(Received for publication, April 4, 1995; and in revised form, June 20, 1995)
From the
The intercellular messenger nitric oxide is produced through the
action of nitric oxide synthases, a class of enzymes that is regulated
by calcium-calmodulin (CaM). In this work, the interaction of CaM with
a 23-amino-acid residue synthetic peptide, encompassing the CaM-binding
domain of constitutive rat cerebellar nitric oxide synthase (cNOS), was
investigated by various NMR methods. Cadmium-113 NMR studies showed
that binding of the cNOS peptide increased the affinity of CaM for
metal ions and induced interdomain cooperativity in metal ion binding
as earlier observed for complexes of CaM with myosin light chain kinase
(MLCK) peptides. By using specific isotopically labeled
[C]methyl-Met and selenomethionine-substituted
CaM in two-dimensional proton-detected
C and
Se NMR studies, we obtained evidence for the involvement
of the Met residues of CaM in the binding of the cNOS peptide. These
residues form two hydrophobic surface areas on CaM, and they are also
involved in the binding of other target proteins. A nitroxide
spin-labeled version of the cNOS peptide caused broadening only for NMR
resonances in the N-terminal half of CaM, showing that the peptide
binds with a C to N orientation to the N- and C-terminal domains of
CaM. pH titration experiments of CaM dimethylated with
[
C]formaldehyde show that Lys-75 (and Lys-148)
experience a large increase in pK
upon
peptide binding; this indicates an unraveling of part of the helical
linker region of CaM upon cNOS peptide binding. Taken together, our
data show that the cNOS and MLCK peptides bind in a closely analogous
fashion to CaM.
The ubiquitous Ca-binding protein, calmodulin
(CaM), (
)is a small acidic protein of 148 amino acid
residues. The protein can regulate many different cellular events by
interacting with almost 30 different target enzymes in a
Ca
-dependent manner (for a review, see Means et
al., 1991; Vogel, 1994). The CaM-binding domains of the CaM
targets are usually contained in a continuous stretch of about 20 amino
acid residues. A large number of CaM-binding domains of various CaM
targets have a tendency to form basic, amphiphilic
-helices,
otherwise these peptides display very low amino acid sequence homology
(O'Neil and DeGrado, 1990; James et al., 1995; Vogel and
Zhang, 1995). The binding of two Ca
ions to each
domain of CaM induces significant conformational changes and exposes
two Met-rich hydrophobic surfaces (Hiraoki and Vogel, 1987; Babu et
al., 1988). The interaction of CaM with peptides encompassing the
CaM-binding domains of various target enzymes occurs mainly via van der
Waals interactions between these two hydrophobic surfaces and the
hydrophobic face of the CaM-binding domain of its targets (Ikura et
al., 1992; Meador et al., 1992, 1993; Zhang and Vogel,
1994b). The Met residues in the hydrophobic surfaces play important
roles by allowing CaM to bind to many targets in a sequence-independent
manner (O'Neil and DeGrado, 1990; Vogel et al., 1990;
Gellman, 1991; Vogel, 1994; Zhang and Vogel, 1994c; Zhang et
al., 1994a). The crystal structure of calcium-CaM reveals a
dumbbell-shaped molecule, where the two globular domains of the protein
are connected by a long, solvent-exposed
-helix (Babu et
al., 1988). This central linker region is very flexible in
solution (Ikura et al., 1991; Spera et al., 1991;
Barbato et al., 1992), and its plasticity is one of the main
reasons why CaM can interact effectively with such a wide spectrum of
target proteins (Ikura et al., 1992; Meador et al.,
1992, 1993; Zhang and Vogel, 1994b). In complexes of CaM with its
binding domains of MLCKs (Ikura et al., 1992; Roth et
al., 1992; Meador et al., 1992) and CaM-dependent kinase
II (Meador et al., 1993), the two globular domains of the
protein essentially retain the same conformation, but in all cases the
central helix of CaM unravels in a specific manner, allowing CaM to
accommodate
-helical amphiphilic CaM-binding domains of different
lengths.
Nitric oxide is a small gaseous messenger molecule which
can regulate diverse physiological processes, such as smooth muscle
relaxation, platelet inhibition, neurotransmission, immune regulation,
and blood vessel dilation (for reviews, see Nathan, 1992; Feldman et al., 1993; Bredt and Snyder, 1994; Nathan and Xie, 1994).
The cellular level of nitric oxide is regulated by the action of nitric
oxide synthases (NOS). There are two distinct isoforms of NOS, a
constitutively expressed brain and endothelial NOS (Bredt and Snyder,
1990; Bredt et al., 1991; Pollack et al., 1991; Sessa et al., 1992) and a cytokine-induced isoform from macrophages
(Stuehr et al., 1991; Xie et al., 1992). The
constitutively expressed NOS (cNOS) are activated by CaM in a
Ca-dependent manner; the inducible NOS are not
further activated by exogenous Ca
-CaM, since they
contain CaM as an integral, tightly bound subunit (Nathan and Xie,
1994). Synthetic peptides encompassing the putative CaM-binding domain
of rat brain cNOS (Bredt et al., 1991) have been shown to bind
to CaM in a Ca
-dependent manner with a 1:1
stoichiometry and a K
of
2 nM (Vorherr et al., 1993; Zhang and Vogel, 1994a). From CD
and isotope-edited FTIR studies, it is known that the cNOS peptide
binds to CaM in an
-helical conformation (Zhang and Vogel, 1994a;
Zhang et al., 1994b). However, further details about the mode
of binding and the location of the cNOS peptide binding site on CaM are
lacking at present.
In the present work, we have used 1D and 2D H,
C,
Se, and
Cd
NMR spectroscopy to study the interaction of the cNOS peptide with CaM.
The involvement of the functionally important Met residues of CaM for
binding the cNOS peptide was studied by
H,
C
HMQC spectroscopy and
H,
Se HMBC spectroscopy.
From the pK
titration behavior of Lys-75,
we have obtained information about the unraveling of the central helix
of CaM upon peptide binding (Zhang and Vogel, 1993). Cadmium-113 NMR
spectra were acquired to study the effect of peptide binding on the
metal-ion binding sites (Vogel and Forsén, 1987;
Ikura et al., 1989). Finally, a paramagnetic nitroxide
spin-labeled cNOS peptide was prepared to map the orientation of the
cNOS peptide in its complex with CaM.
All chemicals (analytical grade) were obtained commercially.
The nitroxide spin labeling reagent
4-(2-bromoacetamido)-2,2,6,6-tetramethylpiperidine-N-oxyl was
purchased from Sigma. The 23-residue cNOS peptide
KRRAIGFKKLAEAVKFSAKLMGQ was obtained and handled as described
previously (Zhang and Vogel, 1994a). Preparations of wild-type CaM, CaM
mutants, isotopically labeled CH
-Met-CaM, and
natural abundance
Se-Met-incorporated CaM have been
described in detail in our earlier reports (Zhang and Vogel, 1993,
1994b, 1994c; Zhang et al., 1994a). Reductive methylation of
the Lys residues in CaM with
C-labeled formaldehyde was
performed according to published procedures; this provides a convenient
means of measuring individual pK
values
in CaM (Huque and Vogel, 1993; Zhang and Vogel, 1993). The
CaM
peptide complexes used in this study were prepared in
essentially the same manner as the complex of CaM with the CaM-binding
domain of MLCK (Zhang and Vogel, 1993).
Cadmium-113 NMR experiments were performed on a Bruker AM400
widebore NMR spectrometer, which was equipped with a 10-mm broadband
probe. All spectra were recorded at a frequency of 88.75 MHz.
Isotopically enriched CdO (94.8%) was purchased from MSD
Isotopes. A 100 mM stock solution of
Cd(ClO
)
(pH
1 in
D
O) was used for the preparation and titration of the
samples. All
Cd NMR samples were prepared in 25% (v/v)
D
O, 100 mM KCl, 20 mM Tris, pH 7.5
± 0.05. For the
Cd
-CaM sample titrated with
cNOS peptide, microliter quantities of a concentrated peptide solution
were added into the sample. The apo-CaM-cNOS peptide mixture was
prepared by gradually adding cNOS peptide to a diluted apo-CaM solution
until a 1:1 ratio was reached. The mixture was then lyophilized and
redissolved in 2.0 ml of buffer. The
Cd NMR acquisition
parameters are as follows: a 45° flip angle, acquisition time 0.14
s, relaxation delay 0.5 or 5 s, sweep width 30 kHz, 8 K data points,
and 70,000 scans. The temperature of all experiments was 298 K. The
total acquisition time for a 0.5 s delay experiment is 12 h, 40 min.
Each FID was zero-filled once and an exponential line broadening of 30
Hz was applied prior to Fourier transformation. All
Cd
spectra are referenced to external 100 mM
Cd
(ClO
)
in D
O.
Figure 1:
Cadmium-113 NMR spectra of
Cd-saturated CaM (0.59 mM protein
concentration, pH 7.5) titrated with cNOS peptide. These spectra were
acquired with a 0.5 s relaxation delay. The ratio of peptide to protein
is indicated in the figure.
Figure 2:
Cadmium-113 NMR spectra of
Cd-saturated CaM (0.53 mM, pH 7.5) and the
1:1 complex with MLCK peptide. These spectra were acquired with a 5-s
relaxation delay, so peak intensity can be compared directly. Note the
obvious line width narrowing in the spectra of the
complex.
We also performed
a titration of the apo-CaM-cNOS peptide mixture with Cd(ClO
)
. Under these conditions,
four
Cd NMR peaks increased simultaneously in intensity
until a 4:1 ratio of metal ions to protein was reached (Fig. 3).
The chemical shifts obtained in this experiment were consistent with
those in Table 1. The parallel increase of the four signals shows
that CaM possesses total positive cooperativity in metal ion binding in
the presence of the cNOS peptide. This is not evident in the absence of
the peptide, but it can be induced by the binding of MLCK peptide, as
well as other targets (Forsén et al.,
1986; Ikura et al., 1989; Vogel, 1994). The line widths of the
various
Cd resonances are listed in Table 2.
Figure 3:
Cadmium-113 NMR spectra of a 1:1 mixture
of apo-CaM and cNOS peptide titrated with Cd (0.82
mM protein concentration, pH 7.5). The relaxation delay used
was 0.5 s. The amount of Cd
added is indicated in the
figure.
Figure 4:
H,
C HMQC NMR
spectra of isotopically labeled
[
C]methyl-Met calmodulin in the
presence (A) and absence (B) of a saturating amount
of the cNOS peptide. Note the large chemical shift dispersion upon
peptide binding (99.9% D
O, 100 mM KCl, pD 7.5, 25
°C).
Figure 5:
H,
C HMQC NMR
spectra of
C-dimethylated wild type calmodulin (A) and the mutant K75R (B) and K148Q (C).
The procedure for isotopic dimethylation of CaM was described in detail
elsewhere (Huque and Vogel, 1993; Zhang and Vogel,
1993).
Figure 6:
pH
titration curves determined for the dimethylated Lys-75 resonance of
Ca-CaM (
) and
Ca
-CaM-cNOS
(
).
Figure 7:
H,
C HMQC NMR
spectra of selectively labeled
[
C]-methyl-Met calmodulin. A,
in the presence of the spin-labeled cNOS peptide; B, after
addition of a 10-fold excess of ascorbic acid. Note that the resonances
for the four C-terminal Met residues have not shifted or broadened
(compare with Fig. 4A). For further explanation see
text.
The synthetic cNOS peptide used in this study has earlier
been shown to contain the complete CaM-binding domain of rat brain cNOS
(Vorherr et al., 1993; Zhang and Vogel, 1994a). NMR studies of
the isolated cNOS peptide have also shown that it can adopt a nascent
-helical structure in aqueous solution that it forms a stable
-helix in aqueous trifluoroethanol (Zhang and Vogel, 1994; Vogel
and Zhang, 1995) and that the
-helix has a typical amphiphilic
structure as seen in many other CaM-binding peptides (O'Neil and
DeGrado, 1990; James et al., 1995). Using CD and
isotope-edited FTIR spectroscopy, we have demonstrated that the cNOS
peptide also adopts an
-helical structure in its complex with CaM
(Zhang et al., 1994b). Here, we have used
H,
C,
Se and
Cd NMR spectroscopic
techniques to further characterize the details of the interaction
between the cNOS peptide and CaM. It is clear from our NMR data that
the cNOS peptide binds to both domains of calcium-CaM in slow exchange
on the NMR time scale(s), since one observes distinct sets of
resonances for the complex and free CaM. These data also show that the
cNOS peptide forms a stable 1:1 complex with CaM, as the spectral
changes in the titrations are completed once the amount of the cNOS
peptide reaches this molar ratio.
Cadmium-113 NMR provides a useful
method for the study of various metalloproteins including a range of
calcium-binding proteins (for reviews see Vogel and
Forsén, 1987; Coleman, 1993). For example, the
binding of metal ions to CaM, its proteolytic fragments, and its
complex with drugs and model-target CaM-binding domain peptides have
been studied in this fashion (Andersson et al., 1983; Thulin et al., 1984; Forsén et al.,
1986; Linse et al., 1986; Ikura et al., 1989; Ohki et al., 1993). Here we have performed titration studies of the
binding of the cNOS peptide to CaM by Cd NMR. The four
Cd resonances in the CaM
cNOS peptide complexes
could be tentatively assigned to the four Ca
-binding
sites of CaM, by comparison to earlier studies (see Table 1).
When the cNOS apo-CaM mixture was titrated with cadmium, four
Cd signals simultaneously increased in intensity, which
showed that cooperativity in metal ion binding is induced between the N
and C domains of CaM in the presence of the peptide. This is likely
what will happen in vivo because CaM always exists in the
presence of CaM-binding proteins in the living cell (Vogel, 1994).
Integration of the
Cd NMR spectra indicated that the
Cd T
relaxation time in the complex is longer
than in
Cd
-CaM. Also the
T
relaxation time for the complex appears to be longer than
that in Cd
-CaM from the obvious line
width narrowing (
) in the complex (Table 2); however, it is important to realize that chemical
exchange processes, rather than T
relaxation generally play
a determining role for the line width of protein-bound Cd
ions (Kördel et al., 1992; Aramini et al., 1995). Because chemical shift anisotropy contributes
significantly to the
Cd relaxation of similar
protein-bound ions at 9.4 Tesla (Kördel et
al., 1992; Aramini et al., 1995), we can calculate the
T
(and line width) values for
Cd
CaM and the
Cd
CaM
MLCK or cNOS complexes,
assuming correlation times of 8.5 and 10 ns, respectively, at 25 °C
and using a chemical shift anisotropy value
= 91
parts/million, as determined for the homologous protein calbindin
(Kördel et al., 1992). If we assume that
the
Cd relaxation can be attributed completely to
chemical shift anisotropy and dipole-dipole relaxation, we can
calculate line widths of 0.87 and 1.02 Hz for CaM and CaM
peptide
complexes, respectively. These values are obviously smaller than the
line widths measured in our NMR spectra, suggesting that some chemical
exchange processes are involved. Because the line widths of the free
Cd
signals did not change significantly upon peptide
addition (Table 2), we do not think that exchange between
protein-bound and free Cd
is involved. A possible
explanation is that some fluctuations exist in the
Ca
-binding sites corresponding to slightly different
Cd
chemical shifts (Vogel and
Forsén, 1987). Be that as it may, our
Cd NMR are consistent with the notion that the
CaM
cNOS peptide complex, as well as the CaM
MLCK peptide
complex, provide a more constrained Ca
-binding
environment than Ca
-CaM.
The binding of CaM with
its target peptides takes place mainly via van der Waals interactions
between the Met-rich hydrophobic surfaces of CaM and the hydrophobic
face of the CaM-target peptides (Ikura et al., 1992; Meador et al., 1992, 1993; Zhang and Vogel, 1994b; Zhang et
al., 1994a). In particular, the polarizability of the Met sulfur
atoms can contribute significantly to the binding affinity (Gellman,
1991). Selectively CH
-Met isotopically labeled
and Se-Met containing CaM were used to study the involvement of the Met
residues in the interaction with the cNOS peptide. The dramatic
chemical shift changes observed for all of the Met and Se-Met residues
in the two hydrophobic surfaces of CaM upon complex formation clearly
indicate that the cNOS peptide also interacts with CaM through
hydrophobic interactions. An analysis of the chemical shift changes of
the Met
H,
C, and
Se resonances
upon binding of the MLCK and cNOS peptides (see Table 3) show
that the changes induced by MLCK and cNOS are not identical. This
behavior is expected, as the different amino acid sequence of these two
target peptides would give rise to distinct bound conformations for the
Met side chains of CaM.
The helical parts of the central linker
region of CaM unravel further upon complex formation (Ikura et
al., 1992; Meador et al., 1992, 1993). It is anticipated
that the linker region of CaM will also unravel when CaM forms a
complex with the cNOS peptide. We have shown earlier that the binding
of a target peptide to the hydrophobic surfaces of CaM will displace
the side chain of Lys-75 from the N-terminal hydrophobic surface; this
process may induce the unraveling of the central -helix (Vogel,
1994). This displacement causes a significant increase in its
pK
(Zhang and Vogel, 1993). Here we have
observed that the pK
of Lys-75 changes
from 9.29 to 9.94 when CaM forms a complex with the cNOS peptide, and
this increase is comparable to what was seen for MLCK (Zhang and Vogel,
1993, see also Table 4). This suggests that the helical parts of
the linker region of CaM also undergo a conformational change upon the
formation of the complex with the cNOS peptide. The
pK
of Lys-148 also increases
significantly when CaM binds to the cNOS peptide, and this again
resembles the results with the MLCK peptide (Zhang and Vogel, 1993). By
analogy to Lys-75, it is believed to be caused by a displacement of the
Lys-148 side chain from an orientation on top of the C-terminal
hydrophobic domain.
In principle, the cNOS peptide can bind to the protein in two orientations. Using a specific nitroxide spin-labeled cNOS peptide derivative, we have shown that the C-terminal part of the cNOS peptide binds to the N-terminal hydrophobic surface of CaM; consequently, the N-terminal portion of the peptide binds to the C-terminal domain of CaM. This orientation resembles the complexes of CaM with the CaM-binding domains of MLCK (Ikura et al., 1992; Meador et al., 1992), CaM-kinase II (Meador et al., 1993), and adenylate cyclase (Craescu et al., 1995). From a sequence alignment of the cNOS peptide with the CaM-binding domains of MLCK, we find that two anchoring hydrophobic residues are found with a spacing of 14 residues, analogous to MLCK, but distinct from CaM kinase II. Moreover, isotope-edited difference FTIR studies have shown that the two domains of CaM retain their secondary structure upon binding the cNOS peptide, as well as the MLCK peptide (Zhang et al., 1994b). Therefore, our data establish that the cNOS peptide binds to CaM in an analogous fashion as the MLCK peptide.