(Received for publication, July 14, 1995; and in revised form, October 5, 1995)
From the
Myosin light chain kinase and peptides from the calmodulin (CaM)
binding domains of myosin light chain kinase (RS-20, M-13), CaM kinase
II, and the myristoylated alanine-rich protein kinase C substrate
protein slowed Ca dissociation from CaM's
N-terminal sites from 405 ± 75/s to 1.8-2.9/s and from
CaM's C-terminal sites from 2.4 ± 0.2/s to 0.1-0.4/s
at 10 °C. Since Ca
dissociates 5-29 times
faster from the N-terminal in these CaM
peptide complexes and both
lobes are required for activation, Ca
dissociation
from the N-terminal would control target protein inactivation.
Ca
binds 70 times faster to the N-terminal (1.6
10
M
s
) than the C-terminal sites (2.3
10
M
s
). In
a 0.6-ms half-width Ca
transient,
Ca
occupied >70% of the N-terminal but only 20% of
the C-terminal sites. RS-20 produced a 9-fold and CaM kinase II a
6.3-fold increase in C-terminal Ca
affinity,
suggesting that some target proteins may be bound to the C-terminal at
resting [Ca
]. When this is the case,
Ca
exchange with the faster N-terminal sites may
regulate CaM's activation and inactivation of these target
proteins during a Ca
transient.
CaM ()is a ubiquitous Ca
binding
protein that consists of a N- and C-terminal lobe, each of which
contains two EF hand Ca
binding sites (see Weinstein
and Mehler (1994) and James et al.(1995) for reviews). The N-
and C-terminal lobes are separated by an 8-turn
-helix, and they
undergo a Ca
-dependent exposure of their hydrophobic
binding pockets, which allows binding and activation of target protein
(LaPorte et al., 1980; Tanaka and Hidaka, 1980). In the
presence of Ca
, both the N- and C-terminal
hydrophobic pockets of CaM bind an amphipathic
-helical peptide
domain within the target protein structure (O'Neil and DeGrado,
1990), and recently the high resolution x-ray and NMR structure of
several Ca
CaM
peptide complexes have been
determined (Ikura et al., 1992; Meador et al., 1992,
1993). Ca
-CaM binding removes a pseudosubstrate
inhibitory domain from many enzymes' active sites, and this
results in the activation of smooth and skeletal muscle MLCK, CaM
kinase II, calcineurin, plasmalemma Ca-ATPase, and cyclic nucleotide
phosphodiesterase (see Kemp and Pearson(1991) and James et
al.(1995) for review).
The CaM binding domains of many CaM
target proteins have been identified, and peptides representing these
domains from skeletal (M-13) and smooth (RS-20) muscle MLCK, CaM Kinase
II (CK), and the MARCKS protein have been synthesized.
These peptides bind CaM with nanomolar affinity as does the entire
target protein (Blumenthal et al., 1985; Lucas et
al., 1986; Payne et al., 1988; Verghese et al.,
1994).
The binding of target proteins, their CaM binding domain
peptides, and hydrophobic drugs all produce dramatic (7-40-fold)
increases in CaM's affinity for Ca (Keller et al., 1982; Olwin et al., 1984; Mills et
al., 1985; Yagi and Yazawa, 1989; Kasturi et al., 1993).
These increases in Ca
affinity might be reflected by
decreases in the rate of Ca
dissociation from the
lower affinity N- and higher affinity C-terminal EF hands of CaM. If
target peptide and protein binding reduces the rate of Ca
dissociation from the CaM
protein (peptide) complex, then
the rate of complex dissociation and inactivation could be delayed
until long after the Ca
transient has subsided. In
this paper, we determined the rates of Ca
exchange
with the N- and C-terminal domains of CaM and examined the effects of
MLCK and several CaM binding peptides on the rates of
Ca
dissociation from the N- and C-terminal
Ca
binding sites of CaM and on C-terminal
Ca
affinity. We relate our results to the mechanism
of CaM's activation and inactivation of its target proteins
during a cellular Ca
transient.
CK peptide was purchased from L.C.
Laboratories (Woburn, MD), RS-20 peptide was from American Peptide Co.
(Sunnyvale, CA), M-13 peptide was purchased from Peptide Technologies
(Gaithersburg, MD). and MARCKS peptide was the generous gift of Dr.
Perry Blackshear (Duke University, Durham, NC). CaM41/75 and CaM85/112
were generously provided by Dr. Zenon Grabarek (Boston Biomedical
Institute, Boston, MA).
The changes in Quin
fluorescence as it dissociates Ca from CaM were
converted to moles of Ca
dissociating from CaM by
mixing increasing concentrations of Ca
(0, 10, 20,
30, 40, 50, and 60 µM) with Quin 2. Quin 2 fluorescence
increased linearly as a function of increasing
[Ca
], allowing us to directly relate a
change in Quin 2 fluorescence to the number of moles of Ca
dissociating per mole of CaM. Calibration curves were performed
at the end of each experiment using the same Quin 2 solutions and
experimental conditions as used in the experiments. The amplitude of
the change in Quin 2 fluorescence was extrapolated from an exponential
fit of the data in Fig. 1A, inset.
Figure 1:
Effect of MLCK and peptides on the
rates of Ca dissociation from the CaM N- and
C-terminal Ca
binding sites as measured by Quin 2
fluorescence. In panel A, the time course of the increase in
Quin 2 fluorescence is shown as Ca
dissociates from
CaM's N-terminal (Fast) and C-terminal (Slow)
Ca
binding sites. The inset shows the time
course of the Quin 2 fluorescence increase that occurs upon
Ca
dissociation from CaM's low affinity
N-terminal Ca
binding sites over 0-20 ms. CaM
(8 µM) + Ca
(60 µM) in
20 mM Hepes buffer, pH 7.0, was rapidly mixed with an equal
volume of Quin 2 (150 µM) in the same buffer at 10 °C.
The 0-2-s trace is an average of four traces fit with a double
exponential (variance < 2.7
10
). The
0-20-ms trace is an average of six traces fit with a single
exponential (variance < 1.6
10
). All
kinetic traces were triggered at time zero; the first 1.6 ms of
premixing is shown, and all traces were fit after mixing was complete.
In panel B, the time course of the increase in Quin 2
fluorescence is shown as Ca
dissociates from
CaM's N- and C-terminal Ca
binding sites in the
presence of MLCK, RS 20, or MARCKS peptide. CaM(8 µM)
+ Ca
(60 µM) and 16 µM of RS-20 or MARCKS peptide was rapidly mixed with an equal volume
of Quin 2 (150 µM) in the same buffer (20 mM Hepes, pH 7.0) at 10 °C. CaM (2 µM) +
Ca
(60 µM) + MLCK (2
µM) was rapidly mixed with an equal volume of Quin 2 (150
µM) in the same buffer, and the signal was amplified to
match the amplitude of the peptide experiments. Each trace is an
average of four to six traces, fit with a double exponential (variance
< 1.1
10
). Control experiments where
Ca
(60 µM) was mixed with an equal
volume of Quin 2 (150 µM) were flat lines. Quin 2 emission
was monitored at 510 nm, with excitation at 330
nm.
Figure 5:
The
time course of the decrease in Ca-CaM-TNS
fluorescence upon Ca
chelation by EGTA and after a
rapid Ca
transient. The time course of the decrease
in TNS fluorescence is shown as EGTA dissociates the
Ca
CaM
TNS complex (trace A) or
when it has been transiently formed by a rapid Ca
transient (trace B). The inset shows the fast
kinetics of these two reactions over 0-20 ms. The EGTA-induced
decrease in TNS fluorescence (traces labeled A (N-EGTA) or A (C-EGTA)) was accomplished by mixing CaM (4
µM) + Ca
(200 µM)
+ TNS (200 µM) in 200 mM Hepes, pH 7.0, with
an equal volume of 2 mM EGTA in the same buffer at 10 °C.
Control experiments where Ca
+ CaM + TNS
was reacted with buffer + Ca
were flat. The
transient occupancy of CaM by Ca
and TNS (traces labeled B (N-Transient) or B (C-Transient)) was
achieved by mixing CaM (4 µM) + TNS (200
µM) + EGTA (2 mM) in 200 mM Hepes,
pH 7.0, with an equal volume of 200 µM Ca
in the same buffer at 10 °C. Control experiments reacting the
above solution with buffer containing EGTA instead of Ca
were flat. TNS fluorescence was monitored through a 510-nm broad
pass filter (Oriel, Standford, CT) with excitation at 370 nm. The
0-1.2-s traces represent an average of six traces fit with a
double exponential curve (variance < 9.8
10
).
The 0-20-ms traces in the inset represent an average of
six traces fit with a single exponential (variance < 3.0
10
). Each kinetic trace was fit after flow had
stopped.
Figure 2:
Effect
of MARCKS peptide on Ca dissociation from CaM and
CaM41/75 C-terminal Ca
binding sites. The upper
traces show the increase in Quin 2 fluorescence associated with
Ca
dissociation from the C-terminal sites of CaM41/75
with and without MARCKS peptide. CaM41/75 (4 µM) +
Ca
(60 µM) ± MARCKS peptide (10
µM) in 20 mM Hepes, pH 7.0, was rapidly mixed
with an equal volume of Quin 2 (150 µM) in the same buffer
at 10 °C. Quin 2 fluorescence was monitored as described in the
legend to Fig. 1. The lower traces show the decrease in
CaM tyrosine fluorescence when CaM ± MARCKS peptide was rapidly
mixed with EGTA. CaM (4 µM) + Ca
(100 µM) ± MARCKS peptide (10
µM) in 20 mM Hepes, pH 7.0, was rapidly mixed
with an equal volume of EGTA (5 mM) in the same buffer at 10
°C. Control experiments where CaM + Ca
were
reacted with buffer + Ca
were flat lines, which
began at the starting amplitude of the EGTA trace. CaM tyrosine
fluorescence was measured through a UV-transmitting black glass filter
(UG1 from Oriel (Standford, CT)) with excitation at 275 nm. Each trace
is an average of five traces fit with a single exponential (variance
< 2.2
10
).
CaM41/75 is a mutant
CaM in which cysteine residues have been introduced at positions 41 and
75 by site-directed mutagenesis. When these two cysteines are
cross-linked by a disulfide bridge, the N-terminal hydrophobic pocket
of CaM cannot be opened in a Ca-dependent manner; CaM
is thereby inactivated and exhibits a greatly reduced
Ca
affinity at its N-terminal Ca
binding sites (Grabarek et al., 1991). (
)When
CaM41/75 (+Ca
) is mixed with Quin 2, the rapid
phase of Ca
dissociation from its N-terminal
Ca
binding sites (which occurs over 0-20 ms) is
not observed. 2 mol of Ca
still dissociate from the
C-terminal sites of CaM41/75 and cause an increase in Quin 2
fluorescence at a rate of 2.4 ± 0.2/s (Fig. 2). Thus,
CaM41/75 allows us to specifically monitor Ca
dissociation from the C-terminal Ca
binding
sites. The addition of MARCKS peptide to CaM41/75 slowed the rate of
Ca
dissociation from its C-terminal Ca
sites from 2.4/s to 0.3/s (Fig. 2). Similar studies with
RS-20, M-13, and MLCK indicated that these peptides/protein reduced the
rate of Ca
dissociation from the C-terminal sites of
CaM41/75 from 2.4/s to 0.07 ± 0.01/s, 0.11 ± 0.02/s, and
0.5 ± 0.02/s, respectively. Similar results were obtained with a
tryptic fragment of CaM, CaM 78-148, which contains only the
C-terminal half of the molecule (residues 78-148). As shown with
CaM41/75, Ca
dissociated from CaM 78-148 at a
rate of 2.3/s, and addition of the MARCKS peptide slowed Ca
dissociation to 0.25/s (data not shown). Thus, these studies with
the C-terminal fragment of CaM and with the N-terminal mutant CaM
(CaM41/75) allowed us to determine the effect of these peptides on
Ca
dissociation from the C-terminal of CaM,
selectively.
Knowing the effect of MLCK and these peptides on the
rates of Ca dissociation from the C-terminal sites of
CaM, it follows (from the data in Fig. 1B) that MLCK,
RS-20, M-13, MARCKS peptide, and C
K peptide slow
Ca
dissociation from the N-terminal Ca
binding sites of CaM from 405/s to 2.3 ± 0.2/s, 1.8
± 0.2/s, 2.6 ± 0.6/s, 2.6 ± 0.3/s, and 2.9
± 0.3/s, respectively. The effects of MLCK and each of these
peptides on the rates of Ca
dissociation from the N-
and C-terminal Ca
binding sites of CaM could,
therefore, be unambiguously assigned as listed in Table 1.
Figure 3:
Effects of RS-20, MARCKS, and
CK peptide on Ca
binding to the
C-terminal sites of CaM or CaM41/75. The Ca
dependence of the increase in tyrosine fluorescence for CaM
(
), CaM41/75 (*), CaM + C
K peptide (
), or
CaM + MARCKS peptide (
) and the Ca
dependence of the increase in RS-20 tryptophan fluorescence in
the presence of CaM41/75 (
) are shown as a function of pCa. Increasing concentrations of Ca
were
added to 1 ml of CaM (1 µM) ± MARCKS (2
µM) or ± C
K (2 µM), or to
1 ml of CaM41/75 (1 µM) ± RS-20 (2 µM)
in 200 mM Hepes, 2 mM EGTA, pH 7.0, at 10 °C to
yield the indicated pCa. CaM and CaM41/75 tyrosine
fluorescence was monitored at 305 nm with excitation at 275 nm. RS-20
tryptophan fluorescence was monitored at 325 nm with excitation at 285
nm. 100% fluorescence corresponds to a
2.4-fold increase in CaM or
CaM41/75 tyrosine fluorescence and a 1.4-fold increase in RS-20
tryptophan fluorescence. Each data point represents an average of three
titrations ± S.E. fit with a sigmoidal
curve.
Since Ca binding to both the N- and C-terminal sites exposes TNS binding
sites, the Ca
affinity at each class of site can be
determined by the Ca
dependence of the increase in
TNS fluorescence. Ca
titrations of TNS in the
presence of CaM41/75 or CaM85/112 (where the exposure of the N- or
C-terminal hydrophobic pocket has been blocked by a disulfide bond,
respectively) were half-maximal at pCa 5.9 and 5.6,
respectively (data not shown). Thus, the Ca
titrations of CaM41/75 with TNS and of CaM or CaM41/75 tyrosine
fluorescence all indicate a C-terminal Ca
affinity of
1.3
10
M at 10 °C.
Ca
titrations of CaM85/112 with TNS indicate a
Ca
affinity of
2.5
10
M at the N-terminal sites. Knowing the rates of Ca
dissociation from the N-terminal (405/s) and the C-terminal sites
(2.4/s), the Ca
association rate (k
) was calculated from k
= k
/K
. These
calculations suggest that Ca
binds to the lower
affinity N-terminal Ca
binding sites at
1.6
10
M
s
and to the higher affinity C-terminal Ca
binding sites at
2.3
10
M
s
. These calculations
suggest that Ca
binds to the N-terminal sites
70
times faster than it binds to the C-terminal sites.
Figure 4:
A
computer simulation of the time course of a Ca transient produced by rapidly mixing Ca
with
EGTA and the time course of the occupancy of the CaM N- and C-terminal
Ca
binding sites by this Ca
transient. A computer simulation of the Ca
transient (
), which is produced when 2 mM EGTA is
rapidly mixed with 200 µM Ca
, was
achieved by using the KSIM program (N. C. Millar) by setting the
initial [Ca
] to 100 µM at time
= 0 and using a measured Ca
off-rate from EGTA
of 0.55/s and a Ca
on-rate to EGTA of 1.3
10
M
s
at 10
°C. The time course of this Ca
transient was
verified by stopped flow, using the rapid Ca
indicator Mg-Fura-2. The trace labeled MF2 (
) shows the
change in Mg-Fura-2 fluorescence (excitation, 380 nm; emission, 510 nm;
trace inverted for comparison) when 1 µM Mg-Fura-2 in the
presence of 2 mM EGTA is rapidly mixed with 400 µM Ca
. This trace is an average of six traces fit
with a single exponential with a variance of <1.7
10
. The simulation of Ca
exchange with
the N- (
) and C-terminal (
) Ca
binding
sites of CaM assumes that Ca
binds to both halves of
CaM as a simple bimolecular process with the Ca
on-
and off-rates given in Table 1for CaM's N- and C-terminal
sites.
Fig. 4also simulates the time course of
occupancy of the N- and C-terminal Ca binding sites
of CaM (using the on- and off-rates shown in Table 1) during this
Ca
transient. This simulation suggests that the
faster N-terminal sites would be 96% occupied at 0.4 ms and that they
would lose this Ca
to EGTA at
240/s. The slower
C-terminal Ca
binding sites would only be 18%
occupied, and they would then lose this Ca
to EGTA at
2.4/s.
Ca binding to both the N- and C-terminal
lobes of CaM exposes hydrophobic sites that bind TNS and cause a large
increase in TNS fluorescence. Fig. 5(trace A) shows
the time course of the decrease in TNS fluorescence when the
Ca
CaM
TNS complex is mixed with EGTA.
Similar to Suko et al.(1985), we observed a biphasic process
in which 54% of the TNS fluorescence decrease occurs at 405 ±
20/s (Fig. 5, trace A (N-EGTA)), and 46%
occurs at 2.1 ± 0.1/s (Fig. 5, trace A (C-EGTA)). These rates correspond to the rates of
Ca
dissociation from the N- and C-terminal sites of
CaM, respectively (as measured by Quin 2), and suggest that both
hydrophobic pockets close and displace TNS as quickly as Ca
dissociates. Thus, these changes in TNS fluorescence provide an
accurate way of following Ca
dissociation from both
the N- and C-terminal Ca
binding sites of CaM.
If
the N- and/or C-terminal Ca binding sites of CaM are
transiently occupied during a rapid Ca
transient (as
in Fig. 4), then TNS fluorescence would increase with
Ca
and TNS binding and then decrease at the rates of
Ca
removal from the N- and C-terminal sites. Fig. 5(trace B) shows the change in TNS fluorescence
when Ca
(200 µM) is rapidly mixed with
CaM (4 µM) and TNS (200 µM) in the presence
of 2 mM EGTA. Ca
binds and produces a rapid
increase in TNS fluorescence, and then TNS fluorescence decreases as a
biphasic process. Most (82%) of this decrease occurs as Ca
dissociates from the N-terminal sites (Fig. 5, trace B (N-Transient)) at 321 ± 33/s, and the remaining
18% occurs as Ca
dissociates from the C-terminal
sites at 2.1 ± 0.1/s (Fig. 5, trace B (C-Transient)). When the amplitudes for the decreases in
TNS fluorescence that occur in the transient occupancy experiments (Fig. 5, trace B) are compared to the amplitudes of the
decreases in the EGTA experiments (Fig. 5, trace A), we
find that 70% of the N-terminal and 20% of the C-terminal
Ca
binding sites are occupied during the transient
occupancy experiments. These data are consistent with the simulation of Fig. 4, where 96% of the N-terminal sites (with a Ca
on-rate of 1.2
10
M
s
) and 18% of the
C-terminal sites (with a Ca
on-rate of 1.2
10
M
s
)
would be occupied during this Ca
transient.
Increasing the [Ca
] produced greater
occupancy of both the N- and C-terminal hydrophobic pockets by TNS.
Thus, Ca
binding is the rate-limiting step and not
the rate of opening of the hydrophobic pocket or TNS binding. These
data confirm the Ca
on-rates we calculated in Table 1and the simulation using these on-rates (Fig. 4),
which shows that the N-terminal Ca
binding sites are
almost fully occupied during a rapid Ca
transient,
while the C-terminal sites, with a 70-fold slower Ca
on-rate, are not.
The observation that the C-terminal sites
were only 20% occupied by Ca during this rapid
Ca
transient was verified using CaM-tyrosine
fluorescence. When CaM (4 µM) and Ca
(200 µM) were mixed with 2 mM EGTA, the
C-terminal tyrosine fluorescence decreased at a rate of
2.1/s as
Ca
dissociated from the C-terminal sites (Fig. 6, CaM + Ca versus EGTA trace). When a rapid
Ca
transient was produced by mixing Ca
(200 µM) with CaM (4 µM) in the
presence of 2 mM EGTA, the tyrosine fluorescence rose to only
23% of the intensity seen with Ca
saturated CaM and
then decayed back at 2.1/s (Fig. 6, CaM + EGTA versus
Ca trace). When the [Ca
] was increased
to 400 µM, the tyrosine fluorescence increased to 42% of
the intensity seen with Ca
saturated CaM. This
suggests that Ca
binding to the C-terminal is the
rate-limiting step and not a slower structural change at the tyrosine
residues. Thus, both the TNS and the tyrosine data indicate that the
N-terminal but not the C-terminal Ca
binding sites
are maximally occupied under the conditions of this rapid
Ca
transient.
Figure 6:
The
failure of a rapid Ca transient to fully occupy the
C-terminal Ca
binding sites of CaM. The time course
of the decrease in CaM tyrosine fluorescence is shown as EGTA
dissociates the Ca
-saturated or transiently occupied
C-terminal binding sites of CaM. CaM (4 µM) +
Ca
(200 µM) in 200 mM Hepes, pH
7.0, at 10 °C was rapidly mixed with 2 mM EGTA in the same
buffer to determine the rate of Ca
dissociation from
the Ca
-saturated C-terminal sites on CaM (CaM
+ Ca versus EGTA trace). The transient occupancy of
CaM's C-terminal sites was tested by rapidly mixing CaM (4
µM) + EGTA (2 mM) in 200 mM Hepes,
pH 7.0, at 10 °C with 200 µM Ca
in
the same buffer (CaM + EGTA versus Ca trace). The CaM
+ Ca versus Ca trace represents the level of tyrosine
fluorescence in Ca
-saturated CaM; it was achieved by
rapidly mixing CaM (4 µM) + Ca
(100
µM) with Ca
(100 µM).
Tyrosine fluorescence was monitored as described in the legend to Fig. 3. Each trace is an average of seven traces fit with a
single exponential (variance < 1.1
10
).
Our studies show that Ca dissociates from
the N-terminal Ca
binding sites of CaM at 405/s and
from the higher affinity C-terminal sites at 2.4/s at 10 °C. These
results agree with the work of Bayley et al.(1984) who have
shown that Quin 2 chelates Ca
from the N- and
C-terminal sites of CaM at 293 ± 93/s and 2.1 ± 0.4/s,
respectively, at 11 °C. Martin et al.(1992) have also
shown that the N-terminal Ca
binding sites lose
Ca
rapidly (389 ± 64/s) while the higher
affinity C-terminal Ca
binding sites lose
Ca
more slowly (11 ± 2.4/s) at 18 °C and
low ionic strength.
Peptide and MLCK binding to CaM reduced the rate
of Ca dissociation from the N-terminal sites from
405/s to 1.8-2.9/s (a 140-225-fold decrease) and from the
C-terminal Ca
binding sites from 2.4/s to
0.1-0.4/s (a 6-24-fold decrease).
Martin et
al.(1985) have shown that the CaM antagonist trifluoroperazine
slows Ca dissociation from the CaM N-terminal sites
from 310/s to 15/s and from the CaM C-terminal sites from 3.6/s to
0.55/s at 13.4 °C. Further, calmidazolium slowed Ca
dissociation to 4/s and 0.2/s for the N- and C-terminal sites,
respectively (data not shown). Thus, these CaM antagonist drugs, which
bind to both the N- and C-terminal hydrophobic pockets, produce similar
decreases in Ca
off-rate as peptide.
The high
resolution structure of CaM complexed with RS-20 (Meador et
al., 1992), CK peptide (Meador et al., 1993),
and M-13 (Ikura et al., 1992) suggest that peptide binding
dramatically alters CaM structure. The central helix of CaM unwinds,
allowing the hydrophobic pockets on the N- and C-terminal lobes to
engulf these peptides. The amphipathic nature of these peptides allows
them to shield the N- and C-terminal hydrophobic pockets of CaM from
solvent (O'Neil and Degrado, 1990) and to stabilize the
Ca
bound state of CaM. This results in
peptide-induced increases in Ca
affinity, which we
see expressed as large decreases in the rate of Ca
dissociation from both halves of CaM.
While all of the
peptides produced similar dramatic decreases in the rate of
Ca dissociation from the N-terminal of CaM, both
RS-20 and M-13 slowed Ca
dissociation from the
C-terminal
3-fold more than either C
K or MARCKS.
Comparison of the high resolution CaM-peptide structures indicates that
there are
40% less contacts between CaM-C
K compared to
CaM-RS-20 (Meador et al., 1993). Furthermore, both RS-20 and
M-13 have an aromatic Trp residue in their N-terminal domain, which
makes extensive contacts with the C-terminal of CaM (Ikura et
al., 1992; Meador et al., 1992). This could explain why
RS-20 and M-13 reduce Ca
dissociation from the
C-terminal to
0.1/s while C
K and MARCKS peptide reduce
Ca
dissociation to only
0.3/s. While every
residue of RS-20, M-13, and C
K form contacts with CaM, the
Trp residue in the N-terminal of RS-20 and M-13 may further stabilize
the Ca
bound state, increase Ca
affinity, and decrease the rate of Ca
dissociation from the C-terminal.
Our results show that the
C-terminal of CaM has a 2.6-fold greater affinity for Ca than the N-terminal of CaM. This agrees with the results of
Minowa and Yagi(1984) who have shown a 3.4-fold higher Ca
affinity at the C-terminal of the molecule. Since the N-terminal
of CaM has a 170-fold faster Ca
off-rate than the
C-terminal but only a 2-3-fold lower Ca
affinity, it follows that the N-terminal of CaM must have a much
faster Ca
on-rate than the C-terminal. Utilizing the K
and Ca
off-rates for the N-
and C-terminal Ca
binding sites of CaM, we calculated
their Ca
on-rates to be 1.6
10
M
s
and 2.3
10
M
s
,
respectively.
Estimates of Ca on-rate from K
/K
could be complicated by
the cooperativity that exists between the two Ca
binding sites in each lobe of CaM. Therefore, it was necessary to
verify the faster Ca
on-rate to the N-terminal sites
by an additional method. Currently, there is no way to directly measure
the Ca
on-rate to the N-terminal Ca
binding sites in a stopped flow apparatus. Our computer
simulations (Fig. 4) were conducted assuming the Ca
off-rates determined by our stopped flow studies and the
calculated Ca
on-rates cited above and in Table 1. They suggest that during a rapid Ca
transient (half-width 0.6 ms), Ca
would occupy
96% of the N-terminal sites and 18% of the C-terminal sites. We have
produced this rapid Ca
transient in the stopped flow
and verified (using CaM and TNS fluorescence) that the N-terminal sites
are >70% saturated by this Ca
transient, while the
slower C-terminal sites are only 20% occupied. Thus, our calculated
Ca
on-rates for the N- and C-terminal sites of CaM
are verified by modeling (Fig. 4) and by the Ca
transient occupancy experiments using CaM-TNS (Fig. 5).
Ca not only binds quickly to the N-terminal
Ca
sites, but it also rapidly exposes the N-terminal
hydrophobic pocket to accommodate TNS and presumably protein binding.
Thus, only the N-terminal Ca
binding sites on CaM can
respond to a very rapid rise and fall in Ca
by
opening and closing their hydrophobic binding pocket. The
Ca
transient (0.6 ms half-width) produced in our
stopped flow experiments is much faster than physiological
Ca
transients, which exhibit half-widths from
milliseconds to minutes. Clearly, during these longer Ca
transients, both the N- and C-terminal Ca
binding sites would be occupied. The rapid
``artificial'' Ca
transient produced in our
stopped flow experiments was designed and used to verify the faster
Ca
on-rate to the N-terminal sites of CaM relative to
the C-terminal sites.
The N-terminal Ca binding
sites of CaM have a
70-fold faster Ca
on-rate
and a
170-fold faster Ca
off-rate than the
higher affinity C-terminal Ca
binding sites.
Therefore, the N-terminal sites of CaM resemble the N-terminal sites of
TnC, which have a fast Ca
on-rate (
2
10
M
s
) and
off-rate (400/s) (Johnson et al., 1979, 1994) compared to the
higher affinity C-terminal Ca
-Mg
sites.
Both the N- and C-terminal lobes of CaM must bind
Ca and expose their hydrophobic pockets to activate
target proteins (Persechini and Kretsinger, 1988). Our Ca
titrations (Fig. 3) indicate that RS-20 and C
K
increase Ca
affinity at CaM's C-terminal
Ca
binding sites so dramatically that these sites may
be 50-80% occupied even at the resting levels of Ca
found in smooth muscle cells (
pCa 6.8, Cornwell and
Lincoln(1989)). Therefore, the higher affinity C-terminal lobe of CaM
may be bound to some target proteins at resting Ca
levels. While this would not result in enzyme activation, the
subsequent rapid binding of Ca
to the N-terminal
sites of CaM would allow it to bind also, resulting in target protein
activation. A similar mechanism exists in skeletal muscle troponin C,
where the higher affinity C-terminal
Ca
-Mg
sites stabilize the troponin
complex at resting levels of Ca
, and the rapid
exchange of Ca
with the faster N-terminal sites
regulates contraction and relaxation (Potter and Johnson, 1981).
Ca dissociates from CaM after cellular free
[Ca
] falls in a Ca
transient. This results in the disruption of most CaM-target protein
complexes and target protein inactivation. Since both halves of CaM are
required for activation, the removal of Ca
from
either the N- or C-terminal Ca
binding site would
result in enzyme inactivation. Our data (Table 1) indicate that
in the presence of MLCK and peptides, Ca
dissociates
from the N-terminal sites of CaM 5-29 times faster
(1.8-2.9/s) than it dissociates from the C-terminal half of the
molecule (0.1-0.4/s). This suggests that as the
[Ca
] falls, the N-terminal half of CaM
would dissociate first, resulting in enzyme inactivation at a rate
slower than 2-3/s (at 10 °C and low ionic strength). At 20
°C, in the presence of RS-20 and KCl, Ca
dissociates from the N-terminal sites of CaM at 4.5/s and from
the C-terminal sites of CaM at 0.18/s. If Ca
dissociation from the N-terminal of CaM controls the disruption
and inactivation of the CaM
MLCK complex, then these events must
occur slower than 4.5/s at 20 °C. Consistent with this, we have
previously shown that EGTA disrupts the CaM-skeletal muscle MLCK
complex at 2/s (Johnson et al., 1981) and the CaM-smooth
muscle MLCK complex at 3.2/s (Kasturi et al., 1993) at 20
°C. Stull et al. (1986) have also demonstrated that EGTA
can inactivate CaM-activated MLCK at a rate of
1/s. Thus,
Ca
dissociation from the N-terminal sites of CaM in
the CaM
RS-20 and CaM
MLCK complexes is fast enough to
control disruption of these complexes and the inactivation of MLCK.
Ca
dissociation from the C-terminal sites at 0.18/s
is too slow to be responsible for the disruption of this complex and
inactivation of MLCK. Thus, Ca
dissociation from the
N-terminal sites of CaM, in the CaM
MLCK complex, appears to
control the rate of enzyme inactivation as the
[Ca
] falls.
Ca dissociates from the N-terminal sites of CaM hundreds of times
more slowly when a target protein is bound (1.8-2.9/s). This
allows a rapid Ca
transient to result in the binding
and presumable activation of a CaM target protein for over
300-400 ms. Thus, the fast rate of Ca
binding
to the N-terminal of CaM allows it to rapidly ``sense'' the
Ca
transient and bind target proteins. The dramatic
increases in Ca
affinity and the reductions in the
rate of Ca
dissociation observed in the CaM-target
protein complex ensures that CaM-activated enzymes can remain active
long after a rapid Ca
transient has subsided.