©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calmodulin Binding of a Peptide Derived from the Regulatory Domain of Bordetella pertussis Adenylate Cyclase (*)

(Received for publication, November 14, 1994; and in revised form, January 20, 1995)

Constantin T. Craescu (1)(§) Ahmed Bouhss (2) Joël Mispelter (1) Eric Diesis (3) Aurel Popescu (4) Maria Chiriac (5) Octavian Bârzu (2)

From the  (1)Institut National de la Santé et de la Recherche Médicale U350, Institut Curie, 91405 Orsay, France, the (2)Unité de Biochimie des Régulations Céllulaires, URA1129, Institut Pasteur, Paris, France, the (3)Service de Chimie des Biomolécules, URA1309, Institut Pasteur de Lille, Lille, France, the (4)Department of Biophysics, University of Bucarest, Romania, and the (5)Institut de Technologie Isotopique et Moléculaire, Cluj-Napoca, Romania

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

This paper reports the solution conformation and calmodulin binding of a 43-residue peptide from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase. The peptide (P) was synthesized and N-labeled at specific amino acids. It binds calmodulin with an equilibrium dissociation constant of 25 nM. Assignment of the NMR spectrum of the free peptide and analysis of the NOE connectivities and secondary shifts of Calpha protons allowed us to identify a 10-amino acid fragment (Arg to Arg) which is in rapid equilibrium between alpha-helical and irregular structures. Titration experiments showed that at substoichiometric molar ratios the two molecules are in intermediate exchange between free and bound conformations. Using N-edited methods we assigned a large part of resonances of the labeled residues in the bound peptide. Analysis of the chemical shift differences between free and bound states shows that the fragment Leu-Ala is the most affected by the interaction. The proton spectra of the calmodulin, in the free and complexed states were extensively assigned using homonuclear experiments. Medium- and long-range NOE patterns are consistent with a largely conserved secondary and tertiary structure. The main changes in chemical shift of calmodulin resonances are grouped in six structural regions both in NH(2)- and COOH-terminal domains. Intermolecular NOE connectivities indicate that the NH(2)-terminal of the bound peptide fragment is engulfed in the COOH-terminal domain of calmodulin. The interaction geometry appears to be similar to those previously described for myosin light chain kinase or calmodulin kinase II fragments.


INTRODUCTION

The complex Ca-CaM (^1)controls the biological activity of more than 30 different proteins including several enzymes, ion transporters, receptors, motor proteins, transcription factors, and cytoskeletal components in eukaryotic cells. It also binds a variety of peptide hormones and toxins with moderate to high affinities. The three-dimensional structure of CaM-Ca complex, solved by x-ray diffraction(1, 2) , presents two globular, mainly helical, domains, each containing two EF-hand motifs and connected by an eight-turn alpha-helix. Much less is known about the spatial structure of target proteins: no crystal structure for a target protein or for a larger CaM-binding domain (free or in complex with CaM) has been described yet. The knowledge, at atomic level, of the molecular interactions between CaM and its targets is, however, necessary for the understanding and controlling the various signal transmission pathways involving CaM.

To overcome this lack of information, homo- and heteronuclear NMR spectroscopic studies were initiated with the aim to characterize the conformational properties of small CaM-binding peptides of 17-26 amino acids derived from target proteins either alone or in complex with CaM (3, 4, 5, 6) . Recently, multinuclear NMR (7) and x-ray (8, 9) experiments were successfully used for a detailed description of a small peptide, derived from target proteins, in complex with CaM. The two methods converged to a similar structure of the complex in which the two interacting molecules changed considerably their structure relative to the isolated state. The peptides assume a helical structure and protrude in a kind of tunnel structure created between the two globular domains of the protein by an unfold and curving of the central helix. However, extrapolation of this interaction model to larger target domains, characterized by a stable tertiary structure, is not straightforward. In particular, the deep embedding of the CaM-binding fragment into the CaM groove requires a high conformational flexibility and relatively weak contacts with the main body of the molecule.

On the other hand, there is some direct evidence for the existence of a conformational adaptability of CaM molecule according to the hydrophobic profile of the target domain. This may give a remarkable plasticity(9) . Also, alternative modes of CaM-target interactions were recently proposed for phosphorylase kinase (10) or cyclic nucleotide phosphodiesterase(11) .

Bordetella pertussis, the etiological agent of whooping cough, secretes a toxin endowed with adenylyl cyclase activity(12) . A striking particularity of bacterial enzyme is activation by CaM either in the presence or absence of calcium ions(13, 14) . The catalytic and regulatory domains of this protein were recently identified and studied in detail(15, 16, 17) . A 20-amino acid peptide (P corresponding to the sequence 235-254), part of the regulatory CaM-binding domain (K = 580 nM), was analyzed for its solution properties by CD and NMR spectroscopy(4) . A small population of peptide organized in alpha-helix is in equilibrium with molecules in random coil structures. The elements of helical structure were centered around Trp, a residue known by mutagenesis experiments to be critical for the activity regulation and CaM binding of the whole protein(18, 19) . The relative low affinity of the peptide suggests that contiguous fragments, in the intact protein, may also contribute to the interaction energy. Therefore, we decided to study the conformational properties of larger fragments derived from the regulatory domain and their interactions with the Ca-CaM complex. Here we describe the conformational properties and interaction with CaM of a fragment of 43 amino acids (P) possessing a much higher affinity for CaM (K = 25 nM) than P. Assignment of NMR spectra and analysis of chemical shifts and NOE connectivities indicate that P has a 10-residue fragment which is organized in alpha-helical structure. Specific N-labeling of the peptide and utilization of heteronuclear experiments enabled us to delimitate the peptide fragment in direct interaction with the protein and its conformational changes. Proton assignments of Ca-CaM-peptide complex and identification of a large number of intraprotein and some intermolecular NOE constraints suggest an interaction model similar to those already proposed for small CaM-binding fragments.


MATERIALS AND METHODS

Chemicals

Protected amino acids were purchased from Bachem. Deuterated water and Tris(d) was obtained from the Commissariat à l'Energie Atomique (France). Bovine brain calmodulin was from Boehringer (Mannheim). N-Enriched glycine was obtained using dipotassium phtalimide method. N-Labeled valine, leucine, norleucine, alanine, and phenylalanine (>98%) were synthesized enzymatically from NH(4)Cl and the corresponding alpha-oxoacids using alanine, leucine, and phenylalanine dehydrogenase and glucose dehydrogenase + glucose as the NADH regenerating system(20) . Details concerning large scale preparation of these amino acids will be published elsewhere.

Peptide Synthesis

Protected peptidyl resin was build up using the conventional solid-phase Boc-benzyl strategy on a paramethyl-benzhydrylamine resin. N-Labeled amino acids were tert-butyloxycarbonylated using di-tert-butyldicarbonate by the Fluka protocol. The peptide was purified by reversed-phase preparative high performance liquid chromatography followed by a preparative ion-exchange chromatography on a sulfoethyl aspartimide column (10 times 100 mm). Peptide purity was checked by analytical reversed-phase high performance liquid chromatography C18 and analytical ion-exchange chromatography. The peptide was acetylated at NH(2) terminus and amidated at the COOH terminus in order to remove any destabilizing interactions with the charged terminal groups. For practical reasons the COOH-terminal Met residue was replaced by the less reactive Nle residue.

Binding Assays

The dissociation constant of CaM in complex with the peptide was determined by competition with the wild-type, active protein(21) . Binding of P to dansyl-CaM was analyzed with a Perkin-Elmer LS-5B spectrofluorimeter maintained at 298 K(17) .

NMR Methods

Samples at a concentration of 1-2 mM were prepared in deuterated Tris buffer 95% ^1H(2)O, 5% ^2H(2)O, or in ^2H(2)O at pH 6.4. pH readings were made with a glass electrode and were not corrected for the isotope effect. NMR spectra were obtained on a Varian Unity 500 NMR spectrometer. Standard methods were used to obtain pure absorption DQF-COSY (22) and phase-sensitive NOESY (23) spectra. Different mixing times were used for the NOESY spectra of the free peptide (between 80 and 150 ms) and the spectra were compared to assess the extent of spin diffusion. TOCSY experiment employed a MLEV-16 pulse sequence for the spin lock and 80 ms mixing time(24) . The residual water resonance was suppressed by a low-power selective irradiation during the relaxation delay and also during the mixing time for NOESY experiments. Usually 512 experiments were done with 96-128 scans and 2048 complex points each. Data processing was made using the FELIX software running on a Silicon Graphics Personal Iris 4D/35. Time data were multiplied by appropriate Gaussian functions in t(2) dimension and by sine-bell functions shifted by /4 in t(1) dimension. The data were zero-filled in both dimensions to give a final matrix of 4 K times 4 K real points.

Two-dimensional heteronuclear experiments, HMQC, NOESY-HMQC, and HMQC-TOCSY used basically the published sequences(25) . The spectral width was 3000 Hz in the nitrogen dimension. The proton spectral width was 3200 Hz in the HMQC experiment and 7000 Hz in the other two-dimensional experiments. N reference was set relative to a NH(4)Cl (3 M in 1 M HCl) solution where the nitrogen resonates at 24.93 ppm from the liquid NH(3)(26) .


RESULTS

The sequence of the peptide P used in the present work is shown in Fig. 1together with the 20-amino acid peptide (P), investigated in a previous study(4) . The sequences were chosen such as to contain Trp, which play an important role in CaM binding by the whole cyclase molecule(18) .


Figure 1: Primary structure of P and P derived from the calmodulin-binding domain of B. pertussis adenylate cyclase. The boxes indicate the N-labeled (at the amino position) residues.



Binding Affinity

The fluorescence emission spectrum of P has a maximum at 350 nm and is not influenced by the presence of Ca or EGTA. Addition of CaM in a slight excess over the peptide (1.2:1), in the presence of 0.1 mM CaCl(2), caused a 1.6-fold increase in total fluorescence intensity and a shift of the spectral maximum to 330 nm, indicating that the indole group is embedded in a more hydrophobic environment. EGTA determined the dissociation of the CaM-peptide complex. The Ca-dependent CaM binding properties of the peptide were confirmed by the reversal of the CaM-dependent activation of B. pertussis adenylate cyclase. The competition binding assays gave a K(d) of 25 nM. On the other hand, titration of fixed concentrations of dansyl-CaM with various concentrations of P and analysis of experimental data according to Scatchard allowed us to determine the K(d) of the complex of dansyl-CaM with the peptide (30 nM).

Resonance Assignment

Almost complete assignment of proton resonances in the spectrum of P was obtained using the standard sequence-specific assignment methodology (27) combined with the main chain-directed strategy(28, 29) . Use of N labeling and heteronuclear experiments were helpful in some cases. As may be seen in Fig. 2, showing the HMQC spectrum of the free peptide, the nitrogen resonances are considerably more dispersed than the corresponding amide proton resonances. In particular, the N resonances of the five Gly residues are all grouped in the high-field region while the Ala resonances are situated on the opposite, low-field region. The chemical shift values of the proton and nitrogen resonances in P at 298 K are given in Table 1.


Figure 2: HMQC spectrum of free P in deuterated Tris buffer (50 mM), pH 6.4, at 298 K. The cross-peaks are labeled with the corresponding amino acid residue.





Peptide Conformation

Two main NMR spectroscopic parameters are generally used to identify and characterize the elements of secondary structure in linear peptides: the pattern of short- and medium-range NOE connectivities and the distribution of CalphaH chemical shift along the sequence. Other parameters such as ^3J coupling constant, temperature coefficients of amide resonances, and ^1H/^2H exchange rate may also be used but proved to be less efficient in our case, due to the intrinsic flexibility of the polypeptide structure and the spectral superposition.

A summary of the short- and medium-range NOE connectivities which are significant for the conformational analysis, is given in Fig. 3. The presence of short- (d(i, i + 1) and medium-range d(i, i + 2) and d(i, i + 3) dipolar interactions is characteristic for alpha helical structures. In a regular alpha-helix the distance between the sequentially amide protons (2.8 Å) is considerably lower than in beta-strand or extended structures (4.2 Å) (27) and gives relatively strong NOE cross-peaks even in cases where the helix is in rapid equilibrium with less ordered structures. On the other hand, observation of d(i, i + 1), of similar intensity, indicates that extended conformations are equally present (d(i, i + 1) distance is 3.5 Å in alpha-helices and 2.2 Å in beta strands). Other medium-range NOE connectivities were unambiguously identified between aromatic and aliphatic side chains: C(1)H and C(3)H (Trp)/CbetaH(3) (Ala). Taken together the NOE information is indicative of a helical structure spanning a fragment of 10-15 amino acids (3-4 helix turns) centered on Trp.


Figure 3: Short- and medium-range NOE connectivities and the chemical shift index for free P. The chemical shift index was calculated as the difference between the actual chemical shift of Calpha protons and the random coil values for a given amino acid type.



Statistical analysis of a large number of NMR spectra revealed that secondary shifts of CH protons, calculated as the difference between experimental and random coil values, are sensitive probes of secondary structure in proteins (30, 31, 32) and linear peptides (33, 34) . Segments of helical structure are always associated with upfield shifts of CH resonances, the mean value being 0.40 ppm(30) . Fig. 3shows the secondary shift distribution, expressed as the chemical shift index (32) for Calpha protons along the sequence. The predicted helix domain (more than four consecutive negative values of the chemical shift index) agrees satisfactorily with the NOE patterns. A conservative interpretation of these results is that the fragment Arg-Arg is significantly organized in alpha-helix while the rest of the molecule is rapidly fluctuating between several irregular conformations. Actually, the regular secondary structure may be longer than this three turn alpha-helix, as suggested by the presence of d(i, i + 1) connectivities (Fig. 3).

An unusual high-field shift (0.70 ppm) was observed for the amide proton of Gly in the COOH-terminal end of the peptide. This may be explained by a specific conformation of the sequence YDGD in which the aromatic ring of Tyr is close to the Gly backbone, as was recently proposed for a similar sequence in bovine pancreatic trypsin inhibitor(35) .

Titration of CaM with P

The low-field region of the proton NMR spectrum of CaM contains several well resolved peaks, corresponding to single amide protons(36) . The effect of increasing amounts of peptide added to the CaM solution may thus be characterized from the evolution of these peaks. As can be observed in Fig. 4, progressive complex formation results in appearance of a new set of peaks, of increasing intensity and the decrease in intensity of the initial peaks, representing the uncomplexed protein. At equimolar ratio one can observe a unique set of resonances which suggests that the complexed protein exists in a single conformation. The final line width is slightly larger than that of CaM alone, due to the increase in molecular mass (29%) by the complexation. At intermediate mixing ratios, the peaks corresponding to complexed and uncomplexed CaM molecules are slightly broader and have intermediate frequencies, indicating that the two molecular species are in intermediate exchange on the NMR time scale. From the chemical shift difference of the high field peaks in Fig. 4we can estimate the lower limit of the exchange rate at about 60 s.


Figure 4: Low-field region of the proton NMR spectra of calmodulin in presence of different amounts of P. Calmodulin solution was 1.25 mM in deuterated Tris buffer (50 mM), 10 mM CaCl(2), pH 6.4, at 308 K. The CaM:peptide ratio is given at the right side of the spectra. The two vertical dashed lines in the high-field side indicates the frequencies of a CaM amide proton at the beginning and at the end of the titration. The vertical dashed line at 10.34 ppm points out the resonance of N1H in Trp of the peptide.



The peak at 10.34 ppm at 1:1 stoichiometry (Fig. 4) behaves differently from its neighbors. In fact we could assign it to the N(1)H of Trp of the peptide; it resonates at 10.18 ppm in the free state. While the protein resonances are practically insensitive to the excess in peptide concentration, this Trp peak is considerably broadened, confirming an intermediate exchange between free and bound peptide molecules.

Peptide Conformational Changes

Fig. 5shows the Nedited proton spectra of P in the free state and in the presence of two different concentrations of CaM. The observed peaks correspond to the 19 amide protons bound to a N atom. The spectrum of the free peptide (bottom) changes considerably in the presence of CaM. At substoichiometric molar ratios some peaks are broadened beyond the detection limit. At equimolar concentrations (Fig. 5, top), these peaks become narrower, enabling observation of the entire edited spectrum. One can observe that peptide binding results in marked changes of chemical shift (of the order of 0.50 ppm) of selected resonances while keeping unchanged other peaks. The line width of the shifted peaks is roughly comparable to that of the calmodulin resonances in the complex (Fig. 4), which is an indication that the bound peptide has the same global tumbling rate as the protein. Observation of a single set of resonances is also indicative for a unique strong binding site and a single conformation of the protein-peptide complex.


Figure 5: One-dimensional N-edited spectra of P in the free state (bottom) and in the presence of calmodulin. The peptide:CaM molar ratio is 1:0.77 in the middle spectrum and 1:1 in the top spectrum. The same physicochemical conditions were used as described in the legend to Fig. 4.



The HMQC spectrum of the peptide in the presence of an equimolar concentration of CaM is shown in Fig. 6. Comparative examination of the heteronuclear spectra in the absence (Fig. 2) and presence of CaM reveals several significant information. 1) The cross-peaks in the complex state are more dispersed, reflecting large conformational and/or environmental changes. 2) A clear difference in intensity divides the cross-peaks in two classes. Eight out of the 19 correlation peaks have a high intensity and the same chemical shift values as in the free state while the low intensity signals are highly shifted in both proton and nitrogen dimensions. The unperturbed peaks correspond to residues situated in the two end fragments of the peptide while the perturbed ones define a peptide site (Leu to Ala), which is sensitive to the complex formation. 3) A large difference in flexibility between the two proton classes, as indicated by a heteronuclear NOE experiment (not shown), means that the perturbed fragment is strongly bound to the protein while the peptide ends have roughly the same molecular flexibility as in the free state.


Figure 6: HMQC spectrum of N-enriched calmodulin-bound P in Tris buffer (50 mM), pH 6.4, at 308 K. The cross-peaks which are not perturbed by the complexation are labeled with the corresponding residue name and number.



Assignment of the proton and nitrogen resonances of the labeled residues in the CaM-bound P was obtained using heteronuclear, N-edited two-dimensional experiments. Assignment of the resonances coming from the noninteracting peptide fragments was readily obtained starting from the known nitrogen frequencies (the same as in the free state) and the HMQC-TOCSY spectrum. The sensitivity of this experiment was too low to allow observation of correlation peaks from the perturbed residues. We used in this case only the NOE-based information. Given the sequence position and the amino acid type of the labeled residues, the analysis of the NOESY-HMQC spectrum enabled us to assign a large part of the resonances originating from the CaM-bound peptide fragment (Table 1). NOE interactions were always observed between sequential amide protons (d) with significant larger intensities relative to the corresponding d. This is a strong suggestion that the dihedral angles in this fragment lie in the alpha-type region.

Additional assignments for non-labeled residues could be obtained from the analysis of a set of homonuclear two-dimensional spectra (DQ-COSY, TOCSY, and NOESY) recorded on the CaM-peptide complex. For instance, the resonance at 10.34, arising during the titration experiment (Fig. 4), gives two strong dipolar connectivities in the aromatic spectral domain, which, together with the typical Trp aromatic spin system observed in the COSY spectrum, allow assignment for Trp side chain. Peptide Tyr was identified in a different way, due to the strong / COSY peak that appears in the aromatic region.

The chemical shift differences between the free and the bound states of the assigned peptide resonances (N, NH, and CalphaH) are shown in Fig. 7. Overall, the largest variations are observed within the interacting domain, previously delimited from HMQC spectra. Nitrogen resonances appear to be the most sensitive probe for the complex formation. The large upfield shifts in the bound peptide, observed for these resonances, may be associated, according to recent data(37, 38) , with a conformational transition toward a helical secondary structure. Proton chemical shifts changes have a lower amplitude and are less regular, probably due to a simultaneous influence of ring-current effects.


Figure 7: Chemical shift differences of peptide resonances between the CaM-bound and free states along the sequence. Plain and dotted rectangles are used for the nitrogen and proton resonances, respectively.



Calmodulin Structural Changes

Almost complete C, N, and ^1H assignment of Drosophila CaM has recently been performed by multidimensional and multinuclear methods(36) . Based on these data and using homonuclear two-dimensional spectra we assigned proton resonances of more than 80% of the protein residues in the spectra of CaM and CaM-peptide complex (supplementary material). In addition to sequential NOE connectivities (d, d, and d) we identified about 200 medium- and long-range intra-protein connectivities. Taken together, these data indicate that the secondary and tertiary structures of the two globular domains are essentially conserved upon complexation. In particular, the d(i,j) NOE interactions between the antiparallel strands of the two small beta-sheets (Thr/Asn and Thr/Asp) involved in the Ca-binding motifs are only marginally perturbed.

One possibility to assess the conformational perturbations in CaM is to compare the chemical shift values of assigned protein resonances in the absence and presence of the target peptide. Fig. 8shows these differences along the primary structure for NH, CalphaH, and one representative proton of the side chain. With the exception of the middle of the central helix (residues 72-79), the present assignment enables uniform probing of the whole sequence. It appears that complexation with P is followed by changes in chemical shift over the entire sequence but with variable amplitudes. Considering only those portions where these changes are larger than 0.1 ppm, one can define six distinct sequence segments which are mostly perturbed by the complexation. The resulting map is similar to that obtained by Ikura et al.(39) although the magnitude of the changes are generally lower in our case.


Figure 8: Proton chemical shift differences (in ppm) between complexed and free CaM plotted against the sequence. The horizontal bars at the bottom of the figure indicate the fragments in which several consecutive values are larger than 0.10 ppm.



When represented on the three-dimensional structure of the protein (Fig. 9) (1) it is readily apparent that the six fragments are clustered in two hydrophobic regions (one in every globular domain), which are mainly populated by the long hydrophobic side chains of Phe and Met residues. Both NH(2)- and COOH-terminal domains are therefore directly involved in the binding of the target peptide as was demonstrated for the myosin light chain kinase (7, 8) and calmodulin-dependent protein kinase IIalpha (9) peptides. In addition, analysis of homonuclear NOESY spectra enabled us to identify several intermolecular contacts between side chain protons of peptide Trp and protons from the COOH globular domain of CaM. Thus C ^3H, C(3)H, and C(2)H (Trp) give NOEs with CH(3) of Leu while C(2)H (Trp) has dipolar connectivities with CH(3) of Val and CH(3) of Ile. From these data it appears that the NH(2) terminus of the peptide is oriented toward the COOH domain of the protein.


Figure 9: Simplified representation of the three-dimensional structure of CaM showing (in black) the amino acids for which the changes in chemical shift induced by peptide complexation are larger than 0.10 ppm. The side chains of the Phe residues are shown in ball-and-stick representation. The figure was drawn with the MolScript software(46) .




DISCUSSION

A better understanding of the various mechanisms by which CaM controls the activity of a number of different proteins requires the characterization of the structural motifs in the target molecules and their interaction with the Ca-CaM complex. The present available data show that, for two small peptides (20-26 amino acids) from myosin light chain kinase (7, 8) and a 25-residue peptide from calmodulin-dependent protein kinase IIalpha(9) , the structure of the complex is globally the same. However, there is evidence that the interaction mechanisms for other target proteins like phosphorylase kinase (10) or cyclic nucleotide phosphodiesterase (11) are significantly different. In addition, recent experiments on yeast genetics (40) revealed that CaM can control different cellular functions by acting on distinct target proteins through distinct molecular mechanisms.

Trying to better simulate the real protein interactions we produced (either by solid-phase synthesis or by overexpression in bacteria) peptide fragments of increasing lengths, derived from the enzyme regulatory domain. The peptide studied here is 43 residues long, almost twice the size of the previously studied target peptides.

Structure of the Target Peptide

The NMR structural study of the free target peptide showed that about a quarter of its primary structure (Arg-Arg) forms an amphiphilic alpha-helix. We found a similar structural organization in the regulatory domain of Bacillus anthracis adenylate cyclase(6) . As is usually the case for the free linear peptides, the folded and unordered structures are in dynamic equilibrium. The helix is centered on the Trp, a residue conserved in the majority of CaM-binding domains and shown to play a critical role in the binding process(41) .

Complex Geometry and Dynamics

Titration experiments showed that at substoichiometric ratios, the interacting molecules are in intermediate exchange, on the NMR time scale, between free and bound forms. Given the equilibrium dissociation constant and the estimated k rate constant one can evaluate that the k rate constant is larger than 2 times 10^9M s. The high value of the k constant indicates that the interaction is mainly controlled by molecular diffusion. Chemical shift and intra-peptide NOE data on the bound peptide are compatible with a stable helical organization. This means that during the complexation process the protein selects those peptides that already have a transitory helical conformation and/or induces the formation and stabilization of the alpha-helix. Consequently, we can assume that the random coil to helix transition of the peptide is a fast, non-limiting process.

Data obtained in this work indicate that binding of P to CaM perturbs significantly only a limited portion of the peptide and have insignificant consequences on both terminal ends. A conservative interpretation of the chemical shift and heteronuclear (^1H-N) NOE results enables us to localize the interacting segment as the contiguous length between Leu and Ala. Therefore, the binding fragment contains the alpha-helix observed in the free peptide, extended by about 10 amino acids toward the COOH-terminal. Interacting peptides of similar sizes from various other targets were found to express almost the entire interaction energy as the parent protein(7, 9, 41) .

Several experimental observations (line width, heteronuclear NOEs, HMQC peak intensities) on the bound peptide indicate a considerable difference in flexibility between residues directly involved in interaction and the rest of the peptide. 40% of the peptide, which is conformationally rigid, has practically the same rotational correlation time as the whole complex while the rest of the peptide retains almost the same degree of mobility it has in the free state. Similar observations of large differences in mobility between different regions of bound peptides were made in peptide-antibody complexes(42, 43) .

The proton resonance assignment obtained here provides a reasonable basis which allows a low-resolution modeling for the geometry of the complex. The large chemical shift changes (up to 0.54 ppm) observed for the indole protons of peptide Trp (particularly, C(3)H, C(2)H, and C(2)H, see Table 1) indicate that the tryptophan side chain is in close contact with one or more aromatic rings of the protein. These changes, together with the observed intermolecular contacts involving Trp, are consistent with a model in which the NH(2)-terminal of the interacting peptide segment is engulfed in the hydrophobic cavity of the COOH globular domain of CaM (close to Leu, Val, and Ile).

We showed that the global folding of the two globular domains of CaM (particularly, the EF-hand motifs) are not significantly perturbed upon complexation. On the other hand, the six regions in the CaM structure which are most affected by the complexation should be in close contact with the peptide. Given the length of the interacting peptide fragment, the central helix of the protein should be bent such as to allow the two globular domains to interact simultaneously with the peptide. Thus, the emerging interacting model is roughly similar to those initially proposed by Persechini and Kretsinger (44) and then determined by Ikura et al.(7) and Meador et al.(8, 9) . The chemical shift perturbations of CaM resonances observed here are less than that of the myosin light chain kinase peptide/CaM, suggesting that the interactions with the target are relatively weaker and/or the complex has a more dynamic structure(45) . The present experimental data do not allow a more detailed characterization of the intermolecular interactions. Extensive labeling (in particular with C) and multidimensional methods would be necessary for a higher resolution structure.

If the model of intermolecular interaction proposed here is also valid for the entire target protein, the CaM binding domain should be situated at the surface of the molecule, in a structural region having a relatively high flexibility. This is in good agreement with experiments carried out in our laboratories showing that Arg is constantly the main site for the proteases attack.

Structure-Activity Relationships

Despite very low sequence similarity, a motif of two hydrophobic, long chain residues, separated by 8 or 12 residues was found in many CaM-binding peptides (Fig. 10)(7, 8, 9) . Our binding fragment has a Trp residue in its NH(2)-terminal end but lacks the second hydrophobic residue, which should interact with the NH(2) CaM domain. Val, which could eventually play this role, is less hydrophobic and is in ``antiphase'' as compared to its counterparts in the high-affinity amphiphilic helices. Due to its remarkable conformational adaptability, CaM still recognize the sequence, but the interaction is considerably weaker.


Figure 10: Sequence comparison of different CaM-binding domains. The continuous line box at the NH(2) terminus indicates the first long-chain hydrophobic residue commonly encountered in these domains. The dashed line boxes label the second hydrophobic side chain necessary for the interaction with the NH(2) domain of CaM. In P Val (V in circle) may play this role but it is less hydrophobic (shorter chain) and is situated on the hydrophilic face of the helix.



The peptide P has a significantly greater affinity (24 times) for CaM than the shorter (20 amino acids) sequence P. As shown by the present results, the interacting segment in P contains several residues in the COOH-terminal portion which are absent in the shorter peptide. This may explain the difference in affinity between these two peptides. Recently, we have designed and overexpressed a larger peptide from the regulatory domain of B. pertussis adenylate cyclase having 29 additional residues at the NH(2)-terminal end (P). (^2)This peptide proved to have a 10-fold higher affinity for CaM than P, suggesting that the additional fragment may form new contacts with the protein. If this is true, we are in the presence of a new interaction mechanism between CaM and a target domain. P was uniformly N-labeled and its interaction with CaM is currently under investigation in our laboratories.


FOOTNOTES

*
This work was supported in part by the Centre National pour la Recherche Scientifique. The results presented in this paper are part of a Ph.D. thesis (of A. B.). 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.

§
To whom correspondence should be addressed: INSERM U350, Institut Curie, Section Biologie, Bâtiment 112, 91405, Orsay, France. Tel.: 33-1-69-86-31-63; Fax: 33-1-69-07-53-27; craescu{at}curie.u-psud.fr.

(^1)
The abbreviations used are: CaM, calmodulin; Boc, butyloxycarbonyl; NOESY, nuclear Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy; DQF-COSY, double-quantum filtered J-correlated spectroscopy; HMQC, heteronuclear multiple quantum correlation spectroscopy; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.

(^2)
H. Munier, A. Bouhss, A.-M. Gilles, N. Palibroda, O. Bârzu, J. Mispelter, and C. T. Craescu, submitted for publication.


ACKNOWLEDGEMENTS

We are indebted to M. Ikura for providing us the assignment of proton resonances of CaM and to K. Soda for providing the enzymes used in the N L-amino acid synthesis. We thank A. Tartar for helpful advice on the synthesis of the peptide and N. Palibroda for determination of N content of L-amino acids.


REFERENCES

  1. Babu, Y. S., Bugg, C. E., and Cook, W. J. (1988) J. Mol. Biol. 204, 191-204 [Medline] [Order article via Infotrieve]
  2. Chattopadhyaya, R., Meador, W. E., Means, A. R., and Quiocho, F. A. (1992) J. Mol. Biol. 228, 1177-1192 [Medline] [Order article via Infotrieve]
  3. Seeholzer, S. H., and Wand, A. J. (1989) Biochemistry 28, 4011-4020 [Medline] [Order article via Infotrieve]
  4. Prêcheur, B., Siffert, O., Bârzu, O., and Craescu, C. T. (1991) Eur. J. Biochem. 196, 67-72 [Abstract]
  5. Roth, S. M., Schneider, D. M., Strobel, L. A., VanBerkum, M. F. A., Means, A. R., and Wand, A. J (1992) Biochemistry 31, 1443-1451 [Medline] [Order article via Infotrieve]
  6. Munier, H., Blanco, F. J., Prêcheur, B., Diesis, E., Nieto, J. L., Craescu, C. T., and Bârzu, O. (1993) J. Biol. Chem. 268, 1695-1701 [Abstract/Free Full Text]
  7. Ikura, M., Clore, M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256, 632-638 [Medline] [Order article via Infotrieve]
  8. Meador, W. E., Means, A. R., and Quiocho, F. A. (1992) Science 257, 1251-1255 [Medline] [Order article via Infotrieve]
  9. Meador, W. E., Means, A. R., and Quiocho, F. A. (1993) Science 262, 1718-1721 [Medline] [Order article via Infotrieve]
  10. Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156-17163 [Abstract/Free Full Text]
  11. Charbonneau, H., Kumar, S., Novack, J. P., Blumenthal, D. K., Griffin, P. R., Shabanowitz, J., Hunt, D. F., Beavo, J. A., and Walsh, K. A. (1991) Biochemistry 30, 7931-7940 [Medline] [Order article via Infotrieve]
  12. Wolff, J., and Cook, G. H. (1973) J. Biol. Chem. 248, 350-355 [Abstract/Free Full Text]
  13. Wolff, J., Cook, G. H., Goldhammer, A. R., and Berkowitz, S. A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3840-3844
  14. Greenlee, D. V., Andreasen, T. J., and Storm, D. R. (1982) Biochemistry 21, 2759-2764 [Medline] [Order article via Infotrieve]
  15. Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A., and Danchin, A. (1988) EMBO J. 7, 3997-4004 [Abstract]
  16. Au, D. C., Masure, H. R., and Storm, D. R. (1989) Biochemistry 28, 2772-2776 [Medline] [Order article via Infotrieve]
  17. Munier, H., Gilles, A.-M., Glaser, P., Krin, E., Danchin, A., Sarfati, R., and Bârzu, O. (1991) Eur. J. Biochem. 196, 469-474 [Abstract]
  18. Glaser, P., Elmaoglu-Lazaridou, A., Krin, E., Ladant, D., Bârzu, O., and Danchin, A. (1989) EMBO J. 8, 967-972 [Abstract]
  19. Oldenburg, D. J., Gross, M. K., Wong, C. S., and Storm, D. R. (1992) Biochemistry 31, 8884-8891 [Medline] [Order article via Infotrieve]
  20. Presecan, E., Ivanof, A., Mocanu, A., Palibroda, N., Bologa, M., Gorun, V., Oargâ, M., and Bârzu, O. (1987) Enzyme Microbiol. Technol. 9, 663-667
  21. Glaser, P., Munier, H., Gilles, A.-M., Krin, E., Porumb, T., Bârzu, O., Sarfati, S. R., Pellecuer, C., and Danchin, A. (1991) EMBO J. 10, 1683-1688 [Abstract]
  22. Rance, M., Sorensen, O. W., Bodenhausen, G., Wagner, G., Ernst, R. R., and Wüthrich, K. (1983) Biochem. Biophys. Res. Commun. 117, 479-485 [Medline] [Order article via Infotrieve]
  23. Kumar, A., Ernst, R. R., and Wüthrich, K. (1980) Biochem. Biophys. Res. Commun. 95, 1-6 [Medline] [Order article via Infotrieve]
  24. Davis, D. G., and Bax, A. (1985) J. Am. Chem. Soc. 107, 2820-2821
  25. Bax, A., Ikura, M., Kay, L. E., Torchia, D. A., and Tshudin, R. (1990) J. Magn. Reson. 86, 304-318
  26. Levy, G. C., and Lichter, R. L. (1979) Nitrogen-15 Nuclear Magnetic Resonance Spectroscopy , J. Wiley & Sons, New York
  27. W ü thrich, K. (1986) NMR of Proteins and Nucleic Acids , John Wiley & Sons, New York
  28. Chazin, W. J., and Wright, P. E. (1987) Biopolymers 26, 973-977 [Medline] [Order article via Infotrieve]
  29. Englander, S. W., and Wand, A. J. (1987) Biochemistry 26, 5953-5958 [Medline] [Order article via Infotrieve]
  30. Szilagyi, L., and Jardetzky, O. (1989) J. Magn. Reson. 83, 441-449
  31. Pastore, A., and Saudek, V. (1990) J. Magn. Reson. 90, 165-176
  32. Wishart, D. S., Sykes, B. D., and Richards, F. M. (1992) Biochemistry 31, 1647-1651 [Medline] [Order article via Infotrieve]
  33. Veitch, N. C., Concar, D. W., Williams, R. J. P., and Whitford, D. (1988) FEBS Lett. 238, 49-55 [CrossRef][Medline] [Order article via Infotrieve]
  34. Bruix, M., Perello, M., Herranz, J., Rico, M., and Nieto, J. L. (1990) Biochem. Biophys. Res. Commun. 167, 1009-1014 [Medline] [Order article via Infotrieve]
  35. Kemminck, J., van Mierlo, C. P. M., Scheek, R. M., and Creighton, T. E. (1993) J. Mol. Biol. 230, 312-322 [CrossRef][Medline] [Order article via Infotrieve]
  36. Ikura, M., Kay, L., and Bax, A. (1990) Biochemistry 29, 4659-4667 [Medline] [Order article via Infotrieve]
  37. Le, H., and Oldfield, E. (1994) J. Biomol. NMR 4, 341-348 [Medline] [Order article via Infotrieve]
  38. Wishart, D. S., Sykes, B. D., and Richards, F. M. (1991) J. Mol. Biol. 222, 311-333 [Medline] [Order article via Infotrieve]
  39. Ikura, M., Kay, L. E., Krinks, M., and Bax, A. (1991) Biochemistry 30, 5498-5504 [Medline] [Order article via Infotrieve]
  40. Ohya, Y., and Botstein, D. (1994) Science 263, 963-966 [Medline] [Order article via Infotrieve]
  41. O'Neil, K. T., and DeGrado, W. F. (1990) Trends Biochem. Sci. 15, 59-64 [CrossRef][Medline] [Order article via Infotrieve]
  42. Cheetham, J. C., Raleigh, D. P., Griest, R. E., Redfield, C., Dobson, C. M., and Rees, A. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7968-7972 [Abstract]
  43. Stanfield, R. L., Fieser, T. M., Lerner, R. A., and Wilson, I. A. (1990) Science 248, 712-719 [Medline] [Order article via Infotrieve]
  44. Persechini, A., and Kretsinger, R. H. (1988) J. Biol. Chem. 263, 12175-12178 [Abstract/Free Full Text]
  45. Fisher, P. J., Prendergast, F. G., Ehrhardt, M. R., Urbauer, J. L., Wand, A. J., Sedarous, S. S., McCormick, D. J., and Buckley, P. J. (1994) Nature 368, 651-653 [Medline] [Order article via Infotrieve]
  46. Kraulis, P. (1991) J. Appl. Crystallogr. 24, 946-950 [CrossRef]

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