From the Division of Physical Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom
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ABSTRACT |
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The specificity of interaction of the isolated N- and C-terminal domains of calmodulin with peptide WFFp (Ac-KRRWKKNFIAVSAANRFK-amide) and variants of the target sequence of skeletal muscle myosin light chain kinase was investigated using CD and fluorescence. Titrations show that two molecules of either domain bind to 18-residue target peptides. For WFFp, the C-domain binds with 4-fold higher affinity to the native compared with the non-native site; the N-domain shows similar affinity for either site. The selectivity of the C-domain suggests that it promotes occupancy of the correct binding site for intact calmodulin on the target sequence. Far UV CD spectra show the extra helicity induced in forming the 2:1 C-domain-peptide or the 1:1:1 C-domain-N-domain-peptide complex is similar to that induced by calmodulin itself; binding of the C-domain to the Trp-4 site is essential for developing the full helicity. Calmodulin-MLCK-peptide complexes show an approximate two-fold rotational relationship between the two highly homologous domains, and the 2:1 C (or N)-domain-peptide complexes evidently have a similar rotational symmetry. This implies that a given domain can bind sequences with opposite peptide polarities, significantly increasing the possible range of conformations of calmodulin in its complexes, and extending the versatility and diversity of calmodulin-target interactions.
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INTRODUCTION |
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Calmodulin is a regulatory protein involved in a variety of Ca2+-dependent cellular signaling pathways. Its importance as a mediator of the second messenger Ca2+ is reflected in its high conservation throughout evolution. This apparently contrasts with its unique ability to interact strongly with and to regulate selectively a variety of proteins (at least 30) without any obvious sequence homology in their calmodulin binding region (see Refs. 1-5 for reviews). Recently, structures of calmodulin and calmodulin-peptide complexes at atomic resolution have been determined (reviewed in Refs. 2, 6, and 7), showing two similar domains with two Ca2+ binding sites each, which for calmodulin in solution are connected by a flexible linker (8-15). The conformational change upon Ca2+ binding to calmodulin exposes those residues that create the binding site for most target proteins (1, 6, 7, 16).
Binding of the Ca2+ saturated form of calmodulin to the
target protein triggers its activation. Despite the wide target range, target affinities are strong (Kd 1 nM) (2-4). The interaction is apparently mediated by both
hydrophobic and electrostatic forces (17). NMR (18) and x-ray
structures (19) of two related Ca2+-calmodulin-target
peptide complexes show that the
-helical MLCK target peptide lies in
a hydrophobic channel composed by the two domains, with the
predominant interactions being those between the N- and C-terminal
domains and the C- and N-terminal portions of the target,
respectively.
Calmodulin can be cleaved by trypsin to generate two half-molecules, i.e. the C-terminal and the N-terminal Ca2+ binding domain (20, 21). The equilibrium (22-24) and kinetic properties (25, 26) of intact calmodulin in the Ca2+ binding and dissociation reactions, as well as the secondary structure (24, 27), are well represented by a summation of the properties of these fragments, suggesting that the two domains are effectively independent structures. The isolated domains are capable of activating target proteins, but the degree of activation varies with the target protein (26, 28-35). In particular, skeletal muscle myosin light chain kinase (sk-MLCK)1 is activated best by a 1:1 mixture of the domains (85% activation compared with calmodulin), but less by either the C-domain (65%) or the N-domain (20%) (31). This activation pattern is reproduced when calmodulin chimeras consisting of two linked N-domains or two C-domains are used (36). Although the C-domain activates target enzymes better than the N-domain in several cases (29-31, 35), this is not always so (31, 33). Therefore, differences in domain sequence and structure may contribute to the versatility of calmodulin's regulatory functions. Binding of the domains to the target enzyme is not necessarily sufficient for activation since isolated domains can inhibit calmodulin-induced activation of enzymes which are not activated by the domain itself (28, 29, 31, 35).
Although the overall structure of the two domains show marked similarities (37), differences between them in sequence and Ca2+ affinity have apparently been well conserved during evolution (38). This points toward possible functional differences of the two domains, which can be further amplified by variations in target sequences. Studies of enhancement of Ca2+ affinities of calmodulin by target sequences suggest that calmodulin which is half-saturated with Ca2+ could bind to the target protein by only one domain without activation, allowing a rapid response in enzyme activity to an increase in Ca2+ concentration (39).
In the present work, we address on a molecular level the specificity of the interaction of the individual calmodulin domains with target peptides derived from the target sequence of sk-MLCK. Previously, spectroscopic studies have been reported on the interaction of the domains with the short peptides melittin and mastoparan (40-42). Here, the binding affinities, the molecular interactions in the complex, and the effects of domain binding on the conformation of the target peptides are investigated using a variety of spectroscopic techniques. The striking finding is that two molecules of either domain bind with good affinity to the 18-residue target peptides. The symmetry of the resulting complexes is considered by comparison with the calmodulin-MLCK peptide structure (2), and is discussed in relation to the known versatility of calmodulin in its specific interactions with a range of target proteins. The feasibility of calmodulin domains to bind with reversed polarity and alternative positions on a target sequence greatly extends the potential range of conformations of calmodulin in its bound form, and provides added diversity to the calcium sensitivity of calmodulin-dependent activation processes.
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MATERIALS AND METHODS |
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Proteins and Peptides-- Drosophila melanogaster calmodulin expressed in Escherichia coli was purified as described previously (43). The purified protein ran as a single band on an SDS-polyacrylamide gel electrophoresis (15% gel; Laemmli system). The tryptic fragments of calmodulin were prepared as described in Ref. 31 with an additional gel filtration step (G75 column) included after the cleavage and before the anion exchange chromatography. For each fragment, no impurities could be detected on SDS-polyacrylamide gel electrophoresis. However, HPLC chromatography showed an impurity of unknown origin in the C-domain preparation corresponding to 2.4% of the protein mass. Mass spectroscopy showed that the predominant fragment for the N-domain preparation was residues 1-75 of calmodulin and, for the C-domain preparation, residues 78-148.
WFFp (Ac-KKRWKKNFIAVSAANRFK-NH2), WF10p (Ac-KKRWKKNFIA-NH2), FFFp (Ac-KKRFKKNFIAVSAANRFK-NH2), FFWp (Ac-KKRFKKNFIAVSAANRWK-NH2), and FW10p (Ac-IAVSAANRWK-NH2) were synthesized on an Applied Biosystems 430A peptide synthesizer and purified by reverse-phase HPLC on a C18 column. All peptides were protected at both termini. Peptide purity was assessed by mass spectrometry, reverse-phase HPLC, and ion exchange chromatography. Concentrations of proteins and peptides were determined spectrophotometrically using the following extinction coefficients: 5690 MFluorescence Measurements--
Uncorrected fluorescence emission
spectra were recorded in UV-transmitting plastic cuvettes or in quartz
cuvettes using a SPEX FluoroMax fluorimeter. Excitation was at 300 nm
for measurements at 50 µM peptide concentration,
otherwise at 290 or 295 nm for the C-domain and at 280 or 300 nm for
the N-domain (bandwidth 0.9 nm). The emission was scanned from 300 or
310 nm to 400 nm (bandwidth 4.3 nm). The temperature was 20 °C and
the buffer 25 mM Tris/HCl, 100 mM KCl, 1 mM CaCl2 at pH 8.0. Data for the fluorescence titrations were obtained either by integrating the spectra in the
region of the largest fluorescence change (300-330 nm) or by
measurements of 30 s in duration at a wavelength in the range 322-334 nm with bandwidth 17 nm. Unless otherwise stated, the dissociation constants are derived from at least three independent titrations.
Circular Dichroism (CD) Measurements--
CD spectra were
recorded in fused silica cuvettes using a Jasco J-600
spectropolarimeter. The measurements were made at 20 °C in 25 mM Tris/HCl, 100 mM KCl, 1 mM
CaCl2 at pH 8.0. Far UV-CD measurements (200-280 nm) with
calmodulin and all peptides as well as with WFFp and isolated domains
were made using 1-mm cuvettes with peptide and protein concentrations
in the range 7-25 µM. With isolated domains and FFFp or
FFWp the measurements were made in a 0.1-mm demountable cuvette at
10-fold higher concentrations. Spectra are presented as the molar CD
absorption coefficient (M) using the molar
concentration of the protein rather than the mean residue weight for
the normalization. In the case of the domain mixture complexes, the
concentration of the complex was used for the normalization to
facilitate the comparison with the spectra of calmodulin (46). The
difference between the CD absorption coefficient at 222 nm in the
presence and the absence of the peptide was used to estimate the number
of residues adopting a helical structure upon complex formation. The
results of three experiments were averaged. The difference was
expressed as molar in peptide (not protein) concentration and compared
with the
M value of fully helical peptides of
different lengths, which were calculated according to Ref. 45.
Data Analysis and Determination of Dissociation Constants-- Titrations were performed by addition of the domain solution to the peptide solution, and recording changes in fluorescence or CD signals deriving from the Trp chromophore of the peptide. Titration curves of WF10p and FW10p peptide with either domain were fitted with a stoichiometry of 1:1, using standard fitting procedures (46). In the case of the long peptides WFFp and FFWp, the titration curves clearly indicated that saturation of the optical signal was achieved close to a stoichiometry of two molecules of domain per molecule of peptide. The simplest model for binding assumes that the optical signal monitors binding of a domain to the Trp-containing portion of the peptide; binding of a second molecule of the domain is revealed only indirectly via the competition of the domain between the two sites. The analysis is based on the known structure of the complex of calmodulin with the sk-MLCK target M13 peptide, in which the C-domain of calmodulin, interacting exclusively with the Trp residue of the peptide, binds predominantly with the (Trp containing) N-terminal portion of the peptide, and the N-domain binds predominantly with the C-terminal portion of the peptide. The two sites on the peptide are therefore designated according to their position in the peptide sequence as site N and site C; in the case of binding to peptide WFFp, binding at site N with KdN, produces an optical signal, and binding at site C with KdC is optically "silent," whereas for peptide FFWp, the optical properties of sites N and C are reversed.
The mechanism for the binding of two molecules of a given domain (D) per molecule of peptide with sites N and C (understood to be oriented N-peptide-C) is shown in Scheme ins;1857s1}1.
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RESULTS |
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Fluorescence Spectra-- Solutions of the free Trp-containing peptides have a fluorescence emission maximum at ~358 nm. The maximum of the enhanced fluorescence emission of all the peptide-domain complexes (Table I) lies between 335 and 338 nm, except for the N-WF10p complex (348 nm), suggesting that the N-domain is less effective than the C-domain in burying the Trp residue of this short peptide in a hydrophobic environment. As discussed in more detail below, two molecules of either domain were found to bind to one molecule of full-length peptide (WFFp or FFWp). Complexes are represented as e.g. X-WFFp-Y, indicating that domain X binds to the N-terminal portion of the peptide and domain Y to the C-terminal portion.
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Near UV CD Spectra--
Near UV CD spectra of the domains in the
absence and the presence of a target peptide, as well as spectra of the
free peptides are shown in Fig. 1. The
free peptides WFFp (Fig. 1A), FFWp (Fig. 1B), and
WF10p (data not shown) have similar near UV CD spectra; below 290 nm,
the signal increases steadily and without fine structure to a
M = 0.4 M
1
cm
1 at 255 nm. Only the FW10p peptide (Fig.
1C) shows a signal above 290 nm.
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Near UV CD Spectra of Complexes with Peptide WFFp--
The near UV
CD difference spectrum of the bound Trp in the C-WFFp-C complex (Fig.
1D) is stronger than that with the shorter WF10p peptide
(Fig. 1D). The Trp signal is also affected by the end
protection of the peptides, the unprotected versions (suffix "u")
showing the weaker signal (39). At 295 nm, the signal decreases in the
order: C-WFFp-C calmodulin-WFFp
C-WFFp-N > calmodulin-WFFu = C-WFFu > C-WF10p > calmodulin-WF10u = C-WF10u. These variations could be due to
relatively localized conformational differences of the Trp residue
relative to the binding pocket and the peptide backbone, and the
effects of different degrees of mobility of Trp and surrounding
residues. We conclude that there is some variation in the binding mode
of the C-domain to the Trp residue in the different peptides.
Near UV CD Spectra of Complexes with Peptide FFWp-- The spectrum of N-FFWp-N has the same form as that of the complex of calmodulin with FFWu (46) (and FFWp; data not shown), but with intensity reduced to 50%, suggesting that the Trp residue is bound similarly in both complexes but with increased mobility within the N-FFWp-N complex. For F10p-N, a significantly weaker signal than for N-FFWp-N is observed, which is not the case when the calmodulin complexes with FFWu and FW10u are compared (39).
C-FFWp-C shows the weakest CD spectrum of the complexes with the long peptides, and this is the only case where the spectrum with the corresponding short peptide FW10p does not show weaker intensity and is significantly different in shape. Therefore, the Trp binding modes are different for the long and the short peptide and are possibly a superposition of spectra of distinct complex conformations.Near UV CD Spectra of Complexes with a 1:1 Domain Mixture-- To find out whether the 1:1 mixture of C- and N-domain binds in a manner similar to that of intact calmodulin, near UV CD spectra were recorded of the mixture with and without peptide WFFp or FFWp. The Trp spectrum of the 1:1:1 complex with WFFp is very similar in shape to that of C-WFFp-C and of the calmodulin-WFFp complex (Fig. 1D). It is estimated that the Trp spectrum of N-WFFp-N contributes less than 15% to the 1:1:1 complex spectrum. Thus, in the 1:1:1 complex, the Trp residue of the WFFp peptide effectively binds only to the C-domain, resulting in the C-WFFp-N complex, whose spectrum closely resembles that of intact calmodulin-WFFp.
In contrast, the spectrum of the 1:1:1 complex of C- and N-domain with FFWp is significantly different from those of N-FFWp-N (Fig. 1E) or the complexes of calmodulin with FFWu (46) and FFWp, but is very similar to that of FW10p-N and, despite a deviation at 295 nm, to the C-FFWp-C spectrum (Fig. 1F). This may suggest that the isolated C-domain can bind (in part) to the unusual Trp containing C-terminal site on the peptide.Binding Stoichiometry and Affinities of Individual Calmodulin Domains for the Target Peptide-- The fluorescence and CD spectra (Fig. 1, D-F) show that each domain binds to the single Trp in the N-terminal portion of WFFp and the C-terminal portion of FFWp and to the short peptides WF10p and FW10p. The fluorescence and CD signals from this Trp residue were used to determine the affinities of the domains for the peptides and the stoichiometry of the complexes.
For the short WF10p and the FW10p peptides, the fluorescence titrations with either domain were fitted well with 1:1 stoichiometry and the Kd values obtained are listed in Table I. For the full-length peptides, titrations of the CD and fluorescence signals with a given domain show that the stoichiometry is clearly greater than 1 (see Fig. 2). These data were analyzed with the two binding site model described under "Materials and Methods." Fig. 2 shows typical examples of two CD titrations (C-domain and WFFp, Fig. 2A; N-domain and WFFp, Fig. 2B) and of two fluorescence titrations at low peptide concentration (C-domain and WFFp, Fig. 2B; N-domain and WFFp, Fig. 2D). The dissociation constants obtained are illustrated in Fig. 3 and listed in Table I together with the corresponding Gibbs free energies, calculated as RT log Kd.
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Far UV CD Spectra of Complexes of Calmodulin or Calmodulin Domains
with Target Peptides--
Secondary structure changes upon WFFp, FFFp,
or FFWp binding to the domains have been monitored using far UV CD
spectroscopy. The domain and the complex spectra were normalized using
the protein concentration, except in the case of the domain mixture,
where the concentration of one domain was used to facilitate the
comparison with the spectra of calmodulin (46). The spectra of the free domains are dominated by the contributions from -helical structures with the characteristic minima near 207 and 222 nm. The spectrum of the
1:1 domain mixture agreed within experimental error with the
corresponding spectrum of calmodulin confirming earlier results for
bovine testis and brain calmodulin (24, 27) that the domain structure
is preserved in the tryptic fragments. Upon addition of the
(unstructured) peptide to the domains, there is an increase in
-helicity consistent with the peptide adopting an
-helical structure in the complex. The conformation of the calmodulin domains is
largely unaffected by binding of the target sequence (18), although
slight structure perturbations have been reported (47). Far UV CD was
measured at different domain:peptide ratios and the change in the CD
spectrum was normalized to peptide concentration. The corresponding
values at 222 nm are collected in Table II together with the change in the number
of helical residues. Calmodulin, the C-domain, and the domain mixture
induce the same high degree of helicity in each peptide, whereas the
N-domain is as effective only with FFFp. The induced helicity tends to be highest for peptide WFFp, decreasing with the number of replacements for FFFp and FFWp. The exception again is the N-FFFp-N complex, which
shows the same induced helicity as N-WFFp-N. Table II shows that the
increased helicity saturates at a 2:1 domain:peptide ratio for peptides
WFFp, FFFp, or FFWp. A 1:1 domain:peptide ratio induces more than 50%
of the maximum helicity, suggesting that binding of the first molecule
of either domain to the long peptides induces more helicity than
binding of the second.
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DISCUSSION |
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The Number of Domain Binding Sites on the 18-residue Peptides WFFp and FFWp-- In the complex of calmodulin with the target sequence of sk-MLCK, the C-domain of calmodulin binds mainly to residues in the N-terminal portion of the target peptide and the N-domain mainly to those in the C-terminal portion (18). This work clearly shows that two molecules of either the C-domain or the N-domain bind to the 18-residue sk-MLCK target peptide WFFp and to the variants FFFp and FFWp. In agreement with this, either domain will bind with 1:1 stoichiometry to the short peptides WF10p or FW10p, which represent the N- and the C-terminal portion of the WFF and FFW target sequences, respectively.
The Hydrophobic Pockets of the Domains--
The maximum of the
enhanced fluorescence at 335-338 nm (Table I) shows that either domain
binds the bulky Trp residue of the peptides, which becomes buried in a
hydrophobic pocket. Generally, the near UV CD Trp spectrum is unique
for each 2:1 or 1:1 domain-peptide complex, and the high intensities
( ± 1-2 M
1 cm
1) are
consistent with strong immobilization of the chromophore. Both static
and dynamic properties of the environment provided by the peptide and
binding pockets of the domains can contribute to the CD signal, and are
therefore characteristic for each complex. We conclude that the binding
mode of the Trp residue depends on its position in the peptide
sequence, the length of the peptide, and the domain to which it is
bound. Near UV CD spectra of complexes of the short peptides WF10p and
FW10p differ from those the 18-residue peptides WFFp and FFWp.
Therefore, the exact binding mode of the Trp residue is not solely
determined by local interactions between Trp and the protein, but is
also sensitive to longer range interactions.
Structural Change of the Peptide--
The far UV CD data show that
calmodulin, the C-domain, and the domain mixture induce a high degree
of helicity in all three peptides: WFFp, FFFp, or FFWp. Compared with
the C-domain, the N-domain is equally effective with FFFp, and
significantly less so with WFFp and FFWp. The helicity induced by the
1:1 mixture of isolated domains is for all peptides close to that
induced by intact calmodulin. The C-domain can replace the N-domain
from its site in the calmodulin complexes without affecting the peptide helicity. On the other hand, replacing the C-domain from its site in
the calmodulin complexes with the N-domain reduces the helicity of
Trp-containing peptides (with an estimated loss of five helical residues). Thus, the C-domain is generally more effective than the
N-domain in maintaining the helicity of the full peptide, as
e.g. in the C-WFFp-C complex. For most complexes, the
induced helicity clusters between 120 and 150 M1 cm
1 corresponding to 13-15
helical residues. For the peptide to become fully helical, the presence
of a Trp residue at position 4 of the target peptide sequence as well
as its interaction with the C-domain is advantageous. Trp-4 does not
have this function of inducing maximum helicity in the interaction with
the N-domain. Thus, the binding pocket of the C-domain and the WFFp
peptide sequence appear mutually optimized to obtain a highly helical target peptide.
The Role of the Individual Domains in the Target Recognition Process-- Both C-domain and N-domain form complexes with either WFFp or FFWp with a stoichiometry of 2 mol of domain/mol of peptide. The N- and the C-domain bind to both sites on the peptide with surprisingly similar affinities (see Table I), with the highest affinity interactions being those between the C-domain and the Trp-containing portion of the WFFp or FFWp target sequences. Similar conclusions were reached from studies of sk-MLCK activation (31); two binding sites for calmodulin domains were found on the enzyme with dissociation constants for the C-domain of KdN = 300 nM and KdC = 20 µM and for the N-domain of KdN = 12 µM and KdC = 3-5 µM (the values are presented according to the nomenclature in this paper from the tentative assignment in the original paper). The Kd values obtained here with a target peptide tend to be 1-2 orders of magnitude smaller than those derived from the enzyme activation studies. This may suggest that binding of the domains to the whole enzyme may require energetically costly disruption of interactions between the calmodulin binding site and other parts of the enzyme (1, 2).
The subtle affinity differences of the two domains for the binding sites on the peptide support the view that the key recognition process between calmodulin and the native target peptide is due to the interaction of the C-domain with the N-terminal portion of the target sequence (31, 39, 48): its preference for the "correct" binding site on the peptide and its higher affinity for this site as compared with the N-domain ensure the correct orientation of the complex. Consistent with this, a 1:1 mixture of the separated N- and C-domains appears to bind to the native target sequence WFFp in the same orientation as intact calmodulin (see "Results"). The resulting C-WFFp-N complex is virtually indistinguishable spectroscopically from the calmodulin-WFFp complex, in terms of the interactions leading to the exact mode of Trp binding and helicity induced in the peptide. This indicates that it is an intrinsic property of the C-domain and N-domain when present together to form a complex of almost identical structure as with calmodulin. Evidence has recently been presented for a potential functional difference between the two domains. At intermediate Ca2+ concentrations (The Role of the Trp Residue in the Target Sequence on the Recognition Process-- The experiments with the FFWp and FFFp variant peptides show the importance of the interaction between Trp-4 of the peptide and the C-domain. Replacing Trp-4 by Phe appears to decrease the helicity in the N-terminal part of the bound peptide, and diminishes the selectivity of that peptide portion for the C-domain. With FFWp, the C-terminal binding site shows the higher affinity for both C- and N-domain. Binding of the C-domain with the C-terminal site of FFWp implies a type of interaction that is not observed in the calmodulin-FFWp complex, which apparently has the same peptide orientation as WFFp (48). This suggests a possible functional role of linking the two domains together in one molecule, which may ensure a defined orientation of the interaction.
Cooperativity and the Energetics of the Domain-Peptide
Interactions--
The formation of the 1:1:1 mixed complex C-WFFp-N
raises the question of co-operativity in the interactions of calmodulin domains with the target sequence. The sum of the total Gibbs free energy change (RT log Kd) for formation
of C-WFFp and WFFp-N as calculated from the values in Table I is 82
kJ/mol and is larger than the corresponding value,
78.9 kJ/mol,
obtained for formation of WFFp-C and N-WFFp. This shows that the
C-WFFp-N complex is energetically preferred, as deduced from the near
UV CD spectra (see "Results"). Simulations using the
Kd values of Table I show that, under the conditions
of the CD experiment with the 1:1 domain mixture, up to 34% of the
complexes would involve "non-native" binding of the peptide Trp to
the N-domain, exceeding the upper limit (<15%) estimated from the
near UV CD data. This suggests a somewhat higher degree of specificity
of the individual domains in the 1:1 C:N-domain mixture for their "native" binding sites on WFFp than is predicted from the
interaction of two identical domains with the peptide. Introducing in
the simulation a positive cooperativity factor of f = 0.5 in forming the C-WFFp-N complex decreases the proportion of
non-native complex to 11%, i.e. consistent with the near UV
CD data. Thus, this factor of 0.5, corresponding to a change of less
than 5% of the free energy change in the interaction, is sufficient to
reconcile the homodomain titrations with the mixed domain near UV CD
results. Thus, although the independent binding site model is generally adequate for the homodomain case, there is some evidence for a small
degree of cooperativity between the complex of WFFp with the mixed N-
and C-domains, and it is likely that this would be further enhanced in
the interaction of calmodulin itself with the target sequence.
Structural Model for the Domain-Peptide Complexes-- The complexes of calmodulin with target peptide M13 (sk-MLCK; Ref. 18) and the RS20 peptide (sm-MLCK; Ref. 19) show an approximately two-fold rotational relationship between the N- and the C-domain of calmodulin as indicated in Fig. 4A. For the interaction of the two copies of one individual domain with the full target sequence, two distinct structural models may in principle be considered. They are presented schematically in Fig. 4 (B and C). It is assumed that the one copy of C-domain that binds to its native binding site on WFFp (colored red in Fig. 4, B and C) does so in its normal orientation as in calmodulin-WFFp. This is supported by the CD results, which give evidence for closely related binding modes for Trp in C-WFFp-C, C-WFFp-N, and the respective complexes with calmodulin (see above). The copy of the domain that binds to the non-native site (shown in green) may in principle do so in an orientation related to the native site either by rotational symmetry (axis out of plane perpendicular to the peptide, Fig. 4B) or by helical symmetry (translation along and rotation around the peptide axis, Fig. 4C). These distinct models have significant implications for the principles of calmodulin-target-sequence interactions. It is therefore important to analyze the similarities of the two domains together with the contacts which each makes with the target sequence.
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ACKNOWLEDGEMENTS |
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We thank Kate Beckingham for advice on expression systems, S. Howell for the mass spectra, P. Fletcher for synthesis of some peptides, and P. Browne for valuable discussion. We thank Dr Tony Wilkinson (University of York) for demonstration of the symmetry of the calmodulin-MLCK peptide complexes.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Wellcome Research Fellowship
044093/Z/95/Z/IGS/ JS/CG.
§ To whom correspondence should be addressed. Tel.: 44-181-959-3666; Fax: 44-181-906-4419; E-mail: p-bayley{at}nimr.mrc.ac.uk.
1 The abbreviations used are: sk-MLCK, skeletal muscle myosin light chain kinase; FFFp, Ac-KKRFKKNFIAVSAANRFK-NH2; FFWp, Ac-KKRFKKNFIAVSAANRWK-NH2; FFWu, unprotected version of FFWp; FW10p, Ac-IAVSAANRWK-NH2; FW10u, unprotected version of FW10p; sm-MLCK, smooth muscle myosin light chain kinase; WF10p, Ac-KKRWKKNFIA-NH2; WF10u, unprotected version of WF10p; WFFp, Ac-KKRWKKNFIAVSAANRFK-NH2; WFFu, unprotected version of WFFp; X-WFFp-Y and related abbreviations, domain-WFFp-complex in which domain X interacts with the Trp-containing N-terminal portion of WFFp and domain Y with the C-terminal portion; M13 and RS20, target sequences of sk-MLCK and sm-MLCK, respectively; HPLC, high performance liquid chromatography.
2 A. Barth, S. R. Martin, and P. M. Bayley, submitted for publication.
3 S. R. Martin, unpublished data.
4 K. Johnson, K. Beckingham, and F. Quiocho, personal communication.
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REFERENCES |
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