From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada
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ABSTRACT |
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Binding of calcium to calmodulin (CaM) causes a
conformational change in this ubiquitous calcium regulatory protein
that allows the activation of many target proteins. Met residues make
up a large portion of its hydrophobic target binding surfaces. In this work, we have studied the surface exposure of the Met residues in the
apo- and calcium-bound states of CaM in solution. Complexes of
calcium-CaM with synthetic peptides derived from the CaM-binding domains of myosin light chain kinase, constitutive nitric-oxide synthase, and CaM-dependent protein kinase I were also studied. The surface exposure was measured by NMR by studying the effects of the
soluble nitroxide spin label,
4-hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy, on the line widths
and relaxation rates of the Met methyl resonances in
samples of biosynthetically 13C-methyl-Met-labeled
CaM. The Met residues move from an almost completely buried state in
apo-CaM to an essentially fully exposed state in
Ca2+4-CaM. Binding of two Ca2+ to
the C-terminal lobe of CaM causes full exposure of the C-terminal Met
residues and a partial exposure of the N-terminal Met side chains.
Binding of the three target peptides blocks the access of the nitroxide
surface probe to nearly all Met residues, although the mode of binding
is distinct for the three peptides studied. These data show that
calcium binding to CaM controls the surface exposure of the Met
residues, thereby providing the switch for target protein binding.
Calmodulin (CaM)1 (1) is
a ubiquitous calcium regulatory protein that is found in all eukaryotic
cells. This small acidic protein (16.7 kDa) can regulate at least 30 different target proteins in a calcium-dependent manner
(1-6). Its target proteins include multiple components in muscle
contraction such as myosin light chain kinases (MLCK), various protein
kinases, protein phosphatase 2B (calcineurin), and nitric-oxide
synthases. CaM responds to an increase in the intracellular
Ca2+ concentration from 10 Another prominent feature that contributes to CaM's flexibility are
the two Met-rich hydrophobic surfaces on the N- and C-lobes of CaM,
which become exposed upon calcium binding to CaM. The majority of the
hydrophobic residues that make up these surface areas are buried inside
the protein in apo-CaM (14, 15). In particular, all Met side chains
involved in target binding are buried in apo-CaM, with the possible
exceptions of Met124 and Met144, which appear
to be partially exposed in the solution structure determined by NMR
(15). Because the interactions between
Ca2+4-CaM and CaM-binding domains in the target
proteins are mainly through Van der Waal's interactions of the side
chains, the importance of the two hydrophobic patches in
Ca2+4-CaM for target protein binding is obvious
(11-13). The Met residues contribute 46% to the solvent-accessible
surface area of these two exposed hydrophobic surfaces in calcium-CaM
(16). In addition, almost all of the Met side chains in
Ca2+4-CaM appear to be involved in binding to
the CaM-binding domain peptides from MLCK, CaM kinase II, caldesmon,
constitutive nitroxide synthase (cNOS), cyclic nucleotide
phosphodiesterase, glycoprotein 41 of simian immunodeficiency virus,
CaM-dependent protein kinase I (CaMKI), and plant glutamate
decarboxylase (2, 11-13, 17-22). Site-directed mutagenesis of the Met
residues of CaM further illustrates the importance of their side chains
in the binding and activation of various target proteins (20, 23-25).
Compared with other aliphatic hydrophobic amino acid side chains, the
Met side chains possess a unique intrinsic flexibility and
polarizability due to the presence of the sulfur atom as noted by
Gellman (26). The flexible and malleable surfaces in
Ca2+4-CaM created by the Met side chains
provide a highly adjustable interaction area that can accommodate the
binding of a wide range of target proteins (1, 2, 4).
4-Hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPOL; also called
HyTEMPO) is a nonperturbing nitroxide-soluble spin label that can be
used for mapping surface-exposed hydrophobic side chains in proteins.
It has been used for the characterization of different protein folding
intermediates, identification of the critical residues involved in
ligand-binding, and simplification of 1H NMR spectra for
assignment purposes (Refs. 27-33; for a review, see Ref. 34). The idea
behind the TEMPOL method is that NMR resonances for residues that are
(or become) exposed on the surface of proteins will be dramatically
broadened by the paramagnetic nitroxide in the surrounding medium (34).
In contrast, the relaxation rate of buried residues will not be
influenced by this probe. As such, the relative surface exposure of
various groups in a protein can be gauged. The method works well for
studying hydrophobic areas on protein surfaces (27, 34). The
combination of high sensitivity proton-detected 1H,
13C heteronuclear multiple quantum coherence (HMQC) NMR
spectra and the availability of the residue-specific assignments for
the nine Met methyl groups in a number of physiologically relevant states of CaM provided us with a unique opportunity to investigate the
Met methyl group exposure in solution for different states of CaM. Here
we have studied apo-CaM, Ca2+2-CaM,
Ca2+4-CaM, the
Ca2+4-CaM-MLCK peptide complex, and complexes
of Ca2+4-CaM with cNOS and CaMKI CaM-binding
peptides. In addition, we have also studied the Met methyl groups in
the separated N- and C-lobes of CaM (TR1C and
TR2C) in the presence and absence of two Ca2+
ions using the same experimental approach. Our results reveal that
changes in the surface exposure of the Met side chains play a key role
in allowing CaM to act as a versatile calcium regulatory protein.
Materials--
TEMPOL was purchased from Sigma. A synthetic
mammalian CaM gene was expressed in Escherichia coli (35,
36). The purification of wild-type CaM and
[methyl-13C]Met-labeled CaM was accomplished
as described before (17, 36). The N-lobe of CaM, TR1C
(residues 1-75), was cloned using the DNA polymerase chain reaction;
the expression and purification of TR1C followed the same
protocol as for wild-type CaM (17, 36). The C-lobe of CaM,
TR2C (residues 78-148), was generated by limited tryptic
digestion of intact CaM in the presence of saturating levels of calcium
(37). Although the purified TR2C fragment may contain three
different fragments of slightly different length (38), the vast
majority was the fragment 78-148, as determined by electrospray mass
spectrometry (data not shown). Two-dimensional 1H,
13C HMQC NMR spectra of the
[methyl-13C]Met-labeled TR2C
fragment (see below) showed only about 20% intensity for the
Met76 resonance compared with the other Met resonances (see
Fig. 4C), in agreement with the outcome of the protein mass
spectrometry. The absence of trace calcium ions in apo-form
preparations of CaM and fragments after Chelex 100 chromatography was
confirmed by one- and two-dimensional NMR spectroscopy. The MLCK
peptide, cNOS peptide, and CaMKI peptide were commercially synthesized by the Core Facility for Protein/DNA Chemistry at Queen's University (Kingston, Ontario, Canada). The preparation of CaM-peptide complexes for NMR analysis has been described before (19, 39). The concentration of CaM was calculated using an extinction coefficient of
EPR--
EPR spectra were acquired at ambient temperature
(22 °C) using a Bruker ER-200D-SRC spectrometer located at the
Department of Chemistry, University of Alberta (Edmonton, Canada). The
parameters used were as follows: modulation amplitude, 1 G; microwave
power, 18 dB; microwave frequency, 9.770 GHz; time constant, 0.2 s. EPR spectra were acquired for 0.08 mM TEMPOL in the
absence or presence of NMR Spectroscopy--
All NMR samples contained 1 mM
[methyl-13C]Met isotopically labeled protein
in 100 mM KCl, 99.9% D2O. The pD was
determined as 7.50 ± 0.05 using a glass electrode; no corrections
for the isotope effects were made. All NMR spectra were acquired on a Bruker AMX-500 spectrometer at 298 K. One-dimensional 1H
NMR spectra were obtained with a sweep width of 6024 Hz and 128 scans.
A 1-Hz line broadening was applied before the Fourier transformation. A
concentrated stock solution of TEMPOL in D2O was prepared,
and 1-µl amounts of the spin label were titrated into the protein
sample. Two-dimensional 1H, 13C HMQC spectra
were acquired as described before (39). Quadrature detection in the F1
dimension was obtained using the time-proportional phase increment
method. The sweep width was 6 ppm in the 1H and
13C dimensions, with the 1H carrier set at
500.1383 MHz and the 13C carrier at 125.7613 MHz. The size
of the HMQC spectra gave a 1024 × 128 real data matrix with eight
scans for each experiment. Spin-lattice relaxation
(T1) measurements were also performed as
described before (39), with a 1024 × 128 real data matrix and 32 scans for each experiment. The different relaxation delays used in the
T1 experiments were 10, 50, 150, 300, 600, 900, 1500, and 2500 ms. NMR spectra were processed on a Bruker X32 data
station using Bruker UXNMR software. Two-dimensional NMR spectra were zero-filled to 1024 × 256, and a 72° sine square window
function was applied before Fourier transformation.
T1 spectra were integrated using an integration
subroutine in UXNMR, and the integration data were fitted to a single
exponential decay curve to give the T1 values
(39). All 1H chemical shifts are referenced to HDO as 4.78 ppm, which is further referenced to DSS (2, 2-dimethyl-2-silapentane-5-sulfonate) as 0 ppm. All 13C
chemical shifts are indirectly referenced to DSS using a converting ratio 13C/1H = 0.251449530 as suggested by
Wishart et al. (40).
EPR Studies of Potential CaM-TEMPOL Interactions--
We first
acquired EPR spectra of TEMPOL in the absence and presence of CaM to
assess whether any specific interactions between TEMPOL and CaM
existed. Such interactions have been reported for other proteins
(e.g. Ref. 31), and they complicate the interpretation of
subsequent NMR experiments, where TEMPOL is used as a general relaxation enhancement reagent. The formation of complexes between the
TEMPOL nitroxide spin label and the protein should lead to a broadening
of the EPR nitroxide signal, accompanied by a commensurate decrease in
the signal intensities (31). As can be seen in Fig. 1, no changes were detected in the EPR
spectra of TEMPOL in the absence or presence of 6 eq of
Ca2+4-CaM. We also did not observe any changes
in the EPR spectra recorded with
TEMPOL/Ca2+4-CaM at a ratio of 6, TEMPOL/apo-CaM at a ratio of Ca2+4-CaM,
Ca2+2-TR1C, and
Ca2+2-TR2C--
Three regions of
the one-dimensional 1H NMR spectra of
Ca2+4-CaM, which were recorded in the absence
and presence of 6 eq of TEMPOL, are shown in Fig.
2A. Several resonances for
aromatic side chains were significantly broadened upon the addition of TEMPOL. These observations are in accordance with the x-ray structure of Ca2+4-CaM, in which four Phe residues are
found in the exposed hydrophobic patches (7, 8). The broadened
upfield-shifted methyl proton resonances are also consistent with this
notion. Val35, Val108, Ile27, and
Ile100 are all part of the two exposed hydrophobic surface
areas in the x-ray structure of Ca2+4-CaM (7,
8). In contrast, the downfield-shifted
Relaxation enhancement effects caused by TEMPOL on the resonances in
NMR spectra can be assessed quantitatively by measuring the enhancement
of relaxation rates in the presence of TEMPOL (28, 30, 32, 34). Because
all resonances (except for Met76) in
1H, 13C HMQC spectra of
[methyl-13C]Met-labeled
Ca2+4-CaM disappeared in the presence of 1 eq
of TEMPOL (data not shown), we decided to use only 0.4 eq of TEMPOL to
study the relaxation effects in Ca2+4-CaM.
Severe line broadening of the resonances of all of the Met methyl
groups with the exception of Met76 can be seen in Fig.
3A. The four N-terminal Met
residues (Met36, Met51, Met71, and
Met72) and the four C-terminal Met residues
(Met109, Met124, Met144, and
Met145) showed an average 53 and 66% increase,
respectively, in their 13C spin-lattice
relaxation rates (R1) (Table
I); this result is in agreement with the
qualitative picture obtained by looking at the line broadening (Fig.
3A). As a control, we also measured the increase of the
R1 relaxation rate for the isolated
[methyl-13C]Met amino acid, and in the
presence of 0.4 eq of TEMPOL the ratio of R1
(0.4 eq)/R1 (0 eq) is 2.07. Thus, the results
obtained with the protein indicate that the Met methyl groups are
almost fully exposed on the surface of
Ca2+4-CaM with the exception of
Met76 (see below).
We also studied the individual isolated lobes of
Ca2+4-CaM using the TEMPOL broadening
experiment. Fig. 3, B and C, shows the
1H, 13C HMQC spectra obtained for
Ca2+2-TR1C and
Ca2+2-TR2C, respectively, in the
absence and presence of 0.4 eq of TEMPOL. By utilizing the similarities
between the NMR spectra of these two protein fragments and intact CaM
in the Ca2+ form as well as the apo-form, the assignment
for the Met methyl resonances could be readily made (Figs. 3 and
4). Table I includes the results obtained
for the R1 relaxation rate measurements for Ca2+2-TR1C and
Ca2+2-TR2C. Clearly, we obtained a
very similar result for the two isolated fragments
Ca2+2-TR1C and
Ca2+2-TR2C, when compared with
intact Ca2+4-CaM (Fig. 3, Table I). These data
imply that Ca2+2-TR1C and
Ca2+2-TR2C have a very similar
exposure for the Met methyl groups as in
Ca2+4-CaM. The broadening effect is slightly
less dramatic, but this possibly reflects the decrease of the
correlation time of TR1C and TR2C compared with
intact CaM. A higher TEMPOL concentration is generally needed to
achieve similar broadening effects for small molecules (34). Be that as
it may, the 13C R1 measurements definitively
show a comparable increase in the spin-lattice relaxation rates in the
presence of 0.4 eq of TEMPOL (Table I). These results suggest that Met
side chains are almost fully exposed on the protein surface in
Ca2+2-TR1C and
Ca2+2-TR2C.
Apo-CaM, Apo-TR1C, and Apo-TR2C--
Next,
we acquired the one-dimensional 1H NMR spectra of apo-CaM
in the absence and presence of 6 eq of TEMPOL (Fig. 2B). In contrast to the results obtained with Ca2+4-CaM
(Fig. 2A), we observed almost no line broadening effects in
the aromatic region and upfield-shifted methyl group region of the
proton NMR spectra upon the addition of TEMPOL (Fig. 2B). This is consistent with the burial of the majority of these side chains
inside the protein as seen in the apo-CaM structure (14, 15).
Because of the lower response to TEMPOL in the NMR spectra of apo-CaM,
two-dimensional 1H, 13C HMQC
spectra of apo-CaM were acquired in the absence and presence of 4 eq of
TEMPOL rather than the 0.4 eq we had used for the
Ca2+-saturated protein. Under these conditions, we observed
a relative lack of broadening for the Met methyl groups in the N-lobe
of apo-CaM. These appear to be completely buried inside the protein (Fig. 4A). This notion is confirmed by the
R1 relaxation rate enhancement data (Table
II). Interestingly, the Met methyl groups in the C-lobe of apo-CaM seem more exposed than those in the N-lobe, as
evidenced by the broader resonances for the C-lobe of apo-CaM (Fig.
4A), as well as a more significant relaxation rate
enhancement (average 69% compared with 3%, Table II). This may be
related to a semi-open conformation adopted by the C-lobe of apo-CaM, as suggested by a computer modeling study (42). It should be noted that
because of the different amounts of TEMPOL added, the quantitative
results presented in Tables I and II cannot be compared as a direct
measure of surface exposure. However, if a 10-fold increase in the
TEMPOL concentration is required to achieve the same relaxation rate
enhancement, the residues are obviously less exposed (34).
Apo-TR1C and apo-TR2C again showed similar
surface exposure properties for their Met methyl groups compared with
their counterparts in intact apo-CaM (Fig. 4 and Table II). Similarly,
we find that the Met methyl groups are fully buried in
apo-TR1C and only partially buried in apo-TR2C.
The spin-lattice relaxation rate enhancement in apo-TR2C on
average is 73% compared with 69% in apo-CaM. These data suggest that
the apo-TR2C alone may also adopt a semi-open conformation
as suggested for the C-lobe in apo-CaM (42).
Ca2+2-CaM--
In order to obtain
information about the Met side chain exposure in the N- and C-lobes of
half-saturated CaM, experiments were performed with only 2 eq of
Ca2+ added to intact apo-CaM. Since there is approximately
a 10-fold difference in the calcium-binding constants of the C- and
N-terminal domains, only the two sites in the C-lobe of CaM will be
fully occupied under these conditions (38). When two Ca2+
ions are added to apo-CaM, the resonances for the methyl groups of
Met109, Met124, Met144, and
Met145 undergo slow exchange on the 13C NMR
time scale, and a set of new resonances for the Ca2+ form
appeared (Fig. 5). However, also the
methyl resonances of N-terminal Met36, Met51,
Met71, and Met72 residues moved to new
positions (Fig. 5), although these effects were much less dramatic than
when the N-terminal half is directly binding Ca2+. The four
Met methyl groups in the C-lobe of Ca2+2-CaM
are all fully exposed; upon the addition of 0.4 eq of TEMPOL, similar
effects as in Ca2+4-CaM were observed for the
C-terminal lobe resonances (data not shown). The addition of 1 eq of
TEMPOL completely broadened these C-lobe resonances. The four Met
methyl groups in the N-lobe of Ca2+2-CaM become
partially exposed, as indicated by the observed relaxation enhancement
in the presence of 2 eq of TEMPOL (Table
III). The N-lobe of
Ca2+2-CaM may maintain a similar structure as
in apo-CaM. However, our data clearly show an increase in the
relaxation rates of the Met36, Met51,
Met71, and Met72 methyl groups. Perhaps a
change in dynamics takes place in the N-lobe upon the binding of two
Ca2+ ions to the C-lobe of apo-CaM; i.e. the
N-lobe of Ca2+2-CaM may be more flexible than
in apo-CaM or apo-TR1C. This change may be transmitted
through the central linker region, after the binding of two
Ca2+ to the C-lobe of apo-CaM (see below). We also observed
some heterogeneity in the Met76 resonance in the
half-saturated CaM (Fig. 5). Additional experiments were also performed
with an equimolar mixture of TR1C and TR2C in
the presence of 2 eq of Ca2+. In this case, the spectra
closely resembled those obtained for Ca2+2-CaM.
While Met resonances of TR2C were fully broadened upon the
addition of 1 eq of TEMPOL, the surface exposure of the Met residues in
TR1C did not change (the average ratio of
R1(2)/R1(0) was 1.12 (Table III), compared with 1.67 in Ca2+2-CaM
(see Table III). This result also makes the possibility unlikely that
the increased exposure in the N-lobe of
Ca2+2-CaM arises from transient binding of
Ca2+ to this
domain.2
Ca2+4-CaM-Target Peptide
Complexes--
When the TEMPOL relaxation enhancement approach was
applied to several Ca2+4-CaM-peptide complexes,
significant differences were observed when compared with
Ca2+4-CaM. The most striking result is that the
majority of the Met resonances are now buried. Almost all Met methyl
groups in the N-lobe and C-lobe of Ca2+4-CaM
have become inaccessible to TEMPOL (4 eq), as would be expected if they
are involved in the complex interface (see Fig.
6 and Table
IV). Interestingly, from the broadening
in the spectra and the R1 relaxation enhancement
data, we noted some small differences between the three complexes (Fig.
6 and Table IV). For example, the methyl groups of Met72
and Met145 are partially exposed in the
Ca2+4-CaM-MLCK peptide complex, with
Met72 being more exposed than Met145 (Table
IV). However, the MLCK peptide still partially covers the
Met72 methyl group, because we still can measure its
R1 rate in the presence of 2 eq of TEMPOL, while
the same resonance in Ca2+4-CaM is unobservable
after the addition of 1 eq of TEMPOL (Table IV). Different results were
obtained for the Ca2+4-CaM-cNOS peptide and the
Ca2+4-CaM-CaMKI peptide complexes. Upon cNOS
peptide binding, Met109 remains partially exposed, while
all other Met residues are fully buried. Only the Met72
methyl group is partially exposed, while all other Met methyl groups
are fully buried in the Ca2+4-CaM-CaMKI peptide
complex. Furthermore, we have found that all Met methyl groups become
completely buried in a complex between Ca2+4-CaM and peptides derived from a plant
glutamate decarboxylase (22).
In this work, we have studied the surface exposure of the Met
methyl groups of CaM in different physiologically relevant states by
using the soluble spin label TEMPOL. TEMPOL has no specific interactions with CaM and thus is suitable to probe the exposed hydrophobic surface in CaM (Fig. 1). In
Ca2+4-CaM, we found that all Met methyl groups
are fully exposed except Met76 (Fig. 3, Table I). This
observation is consistent with the x-ray structures of
Ca2+4-CaM, which indicate that two Met-rich
hydrophobic patches are exposed in Ca2+4-CaM
and ready for target protein binding (7, 8). The result obtained for
the Met76 residue is somewhat surprising, since the x-ray
structure shows that the central The Met methyl groups in the isolated lobes of
Ca2+4-CaM,
Ca2+2-TR1C, and
Ca2+2-TR2C were found to be fully
exposed in the presence of Ca2+ (Fig. 3, Table I). This
observation is consistent with several reports where it was shown that
Ca2+2-TR2C alone or combined with
Ca2+2-TR1C could bind to and
activate some CaM target enzymes (43-46). Interestingly, oxidation of
the Met residues in Ca2+2-TR2C
abolished its capacity to activate the erythrocyte
Ca2+-ATPase, illustrating the importance of exposed
hydrophobic Met side chains in the binding and subsequent activation
process (44).
In the absence of Ca2+, we found that the Met methyl groups
in apo-CaM are fully buried in the N-lobe and partially exposed in the
C-lobe (Fig. 4, Table II). There are no significant differences in the
surface exposure properties for the four Met residues in the C-lobe of
CaM (Met109, Met124, Met144, and
Met145; Table II). For the apo-CaM structure determined by
NMR, it was reported that Met124 and Met144 are
partially exposed, while Met109 and Met145 are
fully buried (15). This result seems to contradict our findings.
However, it is usually difficult to accurately define the side chains
of amino acid residues on the surface of a protein in an NMR structure
calculation (47). The flexibility of many amino acid side chains on a
protein surface leads to averaging of the nuclear Overhauser effects,
which can lead to errors in the structure calculation for such surface
residues (48). In contrast, residues in the core of a protein often
have a unique conformation, and hence they are much better defined in
an NMR structure calculation (47). The difference in the results could also be due to the existence of a semi-open conformation (42). It is
also possible that the conformational averaging processes that may
exist in the C-terminal lobe of apo-CaM contributes to these
measurements by allowing temporary access to the TEMPOL moieties (14,
49). We note, however, that there are almost no changes in the line
width of the methyl protons of Val91, Val105,
and Val108 of apo-CaM in the presence of 6 eq of TEMPOL in
one-dimensional 1H NMR spectra (Fig. 2B); this
suggests that several buried side chains in the C-lobe of apo-CaM are
not accessible to TEMPOL. Therefore, we feel that the most likely
explanation from TEMPOL broadening data is that the C-lobe of apo-CaM
adopts a semi-open conformation with all of the Met side chains
somewhat exposed. The partially exposed Met methyl groups in the
C-terminal lobe of apo-CaM may provide a hydrophobic surface for the
binding of some target proteins in the absence of calcium (42, 50).
When CaM is half-saturated with 2 Ca2+ ions bound to the
C-lobe of CaM, we observed an increased exposure of the Met methyl groups from the N-lobe of CaM (Fig. 5, Table III). These data suggest that information about Ca2+ binding to the C-lobe of CaM
may be transmitted to the N-lobe. Many studies have illustrated the
independent folding of each lobe of calcium-saturated CaM and apo-CaM.
Nevertheless, our results reveal a different conformational transition
involving both lobes of CaM. This phenomenon was also observed by a
fluorescence study using a fluorescent probe attached to
Cys26 in spinach CaM (51). Similarly, Mackall and Klee
reported that, upon the binding of two Ca2+ ions to
apo-CaM, the susceptibility of the central linker region to trypsin
proteolysis increases 10-fold, compared with that in either apo-CaM or
Ca2+4-CaM (52). Also the work by Starovasnik
et al. (53) provided evidence for interlobal communication
in Ca2+2-CaM. The recent work of Shea's group
as well as Bayley's group has provided further evidence for the
existence of an intermediate state in Ca2+2-CaM
(54-56). Although there is no high resolution x-ray or NMR structure
available for Ca2+2-CaM, some differences have
been reported for the N-lobe of skeletal muscle troponin C, a
homologous Ca2+-binding protein (57). Taken together, the
data obtained for Ca2+2-CaM suggest a
structural or dynamic difference between the N-lobe of apo-CaM and the
N-lobe of Ca2+2-CaM. Our data on the exposure
of the Met residues in the N-lobe of Ca2+2-CaM
indicate the presence of a partially exposed form, which is
intermediate between the apo- and calcium-saturated forms. Interestingly, this effect is only observed for
Ca2+2-CaM and not for an equimolar mixture of
two lobes, TR1C and TR2C, in the presence of 2 eq of Ca2+. This suggests that the central linker region of
CaM may play a role in transmitting information about structural
changes between the two domains of CaM.
Upon binding to its target peptides, almost all of the Met methyl
groups become fully buried in Ca2+4-CaM (Fig.
6, Table IV). Our results are consistent with the high resolution
structures of Ca2+4-CaM-peptide complexes
(11-13) as well as site-directed mutagenesis studies (24, 25, 58). The
results obtained with the MLCK peptide are also in accordance with
enzyme assays of the M124L CaM mutant, which is impaired in the
activation of the MLCK enzyme, while mutation of Met72 or
Met145 to a Leu residue had little effect (25, 58). It was
reported that in a series of Met to Gln mutants, only the M124Q mutant of CaM had a seriously reduced activation potential for CaMKI (24). Our
data for the different Ca2+4-CaM -peptide
complexes illustrate the importance of the Met side chains in the
binding interface of CaM with its target proteins. The differences
reported here for three different Ca2+4-CaM
-peptide complexes strongly suggest that the two Met-rich hydrophobic
surface in Ca2+4-CaM can adjust themselves to a
range of target proteins with CaM-binding domains of distinct amino
acid sequences. Thus, the unique properties of Met side chains are
crucial to CaM's versatility in binding and activating so many
different target proteins.
Recently, Howarth et al. (32) conducted a similar study of
the exposure of the Met methyl groups in cTnC, a homologous
calcium-binding regulatory protein. In contrast to CaM, troponin C
binds only one target protein, troponin I. Troponin C is very similar
to CaM both in terms of its amino acid sequence (51% sequence identity and 70% homology (59)) and three-dimensional structure. In addition, it is a Met-rich protein similar to CaM. However, the results obtained
by Howarth et al. (32) are quite different from our results
with CaM. Although these authors did not acquire data for apo-cTnC, the
data for calcium-saturated cTnC clearly showed that the C-lobe Met
methyl groups of cTnC are quite exposed, while in the N-lobe only two
Met residues are somewhat exposed. These results imply that the Met
side chains in cTnC do not become fully exposed upon binding
Ca2+, and hence they are less of a participant in the
exposed hydrophobic binding surface than in
Ca2+4-CaM. The rationale for these differences
was recently revealed by the NMR solution structure of
calcium-saturated cTnC, in which the N-lobe of cTnC is in a closed
conformation compared with the open conformation observed for the
C-lobe of cTnC (60, 61). In addition, in the studies by Howarth
et al. (32), binding of the target cardiac muscle troponin I
peptide to cTnC only protected two Met residues in the C-lobe of cTnC
and had no effect on the Met residues in its N-lobe. This result is in
stark contrast to the results reported here for CaM, where nearly all
Met side chains participate in the binding of the MLCK, cNOS, and CaMKI
peptides (Fig. 6 and Table IV). Clearly, the role of the Met residues
is more important in CaM than in cTnC; it remains to be determined whether the Met residues in skeletal muscle troponin C more closely resemble those obtained for cTnC than for CaM.
Overall, our study demonstrates that the calcium-dependent
control of the exposure of the Met methyl groups in CaM is a major factor in this protein's versatility. Recently, Beatty et
al. (62) showed that very good resolution and sensitivity could be
obtained in HMQC spectra of a protein with a molecular mass of 80 kDa
that was labeled with [methyl-13C]Met. Thus,
the TEMPOL broadening approach can probably be extended to higher
molecular weight complexes of CaM bound to an intact target protein.
Moreover, since Met residues can play an important role in the
protein-protein binding interface of other proteins (63), this approach
can be applied to other protein-peptide or protein-protein systems as well.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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7 to
10
6 M in vivo. CaM's capacity to
bind and activate a diverse range of target proteins originates in its
structure. The x-ray structure of Ca2+-saturated CaM
reveals a dumbbell-shaped molecule with each lobe containing two
"helix-loop-helix" calcium binding motifs, giving a total of four
Ca2+-binding sites (7, 8). The two lobes in
Ca2+4-CaM are connected by a 26-residue highly
exposed
-helix in the crystal structures (7, 8). In contrast,
structural and dynamic information derived by NMR spectroscopy as well
as molecular dynamics simulations indicates that the region between
amino acids 77 and 81 in Ca2+4-CaM is flexible
in solution (9, 10). This central linker region in
Ca2+4-CaM unravels further when a CaM-binding
peptide from a target protein such as MLCK is bound (11, 12). High
resolution x-ray and NMR structures of the complexes between
Ca2+4-CaM and the CaM-binding domain peptides
of MLCK and CaM kinase II clearly indicate the different relative
movement of the two lobes of CaM in the complexes as well as the
bending and unraveling of the central linker region (11-13). This
flexibility of the central linker region in
Ca2+4-CaM is one of the key reasons that allows
CaM to activate so many different systems.
EXPERIMENTAL PROCEDURES
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2761% = 1.8. The
concentrations of the TR1C and TR2C fragments
were determined by using molar extinction coefficients of
258 = 1073 M
1·cm
1 and
276 = 2666 M
1·cm
1, respectively,
which were confirmed by quantitative amino acid analysis.
, 1, or 6 eq of
Ca2+4-CaM. The solution contained 100 mM KCl, pH 7.50 ± 0.05, which was similar to the
conditions used for the NMR experiments except that H2O was
used as the solvent instead of D2O. EPR spectra of TEMPOL
were also obtained in the presence of apo-CaM and the
Ca2+4-CaM-MLCK peptide complex.
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DISCUSSION
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or 6, and the
TEMPOL/Ca2+4-CaM-MLCK-peptide complex at a
ratio of
or 6 (data not shown). Therefore, the outcome of
these EPR studies indicates that there are no specific interactions
between TEMPOL and CaM; consequently, the soluble spin label is
suitable to probe the exposed hydrophobic surfaces and the exposure of
Met methyl groups in CaM (29, 32). Furthermore, in our NMR studies of CaM in the presence of TEMPOL, we also did not observe any chemical shift changes for the Met methyl resonances, which is also consistent with the absence of specific interactions between the spin label and
the protein (see below). Other relaxation enhancement reagents, such as
lanthanide compounds, may give rise to specific interactions with
proteins and chemical shift changes in the protein NMR spectra (41).
Thus, choosing TEMPOL as a reagent to probe the exposed hydrophobic
surface in CaM seems appropriate.
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Fig. 1.
EPR spectra of 0.08 mM TEMPOL in
the absence and presence of 1 or 6 eq of
Ca2+4-CaM. There are no differences among
the spectra, indicating that there are no specific interactions between
TEMPOL and CaM.
-proton resonances between
5.0 and 5.5 ppm are well protected from TEMPOL; these resonances
correspond to the two buried
-sheets between the two
Ca2+-binding loops in each lobe of
Ca2+4-CaM in the x-ray structure (7, 8).
View larger version (18K):
[in a new window]
Fig. 2.
A, one-dimensional 1H NMR
spectra of Ca2+4-CaM in the absence and
presence of 6 eq of TEMPOL, showing the aromatic, downfield-shifted
-proton and upfield-shifted methyl proton resonances. Some of the
resonances are labeled. Note the broadening effects in the aromatic and
methyl proton regions of the spectra. The peak labeled with an
X is an impurity. B, one-dimensional
1H NMR spectra of apo-CaM in the absence and presence of 6 eq of TEMPOL, showing the aromatic, downfield-shifted
-proton and
upfield-shifted methyl proton resonances. Some of the resonances are
labeled. By comparing with Fig. 2A, it is seen that the
broadening effects in the aromatic and methyl proton regions are less
pronounced. One-letter amino acid codes are used.
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Fig. 3.
Two-dimensional 1H,
13C HMQC NMR spectra of Ca2+4-CaM,
Ca2+2-TR1C and
Ca2+2-TR2C in the absence and
presence of 0.4 eq of TEMPOL. A,
Ca2+4-CaM. The assignments were taken from
Siivari et al. (39). Note the significant broadening effects
for all Met methyl resonances except Met76. B
and C, Ca2+2-TR1C and
Ca2+2-TR2C, respectively. The
assignments for Ca2+2-TR1C and
Ca2+2-TR2C were obtained by
comparison with the assignments for Ca2+4-CaM
in A. Again, significant broadening effects are observed in
the two isolated lobes of the bilobal CaM.
Carbon-13 spin-lattice relaxation rate R1 (s1) for
the Met methyl groups in Ca2+4-CaM,
Ca2+2-TR1C, and
Ca2+2-TR2C in the absence and presence of
0.4 eq of TEMPOL
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[in a new window]
Fig. 4.
Two-dimensional 1H,
13C HMQC NMR spectra of apo-CaM, apo-TR1C, and
apo-TR2C in the absence and presence of 4 eq of
TEMPOL. The concentration of TEMPOL used is 10-fold higher than
that used in Fig. 3. A, Apo-CaM. The assignments were taken
from Siivari et al. (39). Selective broadening effects for
the Met methyl resonances from the C-lobe of apo-CaM can be seen. The
Met145 resonance can only be observed at lower contour
level. B and C, apo-TR1C and
apo-TR2C, respectively. The assignments were obtained by
comparing with the assignments of apo-CaM in A. Note that no
broadening effects are observed for apo-TR1C, while they do
occur for apo-TR2C.
Carbon-13 spin-lattice relaxation rate R1 (s1) for
the Met methyl groups in apo-CaM, apo-TR1C, and
apo-TR2C in the absence and presence of 4 eq of TEMPOL
View larger version (12K):
[in a new window]
Fig. 5.
Two-dimensional 1H,
13C HMQC NMR spectra of Ca2+2-CaM
in the absence and presence of 2 eq of TEMPOL. The four Met
resonances from the C-lobe of CaM have identical chemical shift values
compared with Ca2+4-CaM. The four Met
resonances from the N-lobe of CaM have similar (but not identical)
chemical shift values as in apo-CaM. The broadening effects are obvious
for all the Met resonances (except for Met76 in the linker
region). Compare with Fig. 4, A and B.
Carbon-13 spin-lattice relaxation rate R1 (s1) for
the Met methyl groups in apo-CaM, Ca2+2-CaM, and
Ca2+2-TR1C/TR2C in the absence and
presence of 4, 2, and 2 eq of TEMPOL, respectively
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Fig. 6.
Two-dimensional 1H,
13C HMQC NMR spectra of
Ca2+4-CaM-MLCK,
Ca2+4-CaM-cNOS, and
Ca2+4-CaM-CaMKI peptide complexes in the
absence and presence of 4 eq of TEMPOL. A,
Ca2+4-CaM-MLCK peptide complex. The assignments
are taken from Siivari et al. (39). Note the selective
broadening effects on Met72 and Met145. The
Met72 resonance is not observable in the presence of 4 eq
of TEMPOL. However, it is present in the spectra if only 2 eq of TEMPOL
are added (data not shown). B,
Ca2+4-CaM-cNOS peptide complex. The assignments
are taken from Zhang et al. (19). Note the selective
broadening effects for the Met109 resonance. C,
Ca2+4-CaM-CaMKI peptide complex. The
assignments are taken from Yuan and Vogel (unpublished results). Note
the selective broadening effects of the Met72
resonance.
Carbon-13 spin-lattice relaxation rate R1 (s1) for
the Met methyl groups in Ca2+4-CaM-MLCK,
Ca2+4-CaM-cNOS, and Ca2+4-CaM-CaMKI
peptide complexes in the absence and presence of 4 eq of TEMPOL
DISCUSSION
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DISCUSSION
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-helix region is fully exposed (7,
8). Moreover, NMR structural and dynamic measurements have shown that the region between Lys77 and Ser81 is flexible
(9). The absence of significant broadening of the Met76
resonance upon the addition of spin label is probably related to the
fact that TEMPOL is more suitable for probing the hydrophobic surfaces
rather than the charged surfaces in proteins, as noted by Petros
et al. (27). Indeed, there are many charged residues around
Met76 in the amino acid sequence of CaM
(74RKMKDTD80) (7, 8), and these would limit the
approach of the polar uncharged TEMPOL molecule.
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ACKNOWLEDGEMENTS |
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We thank Dr. D. D. McIntyre and G. Bigam for help with the acquisition of the EPR spectra.
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FOOTNOTES |
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* This work was supported by an operating grant from the Medical Research Council of Canada (MRC). The NMR spectrometer used in this study has been funded and maintained with funds provided by MRC and the Alberta Heritage Foundation for Medical Research (AHFMR).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.
Recipient of an AHFMR studentship.
§ An AHFMR Scientist. To whom correspondence should be addressed: Dept. of Biological Sciences, University of Calgary, 2500 University Drive N.W., Calgary, Alberta T2N 1N4, Canada. Tel.: 403-220-6006; Fax: 403-289-9311; E-mail: vogel{at}ucalgary.ca.
2 There are alternative explanations for the observation of the changes in exposure in the N-terminal lobe, upon binding of calcium to the C-terminal lobe of CaM. For example, the addition of more than 2.0 eq of Ca2+ could lead to partial exposure of the N-terminal Met residues. Care was taken to avoid this in our experiments. In addition, when 1.2 eq of Ca2+ were added to apo-CaM, slight changes in the chemical shifts of some of the N-terminal Met resonances could already be seen in the HMQC spectra (data not shown). These subtle changes were missed in original proton NMR studies concerning Ca2+ binding to CaM (38, 64), since the shift change is primarily in the 13C dimension of the HMQC spectra. Interestingly, these changes were largest for residues Met71 and Met72, which are part of the central linker of CaM. It should also be realized that with a approximate 10-fold difference in dissociation constants for calcium binding to the two lobes of CaM, transient binding of calcium to the two N-terminal sites can never be completely excluded. Nevertheless, our suggestion that information about Ca2+ binding to the C-lobe may be transmitted to the N-lobe in Ca2+2-CaM, is in agreement with a number of recent studies.
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ABBREVIATIONS |
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The abbreviations used are: CaM, calmodulin; CaMKI, calmodulin-dependent protein kinase I; cNOS, constitutive nitric-oxide synthase; cTnC, cardiac muscle troponin C; HMQC, heteronuclear multiple quantum coherence; MLCK, myosin light chain kinase(s); R1, spin-lattice relaxation rate; T1, spin-lattice relaxation time; TEMPOL, 4- hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy.
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