green crosses), and their respective fits
(
2) and structural parameters are given in Table
II. As expected, the model for the native
CaM·skMLCK complex is similar to the one proposed previously based on
neutron contrast variation data for the complex with deuterated CaM
(8). Contrast variation allows us to readily distinguish CaM and skMLCK
in the complex, and we therefore were able to unequivocally assign the
smaller ellipsoid to the CaM. The model for the
NCaM·skMLCK
complex indicates individual ellipsoid dimensions similar to those for
the native CaM·skMLCK complex. We therefore conclude that, like
native CaM,
NCaM collapses about the core CaM-binding sequence of
the kinase in this complex. A most significant difference between the
complexes is that bound
NCaM is more centrally located with respect
to the long axis of the kinase than the bound native protein.
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Fig. 2.
Best-fit ellipsoid models (pale
green dots) for the CaM·skMLCK (bottom)
and NCaM·skMLCK (top)
complexes superimposed with ribbon representations of the respective
higher resolution models. The ribbons for skMLCK and
the CaM-binding peptide (MLCK-I) are red and for CaM,
cyan. The pale green dots represent random points
that fill the ellipsoid shapes and are used to calculate model
P(r) functions from which model scattering profiles are
generated. The N terminus of CaM in both models is indicated with
N.
Parameters for the best-fit models to the data and dimensions of the
ellipsoids for CaM·skMLCK and NCaM·skMLCK
NCaM relative to the catalytic cleft in the kinase, we performed
additional calculations using higher resolution structural models. We
modeled the catalytic core of skMLCK using the crystal structure of the
catalytic subunit of the cAMP-dependent protein kinase,
cPKA (Ref. 3, PDB accession number 2cpk) and SwissModel, an automated
homology modeling package (22). The cPKA and skMLCK catalytic cores
have 29.4% amino acid sequence identity and 61% sequence similarity,
making cPKA a good template for homology modeling. For sequences with
30-40% sequence identity, homology modeling can yield structures that
give backbone root mean square deviation (RMSD) values from
experimentally derived structures as low as 1.5 Å (23). To model
bound CaM or
NCaM, we used the NMR structure for CaM complexed with
a peptide based on the core CaM binding sequence in skMLCK (MLCK-I,
Ref. 14, PDB accession number 2bbm), deleting residues 2-8 for the
complex with
NCaM. The skMLCK model does not account for 32 N-terminal residues and 52 C-terminal residues in the kinase, as they
have no corresponding sequences in cPKA. However, of the C-terminal
residues, 23 are accounted for by the core CaM-binding sequence.
Thus, the structural models for the complexes account for ~90% of
the skMLCK sequence.
NCaM or CaM complexes with skMLCK
were generated by positioning the modeled components relative to each
other and calculating the expected scattering profiles using PRPDB (see
"Materials and Methods"). Two approaches were used. The first was
to randomly orient and position the components with respect to each
other while loosely constraining the distance between their centers of
mass to be no more than 60 Å (22,400 models tested). Using this
approach, ideal
2 values (
2 = 1.00) were
obtained for both complexes. In the second approach, the two components
were placed in the same arbitrary relative positions, and the
CaM·MLCK-I component was systematically moved through every position
on a 5 Å grid extending ± 50 Å in each of the x, y, and z
directions (9,261 possible x, y, z coordinates tested). The best-fit
model identified from the 5 Å grid search was further refined by
performing a finer grid search (1Å resolution, ±5 Å in each
direction, 1,331 possible x, y, z coordinates tested). In a final
refinement step, the two components were randomly rotated about their
origins in all directions (125,000 different orientations tested).
Using this approach we also were able to obtain
2 values
of 1.00 for both the CaM·skMLCK and
NCaM·skMLCK complexes. The
relative positions of skMLCK and CaM based on the random search and the
systematic grid search were equivalent, giving RMSD main chain
deviations between the differently derived models of 0.22 and 0.26 Å for the CaM·skMLCK and
NCaM·skMLCK complexes, respectively. Fig.
2 shows the backbone traces of the best-fit higher resolution model for
each complex, overlaid with its corresponding best-fit ellipsoid model.
For both complexes, there is excellent agreement in the positions of
each component determined using the two modeling approaches. Similar to
our previous analysis based on neutron scattering data, the higher
resolution model for the skMLCK component does not completely fill the
ellipsoid dimensions calculated using the x-ray scattering data, which
is to be expected given the N- and C-terminal sequence segments omitted
from the homology model.
NCaM (see Fig.
2). To evaluate the sensitivity of the scattering data to the relative
orientations of the CaM and kinase components, we performed additional
calculations in which they were rotated about the three perpendicular
axes defined by their associated best-fit ellipsoids. The rotations
allowed us to test different orientations of the kinase and CaM
structures while leaving the ellipsoid envelopes unperturbed. At each
rotation angle (sampled in 30° steps) a translation search of ± 5 Å in the x, y, and z directions with a 1 Å step size was performed
to optimize the translation for each rotation angle. The best-fit
obtained after this translational search was considered the best one
for that rotation angle. Fig. 3 presents
a plot of the best
2 values obtained for the calculated
scattering profiles and the experimental data at each rotation angle.
All rotations of the kinase or CaM components away from their positions
in the starting best-fit models result in significantly poorer fits for
both complexes. The sensitivity to rotation angle is greatest for the
kinase component, which shows the narrowest and deepest minimum with
respect to this parameter, presumably because of its more irregular
shape. In contrast, fits for the CaM and
NCaM structures go through shallower and broader minima in
2 as a function of
rotation angle. In the best-fit models for both complexes, the
CaM-binding peptide of MLCK is positioned with its N terminus pointing
toward the kinase, which allows it to be connected with this sequence.
In addition, the N-terminal helix of CaM in the native complex is in
contact with the kinase, as was observed for the model derived from the
neutron contrast variation data (8). The I(Q) functions
derived from the best-fit models for each complex are compared with the
experimental I(Q) and P(r) data in Fig. 1,
B and D, respectively.
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Fig. 3.
Plots of
2 values calculated for the
experimental and model I(Q) profiles as a function of
the rotation angle for skMLCK or CaM components away from the
orientations obtained in the best-fit high resolution models.
A,
2 values calculated for models obtained by
rotating the kinase structure away from the orientation obtained for
the final best-fit model for the CaM·skMLCK complex. Rotations are
about the orthogonal axes; x (
), y (
), and z (
). B,
as in A but for rotations of CaM. C-D, as in
A-B but for rotation of the skMLCK and
NCaM components
of the
NCaM·skMLCK complex. The principal axes for the rotations
are defined using a right-handed system, assigning the y
axis to the longest dimension for each component.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
NCaM bind skMLCK with equal
affinities only the native protein is capable of activating the kinase
catalytic function (16). The inability of
NCaM to activate the
kinase would appear to be due at least in part to its blocking of the catalytic cleft. In this position, it may also fail to disrupt autoinhibitory interactions with the catalytic core. In contrast, a
translocation of bound native CaM exposes the catalytic cleft and
likely also disrupts interactions between the catalytic core and the
autoinhibitory sequence, thus allowing catalysis to proceed. The
precise role of the N-terminal leader sequence in facilitating translocation of bound CaM is not fully resolved. The simplest interpretation is that CaM residues 2-8 facilitate displacement of an
autoinhibitory region flanking the core CaM-binding sequence in the
kinase, allowing translocation of bound CaM to a position at the end of
the C-terminal lobe of the enzyme, thereby allowing substrate binding
to the exposed catalytic cleft.
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FOOTNOTES |
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* This work was performed under the auspices of the Department of Energy (DOE) under contract to the University of California and was supported by DOE/Office of Biological and Environmental Research Project KP1101010 (to J. T.) and by National Institutes of Health Grants GM40528 (to J. T.), HL26043 (to J. T. S.), and DK44322 (to A. P.).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.
§ Present address: Chemistry Dept., University of North Carolina at Charlotte, Charlotte, NC 28223.
** To whom correspondence should be addressed: Bioscience Division, Mail Stop M-888, Los Alamos National Laboratory, Los Alamos, NM 87545. Tel.: 505-6672690; Fax: 505-6672891; E-mail: jtrewhella@lanl.gov.
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.C000857200
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ABBREVIATIONS |
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The abbreviations used are:
CaM, calmodulin;
cPKA, catalytic subunit of the cAMP-dependent
protein kinase;
MLCK, myosin light chain kinase;
NCaM, deletion
mutant of calmodulin missing residues 2-8;
skMLCK, skeletal muscle
myosin light chain kinase;
MOPS, 4-morpholinepropanesulfonic
acid;
AMPPNP, adenosine 5'-(
,
-imino)triphosphate.
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