©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Effects of Myosin Light Chain Kinase and Peptides on Ca Exchange with the N- and C-terminal Ca Binding Sites of Calmodulin (*)

(Received for publication, July 14, 1995; and in revised form, October 5, 1995)

J. David Johnson (1)(§) Christopher Snyder (1) Michael Walsh (2)(¶) Maera Flynn (1)

From the  (1)Department of Medical Biochemistry, The Ohio University Medical Center, Columbus, Ohio 43210 and the (2)Department of Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 4NI, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 CaMbulletpeptide 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 times 10^8M s) than the C-terminal sites (2.3 times 10^6M 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.


INTRODUCTION

CaM (^1)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 alpha-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 alpha-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 CabulletCaMbulletpeptide 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 (C(2)K), 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 CaMbulletprotein (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.


EXPERIMENTAL PROCEDURES

Materials

Quin 2 and Mg-Fura-2 were purchased from Molecular Probes; phenyl-Sepharose CL-4B, TNS, calmidazolium, and EGTA were purchased from Sigma; phenylmethylsulfonyl fluoride was from Research Organics (Cleveland, OH); and hydroxylapatite was from Bio-Rad. All other chemicals were of analytical grade.

C(2)K 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).

Protein Purification

CaM was purified from bovine brain as described previously by Kasturi et al. (1993). MLCK was purified to electrophoretic homogeneity from chicken gizzard smooth muscle as described by Ngai et al. (1984).

Methods

All static fluorescence measurements were performed on a Perkin-Elmer LS5 Spectrofluorometer at 10 °C. Free [Ca] was calculated as described by Robertson and Potter(1984). Kinetic measurements were performed by mixing an equal volume (50 µl) of each reagent together in an Applied Photophysics Ltd. (Leatherhead, UK) model SF.17 MV stopped flow instrument. This instrument had a dead time of 1.6 ms and flow rate of 17 µl/ms. The samples were excited using a 150-watt Xenon arc source at the specified wavelength. Fluorescence emission was monitored through the specified interference filters. The curve fitting program (by P. J. King, Applied Photophysics Ltd.) uses the non-linear Levenberg-Marquardt algorithm.

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 times 10). The 0-20-ms trace is an average of six traces fit with a single exponential (variance < 1.6 times 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 times 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.



Computer Modeling

The computer simulations were performed using KSIM version 1.1 (N. C. Millar, UCLA School of Medicine, Los Angeles). The K(d) of EGTA for Ca at 10 °C and low ionic strength (pH 7.0) was calculated to be 4.23 times 10M from the program of Robertson and Potter (1984). The rate of Ca dissociation from EGTA (k) was determined by stopped flow experiments where EGTA (10 µM) and Ca (20 µM) were mixed with Quin 2 (150 µM) in the same buffer (20 mM Hepes, pH 7.0), at 10 °C. The Quin 2 fluorescence increased at the k of EGTA, 0.55 ± 0.03/s. Our calculated k of 1.3 times 10^6M s (from k = k/K(d)) was in agreement with the value of 9.2 times 10^5M s determined by Smith et al.(1984) at 16 °C in 0.1 M ionic strength (pH 6.8). Modeling of the Ca transient and the Ca occupancy of the N- and C-terminal sites of CaM assumed the following initial concentrations: N- and C-terminal Ca binding sites (4 µM), Ca (100 µM), and EGTA (1 mM), which represent the reaction conditions in the stopped flow experiments of Fig. 5after mixing. The kinetic parameters used for Ca exchange with the N- and C-terminal sites of CaM were those determined as described under ``Results'' and as cited in Table 1.


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 CabulletCaMbulletTNS 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 times 10^5). The 0-20-ms traces in the inset represent an average of six traces fit with a single exponential (variance < 3.0 times 10). Each kinetic trace was fit after flow had stopped.






RESULTS

Ca Dissociation from the N- and C-terminal Ca Binding Sites of CaM

Bayley et al. (1984) have previously used Quin 2 fluorescence to measure the rates of Ca dissociation from CaM. Fig. 1A shows Ca dissociation from CaM's N- and C-terminal Ca binding sites as measured by the increase in Quin 2 fluorescence, which occurs upon Ca binding. Ca dissociated from CaM as a biphasic process with 1.2 ± 0.2 mol of Ca dissociating at 405 ± 75/s (Fig. 1A, inset) and 1.6 ± 0.2 mol of Ca dissociating nearly 170 times more slowly at 2.4 ± 0.2/s. Thus, under the conditions used in our studies, we clearly observe Ca dissociation from both the N- and C-terminal Ca binding sites of CaM.

Effect of MLCK and CaM Binding Peptides on Ca Dissociation from CaM

The effects of MLCK and several CaM binding peptides on Ca dissociation from CaM's N- and C-terminal Ca binding sites were determined using Quin 2 fluorescence. Fig. 1B shows the effect of smooth muscle MLCK, its CaM binding peptide (RS-20), and the MARCKS peptide on Ca dissociation from CaM. In the presence of protein or peptide, the rapid phase of Ca dissociation (405/s) was eliminated. Instead, biphasic Ca dissociation rates of 2.28 ± 0.20/s and 0.39 ± 0.15/s, 1.8 ± 0.2/s and 0.07 ± 0.01/s, and 2.6 ± 0.3/s and 0.3 ± 0.02/s were observed for MLCK, RS-20, and MARCKS peptide, respectively. M-13 peptide, from skeletal muscle MLCK, was similar to RS-20 with biphasic Ca dissociation rates of 2.6 ± 0.6/s and 0.06 ± 0.02/s. C(2)K peptide was similar to the MARCKS peptide with biphasic Ca dissociation rates of 2.9 ± 0.3/s and 0.29 ± 0.03/s. Thus, MLCK and all of these high affinity peptides abolish the rapid rate of Ca dissociation from the N-terminal of CaM and result in 2 mol of Ca dissociating at 1.8-2.9/s and 2 mol of Ca dissociating at 0.1-0.4/s.

Effect of Peptide on Ca Dissociation from the CaM C-terminal Ca Binding Sites

CaM's two tyrosine residues (Tyr-99 and Tyr-138) are in the C-terminal half of the molecule and undergo a large fluorescence increase when Ca binds to the C-terminal sites of CaM (Dedman et al., 1977; George et al., 1993). Fig. 2shows the time course of the EGTA-induced decrease in Ca-CaM tyrosine fluorescence. These data agree with our Quin 2 studies and verify that Ca dissociates from the C-terminal sites of CaM at a rate of 2.1 ± 0.1/s at 10 °C. The addition of MARCKS peptide, which has no Tyr or Trp residues, reduces the rate of Ca dissociation from these C-terminal sites to 0.32 ± 0.02/s.


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 times 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). (^2)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(2)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.

Effects of Temperature and KCl on Ca Dissociation from the N- and C-terminal Sites of CaM in the Presence of RS-20

Since many physiological studies are conducted at higher temperatures and ionic strength, we have examined the effects of RS-20 on Ca dissociation from CaM's N- and C-terminal Ca binding sites at various temperatures in the presence of KCl (90 mM). The addition of KCl to CaM + RS-20, at 10 °C, did not alter the rates of Ca dissociation from either the N- or C-terminal sites from those reported in Table 1. At 20, 30, and 37 °C in the presence of KCl and RS-20, Ca dissociated from the N- and C-terminal sites 2.5, 5.8, and 10 times faster, respectively, than at 10 °C. This suggests a Q of 2.3-2.5 for these processes.

Effects of CaM Binding Peptides on Ca Affinity at the CaM C-terminal Ca Sites

Ca affinity at the C-terminal sites was determined by the Ca-dependent increase in tyrosine fluorescence. Fig. 3shows Ca titrations of CaM in the presence or absence of C(2)K or MARCKS peptide and of CaM41/75 in the presence or absence of RS-20. Ca half-maximally binds to the C-terminal sites of CaM and CaM41/75 at pCa 5.96. In the presence of C(2)K and MARCKS peptide, half-maximal Ca binding occurs at pCa 6.8 and 6.4, respectively. RS-20 shifted the half-maximal binding of Ca to the C-terminal sites of CaM41/75 from pCa 5.96 to 7.0 as monitored by the increase in its Trp fluorescence. These results suggest that CaM and CaM41/75 have the same C-terminal Ca affinities and that RS-20, C(2)K, and the MARCKS peptide produce 9.1, 6.3, and 2.6-fold increases, respectively, in C-terminal Ca affinity. Thus, RS-20, which produces the largest (9-fold) increase in C-terminal Ca affinity, produces the largest (40-fold) decrease in C-terminal Ca off-rate.


Figure 3: Effects of RS-20, MARCKS, and C(2)K 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 (circle), CaM41/75 (*), CaM + C(2)K peptide (), or CaM + MARCKS peptide (up triangle) and the Ca dependence of the increase in RS-20 tryptophan fluorescence in the presence of CaM41/75 (box) 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(2)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.



Determination of Ca Affinity at the CaM N- and C-terminal Ca Binding Sites

The hydrophobic polarity probe TNS undergoes a large fluorescence increase when it binds to the Ca-dependent hydrophobic pockets in both the N- and C-terminal halves of CaM (Suko et al., 1985; Johnson et al., 1986).

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 times 10M at 10 °C. Ca titrations of CaM85/112 with TNS indicate a Ca affinity of 2.5 times 10M 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(d). These calculations suggest that Ca binds to the lower affinity N-terminal Ca binding sites at 1.6 times 10^8M s and to the higher affinity C-terminal Ca binding sites at 2.3 times 10^6M s. These calculations suggest that Ca binds to the N-terminal sites 70 times faster than it binds to the C-terminal sites.

Transient Occupancy of the N-terminal Ca Binding Sites of CaM

We have utilized the slow Ca on-rate of EGTA (Smith et al., 1984) to produce a rapid Ca transient and determined if Ca can bind to the N- or C-terminal sites of CaM and induce TNS binding during a brief Ca transient. A computer simulation of the Ca transient that is produced when 100 µM Ca is instantaneously mixed with 1 mM EGTA is shown in Fig. 4(Ca trace). The free [Ca] decays at 1200/s to an equilibrium value of pCa 7.4, producing a 0.6-ms half-width Ca transient. This Ca transient can be produced and visualized in the stopped flow by following Mg-Fura-2 fluorescence when 200 µM Ca is mixed with Mg-Fura-2 in the presence of 2 mM EGTA (Fig. 4, MF2 trace). Ca bound to Mg-Fura-2 during the dead time and then dissociated at 1300/s, as EGTA chelated Ca. Thus, the simulation and Mg-Fura-2 fluorescence precisely define the time course of this Ca transient.


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 (Delta), 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 times 10^6M 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 (bullet) 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 times 10^4. The simulation of Ca exchange with the N- () and C-terminal (box) 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 CabulletCaMbulletTNS 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 times 10^8M s) and 18% of the C-terminal sites (with a Ca on-rate of 1.2 times 10^6M 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 times 10).




DISCUSSION

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), C(2)K 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(2)K or MARCKS. Comparison of the high resolution CaM-peptide structures indicates that there are 40% less contacts between CaM-C(2)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(2)K and MARCKS peptide reduce Ca dissociation to only 0.3/s. While every residue of RS-20, M-13, and C(2)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(d) 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 times 10^8M s and 2.3 times 10^6M s, respectively.

Estimates of Ca on-rate from K/K(d) 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 times 10^8M 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(2)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 CaMbulletMLCK 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 CaMbulletRS-20 and CaMbulletMLCK 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 CaMbulletMLCK 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK 33727 (to J. D. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medical Biochemistry, The Ohio State University Medical Center, 333 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. Tel.: 614-292-0104; Fax: 614-292-4118.

Supported by a grant from the Medical Research Council of Canada and a Medical Scientist Award from the Alberta Heritage Foundation for Medical Research.

(^1)
The abbreviations used are: CaM, calmodulin; CaM41/75, oxidized calmodulin (Q41C,K75C); CaM85/112, oxidized calmodulin (I85C,L112C); MLCK, myosin light chain kinase; C(2)K, CaM-dependent protein kinase II peptide (MHRQETVDCLKKPNARRLKGAILTTMLA); MARCKS, myristoylated alanine-rich protein kinase C substrate peptide (KKKKKRFSFKKSFKLSGFSFKKNKK); M-13, skeletal muscle myosin light chain kinase peptide (KRRWKKNFIAVSAANRFKKISSSGAL); RS-20, smooth muscle myosin light chain kinase peptide (ARRKWQKTGHAVRAIGRLSS); TNS, 2-ptoluidinylnaphthalene6-sulfonate.

(^2)
Z. Grabarek, personal communication.


ACKNOWLEDGEMENTS

We gratefully acknowledge Cindy Sutherland for purification of MLCK, Dr. Perry Blackshear (Duke University) for the gift of MARCKS peptide, and Dr. Zenon Grabarek (Boston Biomedical Research Institute) for helpful comments on the manuscript and for the gift of CaM41/75 and CaM85/112.


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