(Received for publication, November 14, 1994; and in revised form, January 20, 1995)
From the
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 C
protons allowed us to identify a 10-amino
acid fragment (Arg
to Arg
) which is in
rapid equilibrium between
-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
- and COOH-terminal
domains. Intermolecular NOE connectivities indicate that the
NH
-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.
The complex Ca-CaM (
)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
-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
-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
-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.
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
Cl (3 M in 1 M HCl) solution where the nitrogen resonates at
24.93 ppm from the liquid NH
(26) .
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.
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.
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
helical structures. In a regular
-helix
the distance between the sequentially amide protons (2.8 Å) is
considerably lower than in
-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
-helices and 2.2 Å in
strands). Other
medium-range NOE connectivities were unambiguously identified between
aromatic and aliphatic side chains: C
H and
C
H (Trp
)/C
H
(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
C
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 C
H resonances, the
mean value being 0.40 ppm(30) . Fig. 3shows the
secondary shift distribution, expressed as the chemical shift index (32) for C
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
-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
-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) .
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
, 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
N
1H 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 NH 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.
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
-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 C
H) 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.
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, CH, 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- 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
II
(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
H,
C
H, and C
H (Trp
) give
NOEs with C
H
of Leu
while
C
H (Trp
) has dipolar connectivities
with C
H
of Val
and C
H
of Ile
. From these data it appears that the
NH
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) .
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
II
(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.
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 (
H-
N) NOE results enables us to
localize the interacting segment as the contiguous length between
Leu
and Ala
. Therefore, the binding
fragment contains the
-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
H, C
H, and C
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
-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.
Figure 10:
Sequence comparison of different
CaM-binding domains. The continuous line box at the NH 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
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
-terminal end
(P
). (
)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.