©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of Calmodulin with Its Binding Domain of Rat Cerebellar Nitric Oxide Synthase
A MULTINUCLEAR NMR STUDY (*)

(Received for publication, April 4, 1995; and in revised form, June 20, 1995)

Mingjie Zhang (§) Tao Yuan (¶) James M. Aramini (**) Hans J. Vogel (§§)

From the Department of Biological Sciences, The University of Calgary, Calgary, Alberta T2N 1N4, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

The ubiquitous Ca-binding protein, calmodulin (CaM), (^1)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 alpha-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 alpha-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 alpha-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 approx2 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 alpha-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 ^1H, 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 ^1H,C HMQC spectroscopy and ^1H,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.


MATERIALS AND METHODS

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(3)-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 CaMbulletpeptide 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).

Spin Labeling of the cNOS Peptide

The spin-labeled cNOS peptide in which the single Met residue was specifically reacted was prepared following published procedures (Lundblad and Noyes, 1984; Musci et al., 1988). Briefly, the cNOS peptide was incubated with a 10-fold excess of 4-(2-bromoacetamido)-2,2,6,6-tetramethylpiperidine-N-oxyl for 24 h at room temperature in 0.2 M acetate buffer, pH 3.0. Under these conditions this reagent will react specifically with the sulfur atom of the single Met in the peptide (Musci et al., 1988). The labeled cNOS peptide (yield 50%) was separated from the unreacted cNOS peptide by reverse-phase high performance liquid chromatography; only a single peak was observed for the modified peptide indicating that no side reactions have taken place.

NMR Experiments

All ^1H, C and Se NMR spectra were acquired on a Bruker AMX500 NMR spectrometer equipped with a 5-mm broadband inverse detection probe at 25 °C unless otherwise indicated. A total of 128 scans were collected for the 1D ^1H NMR spectra with a sweep width of 5000 Hz. ^1H,C HMQC spectra were recorded in the phase-sensitive mode (Bax et al., 1983). The two-dimensional natural abundance ^1H,Se HMBC spectrum of the SeMet-CaMbulletcNOS peptide complex was recorded in the magnitude mode (Zhang and Vogel, 1994c). All NMR spectra were processed on a Bruker X32 computer using commercially available Bruker UXNMR software.

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(4))(2) (pH approx 1 in D(2)O) was used for the preparation and titration of the samples. All Cd NMR samples were prepared in 25% (v/v) D(2)O, 100 mM KCl, 20 mM Tris, pH 7.5 ± 0.05. For the Cd(4)-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 mMCd (ClO(4))(2) in D(2)O.


RESULTS

Binding of the cNOS Peptide Induces Total Cooperativity in Metal Ion Binding

Cadmium-113 NMR titration experiments showed that both domains of CaM are affected simultaneously by the binding of the cNOS peptide. It should be noted that only two signals are detected for Cd(4)-CaM in the absence of the peptide; these represent the two cations bound in the two C-terminal sites of CaM; those in the N-terminal part are not detected due to an exchange process that broadens both resonances beyond detection (Forsén et al., 1986). Fig. 1shows the spectra of Cd(4)-CaM titrated with the cNOS peptide; upon addition of the peptide a total of four resonances is detected, as observed earlier with other target-bound CaMs (see Table 1). The spectra reveal that the cNOS peptide binds to Cd(4)-CaM in slow exchange on the Cd NMR time scale. All of the four new resonances increase simultaneously in intensity with the concomitant disappearance of the two signals from Cd(4)-CaM. These four signals can be tentatively assigned to the four Cd ions bound to the four calcium-binding sites in the CaM-cNOS peptide complex, by comparison with values obtained for other complexes (see Table 1). No minor signals are detectable in the spectra, suggesting that the peptide binds in only one orientation. Upon integration of the peaks, we found that the 1:1 complex only equals to 2 equivalents of Cd, as does the Cd(4)bulletCaM complex. It is quite common in Cd NMR that spectra are not fully relaxed if the relaxation delay used is short (Coleman, 1993). Therefore we acquired spectra with a 5 s delay for both Cd(4)-CaM and the Cd(4)bulletCaMbullet cNOS peptide complex at the same protein concentrations. The integration data obtained for these samples showed that the protein-peptide complex equals 3.7 equivalents of Cd, which is close to the expected 4 equivalents; the integrated intensity of Cd(4)-CaM remains 2.0 equivalents indicating a significant change in the T(1) relaxation rate upon the binding of the peptide to the protein. Similar integration results have also been obtained for Cd(4)-CaM titrated with the skeletal muscle MLCK peptide (see Fig. 2).


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(4))(2). 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.





The cNOS Peptide Binds to the Met-rich Hydrophobic Surfaces of CaM

The two hydrophobic surfaces of CaM that are generally involved in the binding of the CaM-binding domains of target proteins are very rich in Met residues (Babu et al., 1988; O'Neil and DeGrado, 1990). Therefore, we studied the interaction between CaM and the cNOS peptide by heteronuclear NMR spectroscopic techniques using CH(3)-Met isotopically labeled CaM (Siivari et al., 1995). Fig. 4, A and B, show the ^1H,C HMQC spectra of the methyl groups of the Met residues from CabulletCaM and the CaMbulletcNOS peptide complex. The assignment of the Met resonances were obtained by using individual Met Leu mutants of the protein (for description see Zhang et al., 1994a; Siivari et al., 1995). With the exception of Met-76 which is part of the central linker of CaM, all the Met residues in the hydrophobic patches undergo significant chemical shift changes upon binding the cNOS peptide (see Table 3). Because of the sensitivity of the Se chemical shift to changes in local environment (Luthra and Odom, 1986), the effects of complex formation on the Met residues in CaM were also studied by Se NMR using CaM with Se-Met incorporated for Met (Zhang and Vogel, 1994c). Again, the binding of the cNOS peptide causes drastic chemical shift changes for the resonances of the 8 Se-Met residues from the two hydrophobic patches (see Table 3). These results demonstrate that the two Met-rich hydrophobic regions of CaM are directly involved in the binding of the cNOS peptide.


Figure 4: ^1H,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(2)O, 100 mM KCl, pD 7.5, 25 °C).





The Conformation of the Central Helix of Ca-CaM Changes upon Binding to the cNOS Peptide

The possibility of conformational changes in the central helix/linker region of CaM upon binding of the cNOS peptide was evaluated by determining the pK changes of Lys-75. It has been shown that the unraveling of the helical part of this region upon complex formation (Ikura et al., 1992) is accompanied by a drastic increase in the pK of this residue (Zhang and Vogel, 1993). Fig. 5shows the ^1H,C HMQC spectra of reductively dimethylated CaM and its K75R and K148Q mutants complexed with the cNOS peptide. From these data, it is immediately apparent that the missing resonances belong to Lys-75 and Lys-148, respectively. pH titration of the C-methylated-CaMbulletcNOS peptide complex allowed us to obtain the pK values of the individual Lys residues of CaM (see Table 4). Fig. 6shows the pH titration curves of Lys-75 in CabulletCaM and in the CaMbulletcNOS peptide complex. Lys-75 displays a significant pK change from 9.29 in CaM to 9.94 in the complex. Similar to what was observed for the CaMbulletMLCK peptide complex, Lys-148 also shows a large pKchange upon complex formation with the cNOS peptide, whereas the other Lys residues only experience minor changes.


Figure 5: ^1H,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(4)-CaM () and Ca(4)-CaM-cNOS (bullet).



The C-terminal Region of the cNOS Peptide Binds to the N-terminal Hydrophobic Surface of CaM

Introduction of nitroxide spin labels covalently attached to ligands provides a useful means of determining the binding orientation of ligands on proteins (see for example de Jong et al., 1988; Penington and Rule, 1992; Folkers et al., 1993; Girvin and Fillingame, 1995). We have utilized a Met nitroxide spin-labeled cNOS peptide in order to map out the orientation of the CaM-bound cNOS peptide with respect to the two domains of the protein. Fig. 7A shows the ^1H,C HMQC spectrum of the CH(3)-Met-labeled CaM complexed with the nitroxide spin-labeled cNOS peptide. Comparing Fig. 7A and 4B, it is clear that the four Met resonances from the C-terminal domains of CaM do not show any chemical shift and line width changes, whereas the Met resonances from the N-terminal domain undergo significant chemical shift as well as line width changes (the contour levels for the three visible N-terminal resonances were about half of those in the C-terminal domain). Most noticeably, a resonance tentatively assigned to Met-72 was broadened beyond detection by the spin label in the cNOS peptide. The chemical shift changes observed for Met-36, Met-51, and Met-71 result from the conformational perturbation induced by the relatively large hydrophobic spin labeling reagent attached to the cNOS peptide, as these persist following the reduction of the spin label (see below). In order to ascertain that the broadening of the resonances of the N-terminal Met residues in Fig. 7A results from the paramagnetic effect of the spin label, we have added ascorbic acid, which can abstract the single electron from the nitroxide (Kosen et al., 1986). Fig. 7B provides the spectrum of the complex recorded 24 h after the addition of ascorbic acid. The reappearance of the Met-72 and the increased intensity for the other N-terminal Met resonances demonstrates that the broadening effect results from the close proximity of the spin label (in panel 7B, the contour levels for Met-36, Met-51, and Met-71 are identical to those in the C-terminal domain). Hence, the C-terminal end of the cNOS peptide binds to the N-terminal hydrophobic surface of the protein.


Figure 7: ^1H,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.




DISCUSSION

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 alpha-helical structure in aqueous solution that it forms a stable alpha-helix in aqueous trifluoroethanol (Zhang and Vogel, 1994; Vogel and Zhang, 1995) and that the alpha-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 alpha-helical structure in its complex with CaM (Zhang et al., 1994b). Here, we have used ^1H, 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 CaMbulletcNOS 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(1) relaxation time in the complex is longer than in Cd(4)-CaM. Also the T(2) relaxation time for the complex appears to be longer than that in Cd(4)-CaM from the obvious line width narrowing (Delta) in the complex (Table 2); however, it is important to realize that chemical exchange processes, rather than T(2) 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(2) (and line width) values for Cd(4)bulletCaM and the Cd(4)bulletCaMbulletMLCK or cNOS complexes, assuming correlation times of 8.5 and 10 ns, respectively, at 25 °C and using a chemical shift anisotropy value Delta = 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 CaMbulletpeptide 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 CaMbulletcNOS peptide complex, as well as the CaMbulletMLCK 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(3)-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 ^1H, 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 alpha-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.


FOOTNOTES

*
This work was supported by an operating grant from the Medical Research Council of Canada (MRC). The NMR spectrometers used have been funded and maintained with funds provided by the Alberta Heritage Foundation for Medical Research (AHFMR) and MRC. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Div. of Molecular and Structural Biology, Ontario Cancer Institute, 500 Sherbourne St., Toronto, Ontario, M4X 1K9, Canada.

Recipient of a studentship from AHFMR.

**
Supported by the National Science and Engineering Research Council of Canada (NSERC).

§§
Recipient of an AHFMR scholarship. To whom correspondence should be addressed. Tel.: 403-220-6006; Fax: 403-289-9311.

(^1)
The abbreviations used are: CaM, calmodulin; 1D, one-dimensional; 2D, two-dimensional; cNOS, constitutive nitric oxide synthase; FTIR, Fourier transform infrared spectroscopy; HMQC, heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond correlation; MLCK, myosin light chain kinase; T(1), spin-lattice relaxation time; T(2), spin-spin relaxation time.


REFERENCES

  1. Andersson, A., Forsén, S., Thulin, E., and Vogel, H. J. (1983) Biochemistry 22,2309-2313 [Medline] [Order article via Infotrieve]
  2. Aramini, J., Hiraoki, T., Ke, Y., Nitta, K., and Vogel, H. J. (1995) J. Biochem. (Tokyo) 117,623-628 [Abstract]
  3. Babu, Y. S., Bugg, C. E., and Cook, W. J. (1988) J. Mol. Biol. 204,191-204 [Medline] [Order article via Infotrieve]
  4. Barbato, G., Ikura, M., Kay, L. E., Pastor, R. W., and Bax, A. (1992) Biochemistry 31,5269-5278 [Medline] [Order article via Infotrieve]
  5. Bax, A., Griffey, R. H., and Hawkins, B. L. (1983) J. Magn. Reson. 55,301-315
  6. Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,682-685 [Abstract]
  7. Bredt, D. S., and Snyder, S. H. (1994) Annu. Rev. Biochem. 63,175-195 [CrossRef][Medline] [Order article via Infotrieve]
  8. Bredt, D., Hwang, P. W., Glatt, C. E., Lowenstein, C., Reed, R. R., and Snyder, S. H. (1991) Nature 351,714-718 [CrossRef][Medline] [Order article via Infotrieve]
  9. Coleman, J. E. (1993) Methods Enzymol. 227,16-43 [Medline] [Order article via Infotrieve]
  10. Craescu, C. T., Bouha, A., Mispelter, J., Diesis, E., Popescu, A., Chiriac, M., and Bârzu, O. (1995) J. Biol. Chem. 270,7088-7096 [Abstract/Free Full Text]
  11. de Jong, E. A. M., Claesen, C. A. A., Dalmen, C. J. M., Harmsen, B. J. M., Konings, R. N. H., Tesser, G. I., and Hilbers, C. W. (1988) J. Magn. Reson. 80,197-213
  12. Feldman, P. L., Griffith, O. W., and Stuehr, D. J. (1993) Chem. Eng. News 71,26-38
  13. Folkers, P. J. M., van Duynhoven, J. P. M., van Lieshout, H. T. M., Harmsen, B. J. M., van Boom, J. H., Tesser, G. I., Konings, R. N. H., and Hilbers, C. W. (1993) Biochemistry 32,9407-9416 [Medline] [Order article via Infotrieve]
  14. Fors é n, S., Vogel, H. J., and Drakenberg, T. (1986) in Calcium and Cell Function (Cheung, W. Y., eds) Vol. VI, pp. 113-157, Academic Press, New York
  15. Gellman, S. H. (1991) Biochemistry 30,6633-6636 [Medline] [Order article via Infotrieve]
  16. Girvin, M. E., and Fillingame, R. H. (1995) Biochemistry 34,1635-1645 [Medline] [Order article via Infotrieve]
  17. Hiraoki, T., and Vogel, H. J. (1987) J. Cardiovasc. Pharm. 10,S14-S31
  18. Huque, M. E., and Vogel, H. J. (1993) J. Protein Chem. 12,693-705
  19. Ikura, M., Hasegawa, N., Aimoto, S., Yazawa, M., Yagi, K., and Hikichi, K. (1989) Biochem. Biophys. Res. Commun. 161,1233-1238 [Medline] [Order article via Infotrieve]
  20. Ikura, M., Spera, S., Barbato, G., Kay, L. E., and Bax, A. (1991) Biochemistry 30,9216-9228 [Medline] [Order article via Infotrieve]
  21. Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256,632-638 [Medline] [Order article via Infotrieve]
  22. James, P., Vorherr, T., and Carafoli, E. (1995) Trends Biochem. Sci. 20,38-42 [CrossRef][Medline] [Order article via Infotrieve]
  23. Kördel, J. Johansson, C., and Drakenberg, T. (1992) J. Magn. Reson. 100,581-587
  24. Kosen, P. A., Scheek, R. M., Naderi, H., Basus, V. J., Manogaran, S., Schmidt, P. G., Oppenheimer, N. J., and Kuntz, I. D. (1986) Biochemistry 25,2356-2364 [Medline] [Order article via Infotrieve]
  25. Linse, S., Drakenberg, T., and Forsén, S. (1986) FEBS Lett. 199,28-32 [CrossRef][Medline] [Order article via Infotrieve]
  26. Lundblad, R. L., and Noyes, C. M. (1984) in Chemical Reagents for Protein Modification , Vol. I, pp. 99-103, CRC Press, Boca Raton, FL
  27. Luthra, N. P., and Odom, J. D. (1986) in The Chemistry of Organic Selenium and Tellurium Compounds (Patai, S., and Rappoport, Z., eds) Vol. I, pp. 189-241, John Wiley, New York
  28. Meador, W. E., Means, A. R., and Quiocho, F. (1992) Science 257,1251-1254 [Medline] [Order article via Infotrieve]
  29. Meador, W. E., Means, A. R., and Quiocho, F. (1993) Science 262,1718-1721 [Medline] [Order article via Infotrieve]
  30. Means, A. R., Van Berkum, M. F. A., Bagchi, I., Lu, K. P., and Rasmussen, C. D. (1991) Pharmacol. Ther. 50,255-270 [CrossRef][Medline] [Order article via Infotrieve]
  31. Musci, G., Koga, K., and Berliner, L. J. (1988) Biochemistry 27,1260-1265 [Medline] [Order article via Infotrieve]
  32. Nathan, C. (1992) FASEB J. 6,3051-3063 [Abstract/Free Full Text]
  33. Nathan, C., and Xie, Q.-W. (1994) J. Biol. Chem. 269,13725-13728 [Free Full Text]
  34. Ohki, S., Iwamoto, U., Aimoto, S., Yazawa, M., and Hikichi, K. (1993) J. Biol. Chem. 268,12388-12392 [Abstract/Free Full Text]
  35. O'Neil, K. T., and DeGrado, W. F. (1990) Trends Biochem. Sci. 15,59-64 [CrossRef][Medline] [Order article via Infotrieve]
  36. Penington, C. J., and Rule, G. S. (1992) Biochemistry 31,2912-2920 [Medline] [Order article via Infotrieve]
  37. Pollock, J. S., Förstermann, U., Mitchell, J. A., Warner, T. D., Schmidt, H. H. H. W., Nakane, M., and Murad, F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,10480-10484 [Abstract]
  38. Roth, S. M., Schneider, D. M., Strobel, L. A., Van Berkum, M. F. A., Means, A. R., and Wand, A. J. (1992) Biochemistry 31,1443-1451 [Medline] [Order article via Infotrieve]
  39. Sessa, W. C., Harrison, J. K., Barber, C. M., Zeng, D., Durieux, M. E., D'Angelo, D. D., Lynch, K. R., and Peach, M. J. (1992) J. Biol. Chem. 267,15274-15276 [Abstract/Free Full Text]
  40. Siivari, K., Zhang, M., Palmer, A., and Vogel, H. J. (1995) FEBS Lett. 366,104-108 [CrossRef][Medline] [Order article via Infotrieve]
  41. Spera, S., Ikura, M., and Bax, A (1991) J. Biomol. NMR 1,155-165 [Medline] [Order article via Infotrieve]
  42. Stuehr, D. J., Cho, H. J., Kwon, N. S., Weise, M., and Nathan, C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,7773-7777 [Abstract]
  43. Thulin, E., Andersson, A., Drakenberg, T., Forsén, S., and Vogel, H. J. (1984) Biochemistry 23,1862-1870 [Medline] [Order article via Infotrieve]
  44. Vogel, H. J. (1994) Biochem. Cell Biol. 72,357-376 [Medline] [Order article via Infotrieve]
  45. Vogel, H. J., and Fors é n, S. (1987) in Biological Magnetic Resonance (Berliner, L. J., and Reuben, J., eds) Vol. VII, pp. 245-307, Plenum Press, New York
  46. Vogel, H. J., and Zhang, M. (1995) Mol. Cell. Biochem., 3-15
  47. Vogel, H. J., Huque, M. E., and Hiraoki, T. (1990) in The Biology and Medicine of Signal Transduction (Nishizuha, Y., eds) pp. 254-259, Raven Press, New York
  48. Vorherr, T., Knöpfel, L., Hofmann, F., Mollner, S., Pfeuffer, T., and Carafoli, E. (1993) Biochemistry 32,6081-6088 [Medline] [Order article via Infotrieve]
  49. Xie, Q.-W., Cho, H. J., Calaycay, J., Mumford, R. A., Swiderek, K., Lee, T. D., Ding, A., Troso, T., and Nathan, C. (1992) Science 256,225-228 [Medline] [Order article via Infotrieve]
  50. Zhang, M., and Vogel, H. J. (1993) J. Biol. Chem. 268,22420-22428 [Abstract/Free Full Text]
  51. Zhang, M., and Vogel, H. J. (1994a) J. Biol. Chem. 269,981-985 [Abstract/Free Full Text]
  52. Zhang, M., and Vogel, H. J. (1994b) Biochemistry 33,1163-1171 [Medline] [Order article via Infotrieve]
  53. Zhang, M., and Vogel, H. J. (1994c) J. Mol. Biol. 239,545-554 [CrossRef][Medline] [Order article via Infotrieve]
  54. Zhang, M., Yuan, T., and Vogel, H. J. (1993) Protein Sci. 2,1931-1937 [Abstract/Free Full Text]
  55. Zhang, M., Li, M., Wang, J. H., and Vogel, H. J. (1994a) J. Biol. Chem. 269,15546-15552 [Abstract/Free Full Text]
  56. Zhang, M., Fabian, H., Mantsch, H. H., and Vogel, H. J. (1994b) Biochemistry 33,10883-10888 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.