(Received for publication, August 13, 1996, and in revised form, October 30, 1996)
From the Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom
The mechanism of dissociation reactions induced by calcium chelators has been studied for complexes of Drosophila calmodulin with target peptides, including four derived from the skeletal muscle myosin light chain kinase target sequence. Reactions were monitored by fluorescence stopped-flow techniques using a variety of intrinsic probes and the indicator Quin2. For most of the complexes, apparently biphasic kinetics were observed in several fluorescence parameters. The absence of any obvious relationship between dissociation rates and peptide affinities implies kinetic control of the dissociation pathway. A general mechanism for calcium and peptide dissociation was formulated and used in numerical simulation of the experimental data.
Unexpectedly, the rate of the slowest step decreases with increasing [peptide]/[calmodulin] ratio. Numerical simulation shows this step could contain a substantial contribution from a reversible relaxation process (involving the species Ca2-calmodulin-peptide), convolved with the following step (loss of C-terminal calcium ions). The results indicate the potentially key kinetic role of the partially calcium-saturated intermediate species. They show that subtle changes in the peptide sequence can have significant effects on both the dissociation rates and also the dissociation pathway. Both effects could contribute to the variety of regulatory behavior shown by calmodulin with different target enzymes.
Calmodulin is involved in the regulation of a range of cellular
functions, usually through its Ca2+-dependent
activation of target proteins (1).
Ca4-CaM1 binds to many target
proteins with high affinity (Kd nM)
and binds peptides derived from the calmodulin binding regions of these
proteins with similar affinities.
The x-ray crystal structure of Ca4-CaM (2-4) shows two
globular domains with similar conformation, each containing two
helix-loop-helix Ca2+ binding sites. Those in the C-domain
have a higher affinity than those in the N-domain, and there is
positive cooperativity between two sites within a domain (5). The
crystal structure shows the two domains separated by an extended
-helix. In solution, this central helix contains a loop (residues
74-82) which allows the calmodulin domains to interact closely with
the peptide (6).
Calcium binding to calmodulin induces a conformational change that
exposes hydrophobic surfaces which comprise the binding site for target
molecules. The solution structure of the complex of Ca4-CaM
with M13, a 26-residue peptide derived from sk-MLCK, has been
determined by NMR (7). The M13 peptide is in an -helical conformation, effectively enclosed by the N- and C-domains of the
calmodulin. The N and C termini of the peptide interact primarily with
the C- and N-domains of calmodulin, respectively, and the Trp-4 and
Phe-17 residues of the peptide appear to play an important anchoring
role. The structures of the complexes of Ca4-CaM with peptides derived from sm-MLCK and CaM kinase II have been determined by
x-ray diffraction (8, 9). The structures of the three complexes are
rather similar, although there are significant differences in the
positions of the peptides and the relative orientations of the
calmodulin domains. In particular, the complex of Ca4-CaM with the CaM kinase II peptide has fewer peptide residues
involved in helix formation and contact with the calmodulin, and
the longer loop in the central helix region (residues 73-83) allows
the domains to move closer together (9). The different structures show how calmodulin can adapt to bind peptides of different sequences, while
maintaining high affinity in the interaction.
In recent work we studied the peptide WFF, which corresponds to residues 1-18 of M13 and contains the major sites of interaction with CaM (7). We have also permuted the sequence of WFF to include either Trp or Phe residues at positions 4, 8, and 17 (peptides FWF, FFW, and FFF: Table I). These peptides bind with high affinity to Ca4-CaM and retain the standard orientation with residues 4 and 17 interacting with CaM C- and N-domains, respectively (10, 11). They are therefore well suited for investigating the effect of controlled structural modifications on the kinetics and equilibria of CaM-target peptide interactions (12, 13).
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In the present work we have measured the dissociation kinetics of seven
Ca4-CaM·peptide complexes (14). The peptides studied are
the four related to M13, the wasp venom peptides mastoparan and
mastoparan X, and a synthetic peptide designed to form a basic amphiphilic -helix (15). The effects of Ca2+ removal
with a chelator were monitored using different fluorescent signals,
including those of the chelator Quin2, peptide Trp, CaM Tyr (Tyr-138 in
Drosophila CaM), and a Trp-containing calmodulin mutant,
T26W (16, 17). Correlation of the kinetics observed with these
different probes allows the deduction of a general mechanism for the
dissociation of Ca4-CaM·peptide complexes. The results
obtained are compared with other studies on the dissociation of
CaM·peptide and CaM·protein complexes (18-24). We show that the
predominant kinetic pathway is sensitive to changes in individual residues of the target peptides and deduce that the rate of the slowest
step is determined by the contribution of a hitherto unsuspected kinetic relaxation mechanism involving the intermediate species Ca2-CaM·peptide, with two Ca2+ ions bound in
the C-domain.
Drosophila melanogaster CaM was prepared as described (10). The T26W SYNCAM mutant was a gift from Dr. J. Haiech. The peptides WFF, FFW, FWF, FFF, and CBP1 (Table I) were synthesized on an Applied Biosystems 430A peptide synthesizer and purified by reverse phase high performance liquid chromatography. The mastoparans were from Bachem. Peptide purity was assessed by mass spectroscopy and high performance liquid chromatography. All solutions were prepared in 25 mM Tris, 100 mM KCl (pH 8). Concentrations were determined using reported extinction coefficients for CaM (25) and calculated extinction coefficients at 259 or 280 nm for T26W and the peptides (26). The affinities of the peptides for CaM were measured by direct fluorometric titration (10) or by competition with a peptide of known affinity.2
Stopped-flow MeasurementsStopped-flow experiments were
performed on a Hi-Tech SF61-MX stopped-flow spectrophotometer. Trp,
Tyr, and Quin2 fluorescence signals were monitored using excitation
wavelengths of 290, 280, and 334 nm, respectively, and emission cut-on
filters of 320, 305, and 370 nm, respectively. Concentrations quoted
are those prior to 1:1 mixing. The instrument dead-time is 3 ms at a
drive pressure of about 4 bar (0.4 MPa). EGTA-induced dissociation was studied by mixing 0.5-2 µM CaM·peptide complex (in 100 µM Ca2+) with 20 mM EGTA. The
observed rates were independent of [EGTA] in the range 1-20
mM. Quin2-induced dissociation was studied by mixing 3 µM CaM·peptide complex (in 20 µM
Ca2+) with 90 µM Quin2. The observed rates
were slightly dependent on [Quin2]. Dissociation induced by the
addition of a silent peptide (CBP1 or FFF) was studied by mixing 0.5-1
µM CaM·peptide complex (in 100 µM Ca)
with excess silent peptide. Finally, association reactions between CaM
and peptide were studied by monitoring Trp fluorescence after mixing of
0.4 µM peptide with 0.4 µM
Ca4-CaM (in 100 µM Ca2+).
For each reaction studied at
least six stopped-flow traces were averaged for nonlinear least squares
analysis. A single exponential was considered satisfactory unless a
better 2 and residual distribution were obtained for a
two-exponential fit. Rate constants were determined independently at
least twice and are reported as mean ± S.D. The EGTA-induced
dissociation of FFF monitored by Tyr fluorescence showed a pronounced
lag phase and was fitted to the equation for the appearance of C in a
first-order series reaction, A
B
C. Reactions showing only a
small lag phase were fitted to a single exponential. Association
reactions were studied under second-order conditions, and approximate
values for association rate constants (kon) were
obtained by comparing the experimental curves with simulations for
kon values in the range 0.1-5 × 109 M
1·s
1.
Numerical simulations were performed using the program KSIM (Runge-Kutta algorithm) and analyzed using the program KFIT (from Dr. N. Millar). Concentrations of complexes present prior to mixing were established by a short presimulation starting at the appropriate total concentrations of peptide and Ca4-CaM. The following assumptions were made for the simulations. All steps involving Ca2+ dissociation were taken to be irreversible because of the large excess of chelator used (27). The Tyr-138 fluorescence of CaM was assumed to change only upon loss of Ca2+ from the C-terminal sites (28). The change in Trp fluorescence of the peptide was assumed to be due to complete dissociation of the peptide from the complex and was not affected by loss of the Ca2+ ions from one of the domains. The behavior of the Trp containing mastoparans is more complicated, since the 1:1 CaM·MasX complex with empty N-terminal Ca2+ sites has a higher fluorescence than the fully Ca2+-saturated complex (29).3 For the CaM T26W mutant, the change in Trp fluorescence is mainly due to the loss of the N-terminal Ca2+ ions (16, 17). Finally, since the Trp signal for dissociation of a Trp peptide and that for the T26W mutant ± Ca2+ are similar, they were made identical in simulations.
Association of Ca4CaM and Peptide
Fluorescence changes on mixing of WFF, FFW, or FWF (0.4 µM) with excess Ca4-CaM (at 20 °C) were
almost complete within the instrument deadtime, indicating a
bimolecular association rate constant (kon) in
excess of 8 × 108 M1
s
1. We therefore studied the association of FWF (0.4 µM) with Ca4-CaM (0.4 µM) at
9.5 °C (Fig. 1D). The
kon value obtained as described under
"Materials and Methods" was 1 ± 0.5 × 109
M
1 s
1. This is similar to a
value of 9 × 108 M
1
s
1 found for the reaction of a fluorescently labeled CaM
with two peptides derived from sm-MLCK (22) and to a value of 2 × 109 M
1 s
1 deduced
from NMR studies on a peptide derived from Bordetella pertussis adenylate cyclase (30). These on-rates are close to the
expected diffusion limit of 2 × 109
M
1 s
1 (31, 32). The high
kon has important consequences for subsequent mechanistic arguments (see "The Kinetic Model"). Although slow processes attributed to conformational changes have been observed in studies of the association of fluorescently labeled CaMs with peptides and proteins (22, 33), no slow processes were observed in the
association reactions studied here. Values of
kon for the interaction of CaM with proteins
tend to be lower (20, 21), but a value of 5.3 × 108
M
1·s
1 was measured for
caldesmon (20).
Calcium Dissociation from Calmodulin
Rates for the dissociation of the C-terminal Ca2+ ions
from Ca4-CaM (Table II) agree well with
previous values (34-36). The rates observed for T26W SYNCAM are two to
three times faster than those for wild-type Drosophila CaM
(Table II) and are somewhat faster than the value of 14 s1 reported for SYNCAM (37). We note, however, that the
slow phase rate observed with T26W only corresponds to some 5% of the
total amplitude; the major signal change reflects dissociation of the N-terminal Ca2+ ions. These small differences may be due to
sequence differences between the proteins (38).
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Dissociation of Ca4-CaM·Peptide Complexes
Fig. 1 shows typical traces for the dissociation of the CaM complexes of the peptides WFF, FFF, and MasX induced by excess chelator. A-C of Fig. 1 are representative of the range of kinetic behavior observed. The complexes clearly show diverse properties, when compared with one another for a given optical parameter or when compared for different optical parameters for the same complex. The results in Table II show rates and relative amplitudes for the complexes studied. Full discussion of these results requires a consideration of possible mechanisms (see "The Kinetic Model"). However, some generalizations can usefully be made here.
The rates observed with the complexes and Quin2 (Table II) are
frequently biphasic, with a fast rate of 5-100 s1 and a
slow rate of 0.5-5 s
1. The total amplitude corresponds
to four Ca2+ ions. The rates are markedly slower than those
observed with CaM alone, where fast and slow rates clearly correspond
to dissociation from N- and C-domains, respectively (27). The presence
of peptide enhances the affinity of both N- and C-domain
Ca2+ sites (see "The Kinetic Model"), and this
assignment of the faster rate from the N-domain is evidently retained
in the presence of peptide. By contrast, for FFW and FFF the Quin2
signals were monophasic, with a small lag phase in the case of
Ca4-CaM·FFF (Fig. 1B). In this case it appears
that a distinction between the N- and C-domain properties cannot be
made.
For those cases showing biphasic Quin2 signals, the EGTA-induced dissociation of the complexes with the Trp-containing peptides also shows biphasic character, with the greater amplitude in the slow phase. The existence of the fast phase in the Trp signal in the case of WFF and FWF is an important observation, with mechanistic significance (see "The Relaxation Step: Experimental Justification"). It raises questions of the fluorescence properties of intermediate species and, more fundamentally, of the effects of coupling individual kinetic steps with different optical parameters. The biphasic kinetics observed in the EGTA dissociation of CaM·MasX is clearly exceptional, since (Fig. 1C) there is an intermediate species of enhanced fluorescence and the two kinetic components are oppositely signed.
The EGTA-induced dissociation of complexes of non-Trp-containing peptides with CaM shows a single phase, usually in the slow range, consistent with the origins of this signal at Tyr-138 in the C-domain of CaM. The EGTA-induced dissociation of Ca4-CaM·FFF shows a lag phase, evidencing the sequential nature of the process.
The T26W mutant was used with all peptides, since it has a significant fluorescent change when Ca2+ dissociates from the N-domain. In general, biphasic and monophasic behavior follow the Quin2 signals for all complexes (except CaM·Mas and CaM·CBP1). The rates are generally similar although CaM·MasX again appears exceptional. The relative amplitudes of biphasic transients are however different, with the fast phase predominating, consistent with the involvement of the N-domain. The signal in the case of complexes of T26W with Trp-containing peptides is composite, with similar contributions from the loss of Ca2+ from the N-domain and the change due to complete dissociation of the peptide. These two contributions overlap and cannot readily be resolved.
There is no obvious relationship between the observed rates and the affinities of the peptides for Ca4-CaM. Thus, for example, EGTA-induced dissociation rates for complexes with Mas or CBP1 are similar, even though the affinity of CBP1 for Ca4-CaM is 2 orders of magnitude higher than that for Mas. Clearly the reactions studied here involve the eventual dissociation of both Ca2+ and peptide from the initial complex, whereas the peptide affinities reflect only the association/dissociation reaction of the peptide from the complex with Ca4-CaM. Thus, the pathway for the chelator-induced dissociation of the peptide appears to be under kinetic, rather than thermodynamic, control. Taken together, the results indicate that the dissociation mechanism is a complex multi-step pathway, with observed rates resulting from the coupling of individual kinetic steps.
The Kinetic Model
The purpose of the kinetic model is to account for the reduction
in the Ca2+ dissociation rates from CaM in the presence of
peptides and to identify steps in the pathway where differences in the
peptides produce significant kinetic effects. The complete kinetic
scheme for the dissociation of Ca2+ and peptide from a
Ca4-CaM·peptide complex becomes unduly complex if the
four Ca2+ ions are considered to dissociate independently.
It is reasonable, however, based on extensive experimental evidence for
the dissociation kinetics of Ca4-CaM, to consider the
Ca2+ ions as dissociating in pairs, one pair from each
domain (34). This assumption results in the Ca2+
dissociation scheme shown in Fig. 2A, where
C represents CaM and the subscripts P,
N, and C represent peptide, and the N- and C-terminal Ca2+ pairs, respectively. Therefore, for
example, CNCP represents the full
Ca4-CaM·peptide complex, and CCP represents
the Ca2-CaM·peptide complex with 2 Ca2+ in
the C-domain.
This kinetic scheme can be simplified given knowledge of the properties
of CaM itself. As shown in Fig. 2A, step 2 of the path involves peptide dissociation from CNCP to produce the
species CNC (i.e. Ca4-CaM).
Dissociation of this species is known to proceed almost exclusively via
steps 3 and 6, and not via steps 9 and
8, as the C-terminal Ca2+ ions dissociate about
one hundred times slower from Ca4-CaM (CNC
) than the N-terminal Ca2+ ions (34). Correspondingly, we
assume that dissociation of the C-terminal Ca2+ pair from
CNCP is always slower than dissociation of the N-terminal pair and have therefore eliminated step 12 and the
subsequent steps 10 and 11. This reduces the
scheme to two competing pathways (Fig. 2B).
In path A the N-terminal Ca2+ ions dissociate
first (step 1), and in path B the peptide
dissociates first (step 2). Path B is simply
steps 2, 3, and 6. For path A, once
the species CCP forms it is possible for either the
peptide to dissociate to form C
C
(step 4),
or the C-terminal Ca2+ ions to dissociate to form
C
P (step 5). Step 4 is a
reversible step and is a relaxation process in the mechanism, as
opposed to a uni-directional irreversible step such as Ca2+
dissociation in the presence of a chelator. However, it is coupled to
unidirectional steps 1 and 6 and is in parallel
with step 5. It is necessary to know the relative rates of
these processes to determine which path will predominate.
The values of
k4 and k
5 can be
estimated, and dissociation via step 4 is found likely to
predominate in certain cases. Constants k
1 and
k
5, the N- and C-terminal Ca2+
dissociation rates, can be estimated for a typical
Ca4-CaM·peptide complex as follows. The interaction of
Ca4-CaM with a peptide may be characterized by Equation 1,
![]() |
(Eq. 1) |
The value of K for Drosophila CaM is 1.45 × 105 M1 (25), and the values of
Kd are known for each peptide (Table II). Values of
Kd
are less well established. Values
of 620 and 25 mM have been reported for the peptides C28W
and C20W from the CaM binding domain of the plasma membrane
Ca2+ pump (40), and values of 80 mM and 5.7 µM have been reported for bovine heart phosphodiesterase
and troponin I (41). Since binding of the peptides WFF and FFW to
apo-CaM cannot be detected by CD at peptide concentrations of 100 µM,2 it is reasonable to assume a
Kd
of more than 1 mM for
the sk-MLCK peptides. For a typical peptide with a
Kd of 1 nM, values of
Kd
in the range 1 to 100 mM would correspond to
(K
/K)4 values in the range
106 to 108 and therefore to
K
/K values in the range 30 to 100. Values in this range have been determined for other peptides (40, 41).
Stoichiometric Ca2+ association constants for CaM in the
presence of the peptides used here show that both N- and C-domain
Ca2+ affinities are enhanced, and consistent with this,
peptide WFF (like other peptides) shows much lower affinity for
Ca2-TR2C (Kd = 76 nM) than
for Ca4-CaM (<0.2 nM) (13). Thus
Ca2+ binding in both domains contributes to the enhanced
peptide affinity, and the peptide affinity for a partially saturated
Ca2-CaM will be less than for Ca4-CaM. The
above calculation can be taken further, assuming that the difference
between K and K is reflected in the
Ca2+ dissociation rates. If the effect of the peptide on
the N- and C-domain Ca2+ affinity is approximately equal,
both the N- and C-terminal dissociation rates are expected to be
decreased by the above factor of 30 to 100. Since the dissociation
rates in the absence of peptide are
700 s
1
(N-terminal) and 8.5 s
1 (C-terminal), we predict
k
1 values in the range 25-7 s
1
and k
5 values in the range 0.3-0.085
s
1. The rate of Ca2+ dissociation from
C
CP (step 5) is therefore predicted to be much
slower than 8.5 s
1 (Table II), the rate of
Ca2+ dissociation from CaM in the absence of peptide
(step 6).
Calculations also show that k4, the peptide
dissociation rate from C
CP, is likely to be at least 10 s
1, owing to the lower affinity of the peptide for CaM
after loss of the N-terminal Ca2+ ions, see above. By
analogy with the kon for the association of FWF
with Ca4-CaM, k4 is likely to be of
the order of 109 M
1
s
1, and hence the re-association reaction of the peptide
becomes significant. Step 4 is reversible, i.e.
it is a relaxation process. The rate of this step will be greater than
k
4, and, since k
4 > k
5, it will exceed
k
5. Hence, dissociation via step 4 should be significant. Even if the effect of the peptide is largely on
the C-terminal Ca2+ sites, both k
4
and k
5 will be reduced, and the relaxation
step 4 and the dissociation step 5 will remain in
competition.
The
reversible nature of step 4 suggests that the observed rates
should be affected by the [peptide]/[CaM] ratio. We therefore measured the dissociation reaction for Ca4-CaM·WFF as a
function of the [WFF]/[CaM] ratio. At [WFF]/[CaM] = 1.05 the
EGTA-induced dissociation of Ca4-CaM·WFF (monitored by
peptide Trp fluorescence) is biphasic with a fast phase rate of 12 s1 and a slow phase rate of 1.5 s
1. The
fast phase accounts for approximately 10% of the total amplitude. At
[WFF]/[CaM] >1.5 the fast phase is no longer observed and the slow
phase rate is reduced (see Fig. 3, A and
B).
Similarly, for Ca4-CaM·FWF, the fast phase (in Trp
fluorescence) accounts for 40% at [peptide]/[CaM] = 1.05 (Table
II) and decreases to 22% at [peptide]/[CaM] = 4.4 (Fig.
3B). Over this range the fast phase rate is unchanged
(32 ± 4 s1; not shown) but the slow phase rate is
again significantly reduced (Fig. 3A). (Note: the fast phase
amplitude for this complex may contain a contribution from a decrease
in the fluorescence of the intermediate Ca2-CaM·FWF
relative to Ca4-CaM·FWF; from the NMR structure of
Ca4-CaM·M13 (7), the Trp-8 of the FWF might locate
between the CaM domains and could be sensitive to the loss of both the
N- and C-domain Ca2+ ions.)
A similar reduction in slow phase rate is seen in the Quin2-induced dissociation of Ca4-CaM·FWF as [peptide]/[CaM] is increased (Fig. 3A). The rate observed with Quin2 is somewhat faster than that for peptide Trp fluorescence (particularly at low [peptide]/[CaM]). This is because a significant amount of Ca2-CaM (in equilibrium with Ca2-CaM-peptide) is produced in the fast phase (13). In the case of the Quin2 measurements, the fast phase rate also remains unaffected and the fast phase amplitude corresponds to two Ca2+ ions at all [peptide]/[CaM] ratios.
In recent work (13) we have studied the dissociation of peptide WF10 (residues 1-10 of WFF) from Ca4-CaM·WF10 and Ca2-TR2C·WF10 (WF10 binds selectively to the C-domain of CaM). In both cases the (monophasic) dissociation rate observed in either peptide-Trp or Quin2 signal decreases as [peptide]/[protein] increases from one to four, also consistent with an analogous relaxation mechanism.
The question then arises of the relative importance of the two
alternatives in Fig. 2A, namely the relaxation mechanism
(steps 1, 4, and 6) and the
intuitively simpler mechanism (steps 1, 5, and
7). Fig. 3, C and D, shows the results
of computer simulations using rate constant set 5A
(Table III) plus k5 = 0, 1, or 3 s
1 (and k7 = 1 × 109 M
1·s
1;
k
7 = 3 × 105
s
1; values selected to reflect the estimated affinity of
the peptides for apo-CaM). As [peptide]/[CaM] increases the
relative contribution of the fast phase to the total amplitude
decreases for all values of k
5 examined (Fig.
3D). This is because at the higher [peptide] the
equilibrium of step 4 is driven further toward
C
CP. The rate of the fast phase remains approximately
constant as [peptide]/[CaM] increases, since it reflects the
coupling to step 1. The rate of the slow phase decreases
significantly (as seen in Fig. 3A) since, as [peptide]
increases, peptide rebinding (k4) competes more
effectively with C-terminal Ca2+ ion loss
(k
6). This is typical of a relaxation
mechanism as formulated for step 4. These computer
simulations are consistent with the behavior observed for dissociation
of Ca4-CaM·WFF and Ca4-CaM·FWF, indicating
the importance of the relaxation step in the overall path A
dissociation pathway. The competing step 5, when included,
reduces but does not eliminate the dependence of the slow phase rate on
[peptide]/[CaM] (Fig. 3C). When the competing step
5 is present the limiting value for the slow phase at
sufficiently high [peptide]/[CaM] will be
k
5 (see Fig. 3C).
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When k4 = 0 the relaxation pathway
(steps 1, 4, and 6) is eliminated. There is then
no fast phase amplitude in Trp fluorescence, and the observed slow rate
is independent of [peptide]/[CaM]. Hence the simple pathway
(steps 1, 5, and 7) is not able to account for
the observed results with the WFF and FWF complexes.
Numerical Simulation: Effects of Individual Constants on the Overall Kinetics of Dissociation
Two limiting cases, with either path A or B
operating in isolation, were simulated using the assumptions outlined
under "Materials and Methods." The rate constants used for the
simulations are given in Table III, and the results of the simulations
are shown in Figs. 4 and 5. The values of
k3 and k
6 were fixed at 700 and 8.5 s
1, the values measured for CaM alone
(Table II). In view of the dominant contribution of pathway 1, 4, 6,
pathway 1, 5, 7 is suppressed in these simulations.
Fig. 4 shows the change in species concentrations observed for a
typical reaction via path A. Curve f shows the release of Ca2+ (monitored as the Quin2 fluorescence change) which is
the sum of step 1 (inverse of curve a,
disappearance of CNCP) plus step 6 (curve
d, appearance of C (apo-CaM)). Because of the sequential nature of the pathway, curve d shows a pronounced
lag. This is seen most clearly in the signal of Tyr-138 (sensitive to
Ca2+ dissociation from the C-domain) which can only be
monitored with a non-Trp-containing peptide. Because curve d
is non-exponential, the summed curve f will also show
complex time dependence, but experimentally it will generally appear as
a double exponential process. The other normal observable peptide Trp
fluorescence is associated with the disappearance of CNCP
and C
CP (i.e. step 1 plus step 4)
and will appear as the inverse of curve e. Again this will
be non-exponential owing to the coupling of the two steps plus the fact
that step 4 is a reversible relaxation process. The species
plot shows the behavior of the intermediate species C
CP
(curve b) and C
C
(curve c) which are involved in this relaxation and also shows how the relaxation rate
of step 4 determines the appearance of C
as step 6 (curve d). In the case of the T26W
mutant, where there is a fluorescence change between species
CNCP and C
CP (step 1) the total
Trp fluorescence change (with a Trp-containing peptide) is the sum of
curve a plus the inverse of curve e, again
analyzing as a double exponential.
Further simulations show the influence of individual rate constants
(Table III) on the form of observable signals. The results for
path A simulations are shown in Fig. 5,
A-D. At low values of k1 all
signals are dominated by a single phase with a rate close to
k
1 (Fig. 5B). At higher values of
k
1, the Ca2+ dissociation signal
monitored by Quin2 is clearly biphasic, with approximately equal
amplitudes in both phases (Fig. 5, A, C, and D; curve a). Generally, the signals for the Tyr
and Trp probes are strongly dependent on the kinetics of the relaxation
step (step 4). Except at very low values of
k
1, the Trp peptide signal is biphasic. The
amplitude of the fast phase is dependent on the value of
k
4, varying from 0.11 to 0.63 for
k
4 values of 20 to 2500 s
1,
respectively (Fig. 5, C and D, curve
c). The rates of the fast processes for both the Quin2 and the Trp
peptide signals are close to k
1 at values of
k
4
100 s
1 (Fig. 5, A,
B, and C). The rates of the slow processes for both the
Quin2 and the Trp peptide signals reflect the convolution of the
peptide relaxation (step 4) with the C-terminal
Ca2+ loss (step 6). Thus, the slow rate does not
correspond directly to a single step in the mechanism. For this reason
the slow rate only changes from 0.5 to 6 s
1 as
k
4 changes from 20 to 2500 s
1
(Fig. 5, C and D). The CaM Tyr signal shows a
small lag phase and is generally monophasic with a slow rate, except at
low k
4 where a biphasic signal results from
the gradual accumulation of the species C
CP (Fig.
5D, curve b). Although the Tyr signal results only from
step 6, the rate is slower than 8.5 s
1, owing
to convolution with the relaxation step (step 4). The T26W
Trp signal with a non-Trp peptide is monophasic, with a rate close to
k
1, directly reflecting the kinetics of
step 1. The T26W Trp signal with a Trp peptide basically
mirrors the peptide signal and thus also reflects
k
1 and k
4. When the peptide signal is biphasic, the T26W signal has the same rates as the
peptide signal but a much greater amplitude in the fast phase (Fig.
5, A and D, curves c and
e). The stability of the species C
CP
determines the kinetics of the relaxation step and is thus critical in
determining the observed kinetics.
Path B, in which the first step is dissociation of the
peptide from CNCP (step 2), must be considered
as a possibility even though the initial step does not involve
Ca2+ dissociation. This is because the species
CNCP is in rapid equilibrium with CNC and
free peptide. The N-terminal Ca2+ ions will dissociate from
Ca4-CaM (CNC
) at 700 s
1 (Table
II) so that loss of Ca2+ (step 3) can compete
effectively with peptide rebinding (step 2). This path is
expected to be the dominant dissociation path if the peptide relaxation
rate (step 2) is faster than N-terminal Ca2+
dissociation (step 1). Path B was simulated by
varying the peptide off-rate, k
2, from 1 to 20 s
1, for a fixed peptide on-rate,
k2, of 109
M
1 s
1, corresponding to
affinities of peptide for Ca4-CaM in the range 1 to 20 nM. The rate constants used are shown in Table III, and the
results of the simulations are shown in Fig. 5, E and
F. The Ca2+ dissociation, as monitored by Quin2,
is monophasic and slow (Fig. 5, E and F,
curve a). The signals from any of the Trp probes are identical and consist of a biphasic trace with a small fast phase (Fig.
5, E and F, curves c, d, and e). The
fraction of the total amplitude in the fast phase varies from about
0.15 to 0.3 for k
2 values of 1 to 20 s
1, respectively. The observed rates do not correlate
directly with single steps of the mechanism and are significantly
decreased by the presence of excess peptide. The simulation is
virtually unaffected if the peptide on-rate, k2,
is slower than 109 M
1
s
1, because then the peptide off-rate,
k
2, must also be slower.
A comparison of the simulations of paths A and B indicates that the Quin2 results are crucial in suggesting which pathway dominates. Biphasic Quin2 signals indicate path A dissociation and allow direct determination of the N-terminal Ca2+ dissociation rate. The T26W results may also be indicative, especially when the signal observed with a Trp peptide is biphasic. Path A is indicated if the relative amplitudes of the fast and slow phases observed with the Trp peptide are reversed when the peptide is studied with T26W. If the relative amplitudes remain unchanged, path B is indicated. It is not possible to see a reversal of the amplitudes if dissociation is occurring via path B, regardless of the relative fluorescence changes of the two Trp probes, peptide Trp and T26W. These simulations indicate how the experimental observables may be expected to deviate from simple mono- or biphasic exponential kinetics owing to the coupling of the individual steps in both of the pathways A and B.
Interpretation of Observed Kinetics
Results obtained with Quin2 and with the T26W mutant suggest which
dissociation pathway is dominant, but it would be useful to know the
relative magnitudes of k2 and
k
1. We attempted to measure
k
2 directly by displacing Trp-containing peptides from their Ca4-CaM·peptide complexes using a
silent peptide (CBP1 or FFF). The observed rate was strongly dependent
on the concentration of the displacing peptide and did not reach a
limiting value, suggesting an associative mechanism, in which the
silent peptide binds to the Ca4-CaM·peptide complex
forming a transient intermediate.
It is possible to estimate a value for
k2 for a particular peptide if the
dissociation constant, Kd, and the association rate
constant, kon, are known. This value can be
compared with an experimentally determined k
1.
As an example, consider Ca2+ dissociation from the
Ca4-CaM·WFF complex (Fig. 1A). The
experimentally determined kon for
Ca4-CaM and FWF suggests that a reasonable estimate for
k2 for the sk-MLCK peptides (20 °C) is 2 × 109 M
1 s
1. The
Kd for WFF is
0.12 nM (Table II), so
that k
2 is calculated (as
kon·Kd) to be
0.25
s
1. For Ca4-CaM·WFF, the dissociation
process observed with Quin2 is biphasic (Table II) with a fast phase
rate of
12 s
1, which can be assumed to reflect
dissociation of the N-terminal Ca2+ ions. Since
k
1 is very much larger than the estimated k
2, it is reasonable that the dissociation of
Ca4-CaM·WFF will proceed predominantly via path
A. Further evidence for this conclusion comes from the observation
that the rates for the EGTA-induced dissociation of
Ca4-CaM·WFF and Ca4-T26W·WFF are similar,
but the relative amplitudes of the fast and slow phases are reversed (Table II). This behavior resembles the simulated data (Fig.
5D) and is typical of dissociation via path A.
The relaxation data (Fig. 3) strongly support the predominant
involvement of path A.
For peptide complexes with a biphasic Quin2
signal, it is possible to estimate k1 in a
similar manner, as the fast phase rate in the Quin2-induced
dissociation. These values are 31, 65, 14, and 8 s
1 for
FWF, MasX, Mas, and CBP1. Using the peptide affinities listed in Table
II and an estimated maximum kon of 2 × 109 M
1 s
1, we
obtain maximum k
2 values of 13, 1.8, 0.6, and
0.01 s
1 for FWF, MasX, Mas, and CBP1. These values
indicate that the peptides MasX, Mas, and CBP1 are likely to dissociate
via path A. The experimental data obtained for these
peptides are consistent with this pathway. The situation is less clear
for the peptide FWF, where k
1 and
k
2 may be comparable and paths A
and B may be in competition. However, the biphasic Quin2
signal, and the reversed relative amplitudes for the biphasic T26W and Trp peptide signals, strongly suggest that Ca4-CaM·FWF
dissociation also occurs via path A. The experimental curves
obtained for FWF (Fig. 1B) closely resemble the simulation
of path A in Fig. 5C, and the relaxation data
again strongly suggest the predominant involvement of path
A.
The EGTA-induced dissociation of the Ca4-CaM·MasX
complex, monitored by Trp fluorescence, has a fast phase corresponding
to an increase in fluorescence (Fig. 1C). Interestingly,
equilibrium fluorescence experiments in which Ca2+ is
titrated into apo-CaM plus MasX show the formation of an intermediate species with high fluorescence at a ratio of 2 mol of Ca2+
per mol of CaM (29).3 Evidence for the existence of an
intermediate Ca2-CaM·peptide species is also seen in
1H NMR studies of calmodulin with mastoparan (39). If this
intermediate species is a complex in which only the C-terminal
Ca2+ sites are occupied (i.e.
CCP), as seems likely, then its existence under
equilibrium conditions is consistent with the proposed path
A mechanism.
For peptides FFW and FFF, with monophasic Quin2 signals (Table II), it
is not possible to say whether path A or B
dominates. For simulations to even approach the kinetic behavior of
these peptides (see Fig. 1B for data on FFF), it was
necessary to assume that the N-terminal Ca2+ dissociation
rate, k1, was approximately 2 s
1. Since maximum values of k
2
are estimated (as described above) to be 2 s
1 (FFF) and
3.2 s
1 (FFW), it is highly likely that paths A
and B operate in competition.
This work has examined the chelator-induced
dissociation of seven Ca4-CaM·peptide complexes. A
striking finding is that substitution or permutation of residues within
the target peptide sequence can have a significant effect not only on
the kinetics but also on the overall pathway for the dissociation
reactions. For all of the complexes studied, except those with FFF and
FFW, we observe biphasic kinetics. This is consistent with a mechanism
in which the first event is loss of the N-terminal Ca2+
ions at a rate which is 10 to 100 times slower than that with CaM alone
(700 s1). There is little obvious relationship between
the rate of this fast process and the affinity of the peptide for
Ca4-CaM. Subsequent dissociation may then occur through
loss of the C-terminal Ca2+ ions, followed by rapid loss of
the peptide. The alternative pathway involves peptide loss from the
complex in which only the C-terminal Ca2+ ions are bound,
followed by loss of the C-terminal Ca2+ ions. Coupling of
these two steps limits the observed slow phase rate to the range 0.5 to
5 s
1, and there is again no strong correlation with
peptide affinity.
By contrast, the Ca4-CaM complexes of FFW and FFF show monophasic kinetics with Quin2 and appear to have unexpectedly slow N-terminal Ca2+ dissociation rates in view of the fact they bind to Ca4-CaM with affinity comparable with that WFF. The slow N-terminal Ca2+ dissociation rates suggest that FFW and FFF may have stronger interactions with the N-domain than other peptides. The FFW and FFF peptides have the same sequence as WFF, except that both have a W4F substitution, and FFW has a F17W substitution. All three peptides appear to bind in the same orientation, with residues 4 and 17 interacting with the C- and N-terminal domains of CaM, respectively (11). It is notable that FFF and WFF behave differently, considering that the C-terminal region of both peptides could interact similarly with the N-domain of the CaM. For FFF, the substitution of the anchoring Trp-4 residue may confer a somewhat different conformation on the Ca4-CaM·peptide complex. Direct measurement of Ca2+ binding to apo-CaM plus FFF confirms the enhancement of the N-domain Ca2+ affinity.2
For these M13-related peptides and for CBP1 the rates observed with T26W CaM are similar to, although somewhat faster than, the rates observed with Drosophila CaM. For the mastoparans, however, the rates observed with T26W CaM are significantly faster (Table II). This suggests that the interaction of these peptides with calmodulin may have been significantly affected by the mutation in the N-domain, particularly for Mas itself. There is evidence that the mastoparans bind more strongly to the C-domain of CaM than the N-domain (42), and it has been suggested that they bind exclusively to the C-domain in the 1:1 complexes (43). Our results suggest that the mastoparans interact significantly with both domains of CaM, supporting the results obtained with Ca2+ binding site mutants of CaM (44).
Rates for the chelator-induced dissociation of several CaM-peptide and
CaM·protein complexes have been reported. For example, values of 2 and 1 s1 have been reported for the complexes of CaM with
intact sk-MLCK (18) and with the RS-20 peptide from the CaM binding
domain of sm-MLCK (19). Rates of 140, 12.1, and 1.1 s
1
have been reported for the Quin2-induced dissociation of CaM-melittin (45); the two slower rates, which accounted for
90% of the total
amplitude, were attributed to N- and C-terminal Ca2+
dissociation, respectively. Snyder et al. (46) report that the binding of an (unspecified) amphipathic peptide reduces the N-terminal dissociation rate by a factor of 150 and the C-terminal rate
by a factor of 8. Calmodulin labeled at Cys-27 with the fluorescent probe MIANS has been used to study the EGTA-induced dissociation of the
CaM complexes with the proteins caldesmon, calponin, and sm-MLCK. The
rate determined for the calponin complex (Kd
nM) was 1 s
1 (21). The rates for the
complexes with sm-MLCK (Kd = 1.1 nM) and
caldesmon (Kd = 108 nM) were 3.5 and
13.5 s
1 (20). The observed kinetics in these systems,
namely a slow rate in the range 1-5 s
1 (except for the
complex with caldesmon which binds with low affinity), are similar to
the results in Table II suggesting that the dissociation mechanism
outlined here may also be valid for a range of peptides and
proteins.
Two new stopped-flow studies have appeared recently. Persechini
et al. (23) reported biphasic rates for Quin2-induced
dissociation reaction of Ca4-CaM·M13 of 1.9 and 0.15 s1 (25 °C). These are approximately 5- and 10-fold
slower than we measured for WFF (Table II). However, the corresponding
rates for the N- and C-terminally protected WFF peptide are 3.5 and 0.4 s
1.2 Values of 17.7 and 1 s
1
were reported for dissociation of Ca4-CaM·nPEP, where
nPEP is the target sequence of neuronal nitric oxide synthase.
Interestingly, the corresponding experiments with both intact enzymes
show fast rates >1000 s
1 (but with different
amplitudes). These results indicate that the partially saturated
CaM·enzyme complex can exert a significant functional role, which is
apparently different in the two systems, being active for sk-MLCK but
inactive for neuronal nitric oxide synthase. Johnson et al.
(24) also provide evidence for the prior dissociation of the N-domain
Ca2+ ions from CaM complexes with target peptides (from
CaM-dependent protein kinase II, peptides RS20 and M13 from
sm- and sk-MLCK, and MARCKS peptide) and deduced that the corresponding
intermediate partially saturated CaM·enzyme species could function in
a manner similar to the action of troponin C on skeletal muscle.
A second striking feature is the
important role of the intermediate species
Ca2-CaM·peptide with two Ca2+ ions in the
C-domain. Numerical simulations show that a potentially important
factor in determining the observed dissociation kinetics is the
affinity of the peptide in this intermediate. If we assume that the
increase in Ca2+ affinity for CaM on peptide binding is
fully reflected in changes in the Ca2+ dissociation rates,
the extent to which Ca2+ binding to each of the N-terminal
sites is enhanced by the peptide is calculated as
700/koff, where koff is
the estimated dissociation rate for the N-terminal sites in the
presence of the peptide. The factor by which peptide binding is
enhanced by binding of the N-terminal Ca2+ ions is then
(700/koff)2, and the value of the
dissociation constant for interaction of the peptide with the
Ca2-CaM species is Kd (INT) = Kd·(700/koff)2.
Using the values measured for the fast phase of Quin2-induced dissociation as our estimate of koff, we
calculate values of Kd (INT) of 600 nM (WFF), 3.3 µM (FWF), 100 nM
(MasX), 700 nM (Mas), and 40 nM (CBP1) (Note:
For FFW and FFF we estimate values of 100 and 60 µM using
an upper limit for koff of 4 s1).
The Kd (INT) values calculated for MasX
and WFF are consistent with values measured for dissociation constants of these peptides interacting with TR2C, the C-terminal tryptic fragment of CaM, of <200 nM for MasX (42), and of
700
nM for WFF (13).
These values of Kd (INT) are consistent with the observations that the species Ca2-CaM·peptide is an important intermediate in the major pathway for the response of the complex to removal of Ca2+. The existence of the relaxation step also suggests a mechanism for regulating the CaM-target sequence interaction in terms of the fast reversible exchange between the species Ca2-CaM·peptide and Ca2-CaM. This is in contrast to the obligatory removal of Ca2+ ions from the C-domain, as implied by the step 5/step 7 mechanism. This exchange reaction could be important in the regulation of the typical CaM-enzyme system where, owing to the proximity of the target sequence, the effective concentration of the target is enhanced (47), hence stabilizing the intermediate complex. This species could then serve to ensure attachment of CaM to the target protein via the C-domain, with enzyme activation requiring binding of Ca2+ to the N-domain to enable its interaction with the target (12, 13). It is notable that this mechanism would be possible for target sequences which followed path A (rather than B), a distinction which is evidently conferred at least in part by the characteristics of the individual target sequences.
In conclusion, this study shows that the dissociation reactions of Ca4-CaM·peptide complexes in the presence of Ca2+ chelators are perhaps more complicated than has previously been appreciated, but they are nonetheless capable of being resolved into a number of specific components. Irrespective of the detailed molecular mechanism, it is clear that, in general, observed dissociation rate constants are related only indirectly to the kinetics of individual steps in the Ca2+ dissociation processes. For the peptides studied, the predominant dissociation path appears to be loss of the N-terminal Ca2+ ions followed by loss of the peptide in a relaxation process and is indicated by biphasic Quin2 and EGTA kinetics. An alternative dissociation path involving initial loss of the peptide may also be important for some peptides. The results indicate that subtle changes in the peptide sequence can have significant effects on the dissociation kinetics and therefore on the relative importance of different pathways. Extrapolating to the biological function of CaM with target proteins, this diversity suggests that different intermediate states such as Ca2-CaM·peptide may be generated which can modulate either enzymatic activity or kinetic properties related to the specific regulatory processes involved, in a way which is highly dependent on the specific target sequence.
We thank Dr. K. Beckingham for supplying the Drosophila calmodulin clone, Peter Fletcher for synthesizing the peptides, and Peter Browne for purifying the calmodulin and peptides. We also thank Dr. J. Haiech (Marseille, France) for providing the SYNCAM mutant and Dr. N. Millar for providing the computer programs KSIM and KFIT.