A Mechanism for Calmodulin (CaM) Trapping by CaM-kinase II Defined by a Family of CaM-binding Peptides*

M. Neal WaxhamDagger §, Ah-lim Tsai, and John A. Putkey§parallel

From the Departments of Dagger  Neurobiology and Anatomy,  Internal Medicine, and parallel  Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, Texas 77030

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Autophosphorylation of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) induces a striking >1,000-fold increase in its affinity for CaM, which has been called CaM trapping. Two peptides modeled after the CaM binding domain of CaM-kinase II were previously shown to kinetically resemble CaM binding to phosphorylated and dephosphorylated forms of the enzyme, thus providing a model system with which to define the molecular basis of CaM trapping. In this report, the specific contribution of each amino acid to the rates of association and dissociation, and the overall Kd of CaM binding to CaM-kinase II was determined using an overlapping peptide family, and a fluorescently labeled CaM. The association rate constants were similar for the entire family of peptides and ranged from 8 × 107 to 32 × 107 M-1 s-1. In contrast, the dissociation rate constants for the peptides varied by >3500-fold and ranged from 0.26 to 7 × 10-5 s-1. These rate constants yield overall Kd values for binding CaM to the peptides that range from 2 × 10-9 M to 2 × 10-13 M. Extending the low affinity CaM-binding peptide, CKII(296-312), to include 293Phe-Asn-Ala295 provided the single largest contribution to the decreased dissociation rate constant, 1,300-fold. It was further shown using Ala-substituted peptides that the basic residues 296Arg-Arg-Lys299 were also essential for slow CaM dissociation; however, their contribution was realized only when 293Phe-Asn-Ala295 were present. These results suggest a plausible model in which autophosphorylation of CaM-kinase II leads to a conformational change in the region of 293Phe-Asn-Ala295 which makes these residues accessible for binding to CaM. As a consequence of these changes, further CaM contacts with 296Arg-Arg-Lys299 are established leading to high affinity CaM binding or "CaM trapping."

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II, EC 2.7.1.123)1 is a well studied multifunctional Ser/Thr-directed protein kinase that regulates numerous biochemical and cellular processes (1). CaM-kinase II is activated by binding Ca2+/CaM, which leads to an increase in affinity for Mg2+/ATP and autophosphorylation at a specific Thr residue (Thr286)2(1). Ca2+/CaM can be removed from the autophosphorylated enzyme, and it will continue to phosphorylate substrate proteins, although at a reduced rate (1). Additionally, it was recently shown that autophosphorylation of Thr286 also leads to a change in the affinity of the enzyme for Ca2+/CaM (2, 3). This phenomenon was termed "CaM trapping" by Meyer et al. (2) to convey the point that the increase in CaM binding is robust with the affinity increasing by several orders of magnitude.

Synthetic peptides have proved to be useful models for elucidating the mechanistic details of CaM binding to CaM-kinase II (4-8). We recently showed that synthetic peptides can kinetically mimic low affinity (unphosphorylated) and high affinity (phosphorylated) binding of CaM-kinase II to CaM (9). The low affinity peptide encompassed amino acids 296-312, while the high affinity peptide encompassed residues 290-314. Since the high affinity peptide, CKII(290-314),3 did not contain the sequences surrounding the autophosphorylation site (Thr286), we hypothesized that autophosphorylation alters the conformation of the enzyme such that new points of contact with Ca2+/CaM are exposed, leading to the increased CaM-binding affinity of the enzyme. Residues in CaM-kinase II, which likely interact with CaM and lead to high affinity binding, were identified based on the crystal structure of Ca2+/CaM bound to CKII(290-314) (10). This structure shows that amino acids 293-310, with the exception of Asn294, are in direct contact with Ca2+/CaM. We thus proposed that residues 293Phe-Asn-Ala295, which are not present in the low affinity peptide CKII(296-312), were necessary for high affinity binding of peptides to CaM.

In the present study, we identify and quantitate the contribution of individual amino acids to the increased CaM-binding affinity by utilizing a family of synthetic peptides that mimic the sequence of CaM-kinase II. The data show that the peptides exhibit a range of affinities for CaM that span four orders of magnitude (10-9 to 10-13 M). This is analogous to the change in the affinity of CaM-kinase II for CaM following autophosphorylation. The data further reveal that the 293Phe-Asn-Ala295 motif is necessary but insufficient for high affinity CaM binding, and that it appears to act in synergy with the basic sequence of amino acids 296Arg-Arg-Lys298. Based on these data, we propose a four-step model to explain the transition of CaM-kinase II from a low to a high affinity CaM-binding enzyme.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression, Purification, and Mutagenesis of Calmodulin-- These procedures were identical to those described previously (9). Briefly, the Lys residue at amino acid position 75 of the cDNA encoding chicken CaM was changed to a Cys residue to create CaM(C75). The mutation was verified by sequencing. The protein was then expressed in bacteria from a plasmid driven by the lambda  PL promoter. Purification of CaM(C75) from the soluble fraction of lysed bacteria was as described previously (11). The final product was greater than 95% pure when a fraction was analyzed by Coomassie Blue staining following SDS-polyacrylamide gel electrophoresis.

Labeling of CaM(C75) with Acrylodan and IAEDANS-- Acrylodan (Molecular Probes) was dissolved in N,N'-dimethylformamide at a concentration of 100 mM. CaM(C75) at a concentration of 2-2.5 mg/ml was made 5 mM in dithiothreitol and then desalted into 50 mM MOPS, pH 7.5, and 0.5 mM CaCl2 with 6 M urea. Acrylodan was added in a 2-fold molar excess and allowed to react overnight at room temperature followed by exhaustive dialysis against 50 mM MOPS, pH 7.5. Final protein concentrations were determined using the BCA assay (Pierce) with CaM as the standard. The amount of bound probe was determined by absorbance spectroscopy using an extinction coefficient at 391 nm of 20,000 M-1 cm-1. Probe to protein ratios were 0.9 to 1.0. The labeling of CaM(C75) with IAEDANS was described in Putkey and Waxham (9).

Synthesis and Purification of Peptides-- Peptides were synthesized either by the Analytical Chemistry Center at the University of Texas Medical School at Houston or by Research Genetics Inc. All peptides were purified by reverse-phase high performance liquid chromatography, and the final products were analyzed by mass spectroscopy. Peptides were quantified by absorbance at 215 nm.

Rate Constant Measurements-- Determination of slow dissociation rates were made using a PTI QuantaMaster spectrofluorimeter. Fluorescence excitation and emission maxima for CaM(C75)ACR were 370 and 470 nm, respectively. Slit widths were typically maintained at 1-2 and 5 nm for excitation and emission, respectively. Addition of components were made manually to a cuvette with a 1-ml volume. The standard buffer for fluorescence experiments was 25 mM MOPS, pH 7.0, 150 mM KCl, and 0.1 mg/ml bovine serum albumin. CaM(C75)ACR was used at a concentration of 0.06 µM. For measuring CaM dissociation, peptide was mixed at a saturating concentration with CaM and Ca2+ (0.5 µM final concentration). After measuring emission for 2 min, a 50-fold excess of unlabeled CaM was added to the cuvette. To minimize photobleaching during extended data collection periods, a computer-controlled rotating cuvette turret was utilized to expose a given sample to incident light for a 2-s data collection period every 60 s. A cuvette containing the same sample but lacking excess CaM served as a monitor for photobleaching and variations in lamp intensity in each of these experiments. All such measurements were performed at least two times with each peptide, and the data were fit with a single or double exponential function and averaged to determine the value of koff for CaM.

On rates and fast off rates were measured using an Applied Photophysics Ltd. (Leatherhead, UK) model SV.17 MV sequential stopped-flow spectrofluorimeter with a dead time of 1.7 ms. Excitation was at 370 nm with 5-nm slit widths, and emitted light was collected using a 470-nm cutoff filter. Samples were excited with a 150-watt xenon lamp. Off rates were determined by rapid mixing of equal volumes of solutions from two syringes. Typically, one syringe contained the standard buffer (without EGTA) including 200 µM CaCl2 with CaM(C75)ACR at a concentration of 0.26 µM and an equimolar peptide which achieved maximal fluorescence intensity. The second syringe contained excess unlabeled CaM (10 µM) in the same buffer. Data from five to six injections were averaged and then fit with a single exponential function. All stopped-flow measurements were made at 4-6 °C. On rates were determined in a similar fashion. One syringe was filled with 0.25 µM CaM(C75)ACR in standard buffer containing 200 µM CaCl2, and the second syringe contained peptide in the same buffer. The peptide concentration was varied from 0.25 to 8 µM in 2-fold steps. Data from five to six injections were averaged for every peptide concentration, and each average was then fit with a single exponential. On rate constants were calculated for each peptide by determining the slope of a plot of peptide concentration versus observed association rate, where kobs = kon[peptide] + koff. A minimum of four peptide concentrations were included in each plot.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Characterization of CaM(C75)ACR-- We previously established that CaM(C75)IAE, which has an IAEDANS fluorescent probe covalently coupled to a single Cys residue at position 75, proved a useful tool to measure CaM dissociation rates from synthetic peptides and holoCaM-kinase II (9). However, preliminary experiments (data not shown) showed that the fluorescence quantum yield from IAEDANS-labeled CaM was insufficient to determine association rates using dilute CaM solutions. We therefore coupled acrylodan, a fluorescent probe with higher quantum yield than IAEDANS, to CaM(C75) to produce CaM(C75)ACR.

The emission spectra for CaM(C75)ACR in the presence and absence of Ca2+ and the CaM-kinase peptide CKII(290-312) are shown in Fig. 1. Addition of Ca2+ to apoCaM(C75)ACR induces an ~90% increase in fluorescence intensity. Subsequent addition of CKII(290-312) results in a further increase in fluorescence intensity of ~185% relative to apoCaM(C75)ACR and a slight blue shift in the peak of fluorescence intensity (lambda max approximately 470 nm). The increase in fluorescence intensity due to peptide binding likely involves two mechanistic steps with distinct kinetic parameters. First, binding of the peptide to CaM must occur (k1) followed by a subsequent direct or indirect change in the microenvironment of the fluorescent probe (k2). In these studies, we assume that k2 is much faster than k1. Thus, all time-dependent changes in fluorescence measured in the experiments described below are assumed to reflect the rate of peptide association or dissociation.


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Fig. 1.   Characterization of changes in emission spectra of CaM(C75)ACR by binding Ca2+ and peptide. CaM(C75)ACR (1 µg; 0.06 µM final concentration) was added to 1 ml of 25 mM MOPS, pH 7.0, 150 mM KCl, 0.1 mM EGTA, and 0.1 mg/ml BSA, and an emission spectrum was obtained between 400 and 600 nm (trace labeled -Ca2+). CaCl2 (0.5 mM final concentration) was added, and a second scan was obtained (+Ca2+). Finally, an excess of CKII(290-312) was added (5 µM final concentration), and a third scan was taken (+Ca2+/+CKII(290-312)). The samples were excited at 375 nm at room temperature. The excitation and emission bandwidths were 2 and 2 nm, respectively. Each spectrum is the average of three scans. Fluorescence measurements in all figures are given in relative fluorescence units.

The Ca2+ and peptide-induced increase in fluorescence intensity and shift in lambda max are qualitatively similar to those seen for IAEDANS-labeled CaM(C75) (9). As expected, on a molar basis, CaM(C75)ACR provided a fluorescent indicator that was approximately 6-fold more sensitive than CaM(C75)IAE.

CaM(C75)IAE does not alter the activation of CaM-kinase II when compared with unmodified CaM (9). CaM(C75)ACR was similarly tested for its ability to activate CaM-kinase II. The half-maximal activation of the enzyme was determined to be 34 nM for CaM and 110 nM for CaM(C75)ACR. There was also a 15% decrease in Vmax utilizing CaM(C75)ACR compared with CaM. Thus, CaM(C75)ACR was about 3-fold less potent at activating the enzyme than CaM or CaM(C75)IAE. This decreased effectiveness could be due either to decreased binding affinity or decreased capacity to activate the enzyme and is possibly due to the charge difference of the acrylodan moiety in relation to IAEDANS. Meyer et al. (2), reported that randomly dansylated CaM had an approximately 3-fold lower binding affinity than CaM when assayed by competitive binding studies. Dansylated CaM was used successfully in their studies to measure the association and dissociation rates of CaM from CaM-kinase II.

To address the issue of binding affinity in more detail, the rate of dissociation of CKII(296-312) from CaM(C75) was determined using CaM(C75)IAE or CaM(C75)ACR (Fig. 2). The dissociation rate for CaM(C75)IAE was 0.14 s-1 and the rate for CaM(C75)ACR was 0.16 s-1. Similar experiments were accomplished with several other peptides and no significant differences were detected in the dissociation rates using CaM labeled with the two different fluorescent probes (data not shown). We conclude that CaM(C75)ACR can be reliably utilized to measure the rates of association and dissociation between CaM and synthetic peptides that mimic the CaM-binding domain of CaM-kinase II.


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Fig. 2.   Measurement of CaM dissociation rate constants from peptide associated with CaM(C75)ACR and CaM(C75)IAE using stopped-flow kinetics. Panel A shows the time course for CaM dissociation from CKII(296-312) from CaM(C75)ACR using a stopped-flow fluorimeter. CaM(C75)ACR (0.2 µM) and CKII(296-312) (approx 0.1 µM) in 25 mM MOPS, pH 7.0, 150 mM KCl, 0.1 mg/ml BSA, 0.5 mM CaCl2 were rapidly mixed with CaM (10 µM) in the same buffer without peptide at 4-6 °C. Excitation was at 370 nm (5-nm bandwidths), and emission was monitored using a 470-nm cutoff filter. Panel B shows a similar experiment performed with CaM(C75)IAE. CaM(C75)IAE was used at 0.6 µM and CKII(296-312) was 0.1 µM. For measuring dissociation, CaM was elevated to 30 µM in the second syringe to maintain the same 50-fold excess as in panel A. The excitation was at 340 nm (5-nm bandwidth), and the emission was monitored using a 450-nm cutoff filter. Each curve represents the average of five to six exchange reactions, and only every third data point is presented for clarity. The solid lines and indicated rate constants were derived by fitting the experimental data to the single exponential equation F = (Finitial × e-kt) + Ffinal. Note that the dissociation rates determined for CaM(C75) labeled with the two fluorescent probes are not significantly different and that CaM(C75)ACR provided significantly greater (approximately 6-fold) signal.

Determination of the Association Rates between CaM(C75)ACR and Synthetic Peptide Mimicking the CaM Binding Domain of CaM-kinase II-- We previously showed that CKII(296-312), with a C-terminal Gly-Cys extension, exhibited a CaM dissociation rate constant of 5.0 s-1, while CKII(290-314) exhibited a dissociation rate constant of 3.4 × 10-5 s-1 (9). From this data, we postulated that residues immediately N-terminal from amino acid 296 play a dominant role in the decreased rate of dissociation. Specifically, we hypothesized that amino acids 293Phe-Asn-Ala295 (see Table I) contributed to the large decrease in off rate. We therefore produced a family of overlapping peptides to test specifically the relative contribution of each amino acid between 290 and 296 to the overall affinity of the peptides for CaM. We used Asn312 as the common C-terminal amino acid for all peptides in this study.

                              
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Table I
Summary of kinetic parameters and derived equillibrium constants
Experimental data, such as that shown in Figs. 3 and 4, were fit to the exponential equation F = ((F1 × e-k1) + (F2 × e-k2)) + F1, where F is the observed fluorescence intensity at time, t, which were measured at room temperature. The dependent variables F1 and k1, and F2 and k2 are the overall change in fluorescence and rate constants for two exchange components. Two rate constants are shown only for data that best fit a two-component equation. The numbers in parentheses indicate the percent of the total change in fluorescence due to the minor component. Kd and Delta G0 values were calculated using the rate constant for the major component. All rate constants were measured at 5 °C except the off rates for CKII(293-312), CKII(292-312), CKII(291-312), and CKII(290-312)

Utilizing stopped-flow techniques, we first determined the rate of association of each peptide with CaM by monitoring the time-dependent increase in fluorescence intensity upon peptide binding to CaM(C75)ACR. At least five concentrations of each peptide, typically ranging from 0.25 to 8 µM, were rapidly mixed with CaM(C75)ACR and the second order rate constant (kon) was obtained from the slope of the secondary plot. A representative example of this type of analysis is shown for CKII(293-312) in Fig. 3. Rates of up to approximately 600 s-1 could be reliably measured.


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Fig. 3.   Measurement of CaM association rate constants with peptides using stopped-flow kinetics and determination of kon. Panel A shows the time course for CaM association with CKII(293-312) using a stopped-flow fluorimeter. CaM(C75)ACR (0.25 µM) in 25 mM MOPS, pH 7.0, 150 mM KCl, 0.1 mg/ml BSA, 0.5 mM CaCl2 was rapidly mixed with different concentrations of CKII(293-312) at 4-6 °C. Excitation was at 370 nm, and emission was monitored using a 470-nm cutoff filter. Each curve represents the average of five to six reactions, and the data from 0.25 to 4 µM peptide is shown. Every third data point is shown. The solid lines indicate the fitting to the single exponential equation F = (Finitial × e-kt) + Ffinal. The inset shows the linear relationship between the concentration of CKII(293-312) and the rate constants as determined for each peptide concentration.

Table I summarizes the association rate constants derived for the entire family of peptides. Association rate constants for the native peptides did not vary greatly, ranging from 8 × 107 to 32 × 107 M-1 s-1. These values are similar in magnitude to the association rate constant of 88 × 107 M-1 s-1 (measured at 21 °C) reported by Torok and Trentham (12) for the binding of fluorescently labeled CaM to a peptide that mimics the CaM binding domain of myosin light chain kinase. These on rates are also generally consistent with the results of Meyer et al. (2) who reported the association rate constant for dansylated CaM with CaM-kinase II at 30 °C was ~5 to 15 × 107 M-1 s-1, depending on the phosphorylation status of the enzyme. Together these data suggest that the affinity of CaM for various target peptides is primarily determined by differences in off rates. Somewhat slower association rates were measured for the Ala-substituted peptides, CKII(296-312)* and CKII(293-312)* (Table I). This could be due to altered conformations of the free form of these peptides induced by the hydrophobic N-terminal amino acids. Nevertheless, both these mutant peptides exhibited linear concentration-dependent increases in association rates.

Determination of the Dissociation Rates between CaM(C75)ACR and Synthetic Peptide Mimicking the CaM Binding Domain of CaM-kinase II-- Rates of dissociation of synthetic peptides from CaM(C75)ACR were by monitoring the rate of decrease in fluorescence as CaM(C75)ACR bound to peptide is exchanged for unlabeled excess CaM. Stopped-flow or conventional fluorimeters were used as dictated by the overall time necessary to achieve complete exchange of peptides with CaM(C75)ACR. For technical reasons, experiments using the conventional fluorimeter were performed at room temperature rather than at 5-6 °C, the temperature at which all of the stopped-flow data were collected. The elevated temperature would be expected to enhance the dissociation rates from 2- to 4-fold, which should be taken into account when making comparisons between the dissociation rate constants calculated for the stopped-flow data versus the conventional fluorimeter data.

Fig. 4 shows typical data for peptides that exhibit fast and slow dissociation rates. Table I summarizes the rate constants for all peptides. The dissociation rates for CKII(296-312) and CKII(295-312) were fast and essentially identical (0.26 s-1 versus 0.25 s-1), indicating that Ala295 by itself has little effect on CaM binding affinity. Addition of Asn294 in CKII(294-312) and 293Phe-Asn294 in CKII(293-312) resulted in 60- and 1000-fold decreases in the CaM dissociation rate, respectively, relative to CKII(295-312). This is a minimal estimate since the dissociation rate for CKII(295-312) would be 2-4-fold faster if measured at 22 °C. Further extension of the peptides N-terminal to Phe293 resulted in an additional slight decrease in dissociation rate constants relative to CKII(293-312). Thus, the slow CaM dissociation rate of phosphorylated CaM-kinase II can be largely mimicked by CKII(293-312), and requires 293Phe-Asn-Ala295.


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Fig. 4.   Measurement of CaM dissociation rates from peptides using stopped-flow kinetics and conventional fluorimetry. Panel A shows the time course for CaM dissociation from CKII(296-312). CaM(C75)ACR (0.2 µM) in 25 mM MOPS, pH 7.0, 150 mM KCl, 0.1 mg/ml BSA, 0.5 mM CaCl2 and peptide (at approximately 0.6 µM) were rapidly mixed with CaM (10 µM) in the same buffer without peptide at 4-6 °C. Excitation was at 375 nm (5-nm slit widths), and emission was monitored using a 470-nm cutoff filter. The data represent the average of five to six exchange reactions, and only every third data point is presented for clarity. The solid line is the single exponential fit. Panel B shows the time course for CaM dissociation from CKII(290-312). CaM(C75)ACR (0.2 µM) in 25 mM MOPS, pH 7.0, 150 mM KCl, 0.1 mg/ml BSA, 0.5 mM CaCl2, and peptide (at approximately 0.6 µM) were manually mixed with CaM (10 µM) in the same buffer without peptide. Data were obtained by sampling for a period of 2 s every 60 s, and only every third data point is shown for clarity. Excitation and emission were monitored at 375 and 475 nm, respectively; slit widths were 2 and 10 nm, and experiments were performed at room temperature. A cuvette to which there was no added excess CaM served as a monitor for photobleaching in each of these experiments. All such measurements were performed at least two times with each peptide, and the data were fit with a single or, in the example shown, double exponential function and averaged to determine the value of koff for CaM.

The raw data for peptides CKII(292-312), CKII(291-312), and CKII(290-312) were best fit by a 2-exponential as indicated in Table I. Interestingly, similar experiments using the phosphorylated form of CaM-kinase II (data not shown) also yielded two CaM dissociation constants of 6.2 × 10-4 s-1 and 30 × 10-4 s-1. The mechanistic basis for the two rate constants is not clear, yet it appears that CaM-kinase II peptides can mimic, at least kinetically, multiple CaM binding characteristics of the intact protein.

Calculated Kd and Delta G Values for the Family of CaM-binding Peptides-- Having experimentally determined the association (kon) and dissociation (koff) rate constants for each peptide, we calculated the Kd for each member of the family (Kd = koff/kon). These calculations are summarized in Table I. The Kd values for the peptides with native sequence ranged from 2.0 × 10-9 for CKII(296-312) to 2.0 × 10-13 M for CKII(291-312). It is clear that the largest increase in affinity comes from the addition of the 293Phe-Asn-Ala295 sequence, producing a three-order of magnitude decrease in Kd. However lengthening the peptide further to include the two Lys residues N-terminal to 293Phe-Asn-Ala295 provide for an additional order of magnitude increase in affinity.

The Role of 296Arg-Arg-Lys298 in the Dissociation Rate of CaM-- The crystal structure of CaM-bound CKII(290-314) shows that positively charged side chains of the basic amino acids 296Arg-Arg-Lys298 appear to form electrostatic interactions with acidic residues in the N-terminal domain of CaM (10). Since these basic residues are adjacent to 293Phe-Asn-Ala295, we previously hypothesized that the predominantly hydrophobic interactions between this triplet and CaM may alter the conformation of the CaM-peptide complex such that electrostatic interactions could form between CaM and 296Arg-Arg-Lys298 (9). Such interactions may contribute to the decrease in Delta G0 necessary for CaM rapping.

To specifically test this hypothesis, the basic amino acids 296Arg-Arg-Lys298 were replaced with Ala residues within the context of CKII(296-312) and CKII(293-312). Alanine substitutions in CKII(296-312) have relatively little effect and cause a 6-fold decrease in affinity due to association and dissociation rates that are 20- and 4-fold slower, respectively, than for the corresponding native peptide (see Table I). Substitution of 296Arg-Arg-Lys298 with Ala residues in CKII(293-312) causes a dramatic 3000-fold decrease in affinity for CaM, due to a 6-fold slower rate of association, and a dissociation rate that is 500-fold faster that the native peptide. In total, these data show that the absence of either Arg-Arg-Lys298 or 293Phe-Asn-Ala295 are sufficient to eliminate the high affinity binding of CKII peptides to CaM.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We showed previously that the rate of dissociation of the CaM-kinase peptide CKII(290-312) from CaM was at least 1000-fold slower than the shorter peptide CKII(296-312) (9). Thus, these peptides provided a kinetic model for high and low affinity binding of CaM to the phosphorylated and unphosphorylated forms of CaM-kinase II, respectively. The crystal structure of CaM bound to CKII(290-314) indicated a plausible explanation for these findings. CKII(296-312) lacked 3 amino acids (293Phe-Asn-Ala295), of which both Phe293 and Ala295 interact with residues in CaM (10). Based on these observations we proposed that 293Phe-Asn-Ala295 were required for high affinity binding of CaM-kinase II peptides to CaM. Their contribution to the overall decrease in free energy of the high affinity complex would be either through direct stabilizing interactions with CaM and/or by promoting a conformation that allows CaM to form stable interactions with other residues on the peptide, specifically, the adjacent 296Arg-Arg-Lys298. This working model was tested in the current manuscript by kinetic analysis of rates of association and dissociation, and the overall Kd for the binding of CaM to a family of peptides.

The data demonstrate an extremely high affinity association between CaM and the longer peptides (Kd approx  10-13 M), yet previous enzyme inhibition studies using similar peptides have reported Ki or IC50 values of approx 10-8-10-9 M (4, 6). While competitive enzyme inhibition assays provide useful information on the relative affinity of peptides for CaM, these assays essentially measure the amount of peptide required to reduce the free CaM levels to a value equal to its Kd for the marker enzyme. Thus, the observed Ki or IC50 for peptides will never be lower than this Kd. Fluorescently labeled CaM coupled with kinetic measurements provides a means to directly measure binding constants for high affinity association of CaM with a variety of peptides and proteins.

The data presented here clearly demonstrate that Phe293 and Asn294 are necessary for high affinity binding of CaM-kinase II peptides to CaM. The increase in affinity discovered for peptides of increasing length was dominated by decreased rates of CaM dissociation. Addition of Ala295 to the core peptide CKII(296-312) had no discernible effect on CaM-dissociation. However, addition of Asn294 and Ala295 produced a 40-fold increase in binding affinity. This was somewhat surprising in that Asn294 was disordered in the crystal structure and presumably does not make direct contacts with CaM. Addition of 293Phe-Asn-Ala295 produced the single most substantial increase in binding affinity, increasing over 1,100-fold from the core peptide CKII(296-312) and 28-fold over CKII(294-312). Further extension of the peptide in the N-terminal direction produced measurable but much smaller increases in CaM binding affinity.

A second feature of our hypothesis was that amino acids 296Arg-Arg-Lys298 are also important for high affinity binding of CKII peptides to CaM. This is based on 1) their proximity to 293Phe-Asn-Ala295, 2) the fact that these residues do indeed form electrostatic interactions with clusters of acidic residues in CaM (10), and 3) that analogous basic residues are found in the N-terminal end of CaM-binding peptides from myosin light chain kinase. Table I shows that conversion of 296Arg-Arg-Lys298 to Ala within the context of CKII(296-312) has little effect on overall affinity. In contrast, the same change made in CKII(293-312) decreases overall affinity by several orders of magnitude. From these observations, we conclude that potential electrostatic interactions between CaM and CKII peptides are not established with CKII(296-312), even though 296Arg-Arg-Lys298 is present in the peptide, and that 293Phe-Asn-Ala295 is necessary for 296Arg-Arg-Lys298 to form the appropriate contacts with CaM for high affinity binding. Formation of such electrostatic interactions may also be responsible for the 40-fold increase in CaM binding affinity upon extension of the CKII peptides to include Asn294. Even though Asn294 is not ordered in the crystal structure it may shield 296Arg-Arg-Lys298 from water and increase the probability that these amino acids can form electrostatic interactions with CaM. In total, our data demonstrate that both 293Phe-Asn-Ala295 and 296Arg-Arg-Lys298 are necessary and act in synergy to produce high affinity CaM binding.

Our data are consistent with a recent report by Chin and Means (14) who systematically converted the nine Met residues in CaM to Gln. They showed that substitution of only Met124 produced a decrease in the maximal activation of CaM-kinase II. This alteration, and others, also produced significant increases in the amount of CaM required for half-maximal activation of the enzyme. Met124 is particularly significant in the context of our findings since this residue contacts Phe293, Ala295, and Leu299. It is possible that following autophosphorylation of Thr286, interaction of Phe293 and Ala295 with Met124 in CaM allows alignment of other residues in the C and N terminus of CaM to form stable contacts with 296Arg-Arg-Lys298.

Our data have implications with respect to a previous report which showed that amino acids 291Lys-Lys-Phe-Asn294 are necessary to maintain CaM-kinase II in the fully inhibited state. Cruzalegui et al. (15) reported that deletion or replacement of 291Lys-Lys-Phe-Asn294 confers partial Ca2+/CaM-independent activity to CaM-kinase II. Further, C-terminal truncation of CaM-kinase II up to, but not including, 291Lys-Lys-Phe-Asn294 inactivated the enzyme. Further truncation to remove 291Lys-Lys-Phe-Asn294 produced an active form of the enzyme. Apparently in the basal state, these residues are in contact with residues in the catalytic region providing a portion of the autoinhibitory domain of the enzyme. As such, one could speculate that 291Lys-Lys-Phe-Asn294 would not be available for simultaneous binding to CaM. Only upon binding CaM and autophosphorylation of Thr286 would these residues become available for CaM interactions. This hypothesis is supported by proteolysis studies, which showed that autophosphorylation of Thr286 exposes the region surrounding Phe293 to proteolysis (16, 17).

It is logical to speculate that the high and low affinity CaM-binding peptides, as well as the phosphorylated and unphosphorylated forms of CaM-kinase II, will have differential and biologically significant effects on the Ca2+-binding properties of CaM. This follows from a series of studies showing that CaM binding to different targets differentially increase the affinity of CaM for Ca2+ (18-20). This observation has led some to speculate that stable intermediates of CaM bound to targets might persist even at resting intracellular Ca2+ (for a formal kinetic scheme, see Brown et al. (13)). If the high affinity association of CKII(290-312) is reflected in a significantly slowed off rate of Ca2+ from CaM, then it seems possible that Ca2+ would not fully dissociate from CaM when intracellular Ca2+ concentration returns to basal levels, leaving the enzyme in a CaM-bound state. A corollary to this mechanism would be that the proportion of cellular CaM-kinase II that exists in the Thr286-phosphorylated state (high CaM affinity) may exist with CaM physically associated with its CaM binding domain. As proposed (2), the abundance of CaM-kinase II in neurons coupled with its capacity to bind CaM with high affinity might not only maintain the enzyme in an active state, but also regulate the availability of CaM for other CaM-dependent enzymes. The potential for these scenarios will be explored by determining whether CKII(296-312) and CKII(290-312) induce different degrees of increase in the Ca2+-binding affinity of CaM.

Based on these data and the literature reports discussed above, a model for the transition of CaM-kinase II from a low to high affinity CaM-binding form can be proposed to include the following steps. 1) First, Ca2+ binds to CaM, inducing a conformational change that produces an initial interaction with the core CaM-binding domain of CaM-kinase II. A reasonable prediction from the crystal structure (10) is that the critical residues in the core are likely Leu299 and Leu308; 2) Ca2+/CaM-binding induces intersubunit autophosphorylation of Thr286; 3) the phosphorylation of Thr286 exposes 293Phe-Asn-Ala295, and possibly 290Leu-Lys-Lys292, which are then available to form additional contacts with CaM; and 4) formation of these contacts lead to further conformational changes permitting CaM to interact with 296Arg-Arg-Lys298 which provides for the highest affinity CaM-binding or CaM-trapping.

Step 3, and possibly 4, above may also be important for CaM-kinase II to exhibit maximal enzymatic activity. By interacting with CaM, the area surrounding 293Phe-Asn-Ala295 would not be available to interact with and inhibit the catalytic domain (15). This model also provides a possible explanation for why the Ca2+/CaM-independent form of the enzyme expresses only a portion of maximal activity, typically 20-80% (1). When Ca2+/CaM is removed following autophosphorylation, with for example EGTA, the area surrounding 293Phe-Asn-Ala295 would again become free to interact with the catalytic domain. Thus, the enzyme expresses sub-maximal activity in the Ca2+/CaM-independent state because the autoinhibitory domain has partially re-established itself with the catalytic domain. Analyzing these possibilities in the context of native and mutated forms of CaM-kinase II are currently underway.

    ACKNOWLEDGEMENTS

We thank Andy Hudmon, Steve Kolb, Bryan Davis, Wen Liu, and Dr. Don Blumenthal for providing reagents and thoughtful discussion during the course of these studies. We also are grateful to Katayun Irani for performing some of the dissociation rate determinations.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants NS26086 (to M. N. W.), GM44911 (to A.-L. T.), and HL45724 (to J. A. P.) and Robert A. Welch Foundation Grant AU1144 (to J. A. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 713-500-6061; Fax: 713-500-0652; E-mail: jputkey{at}utbmb.med.uth.tmc.edu.

1 The abbreviations used are: CaM, calmodulin; CaM(C75), CaM(K75C); CaM(C75)IAE, IAEDANS-labeled CaM(C75); IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)napthalene-1-sulfonic acid; CaM(C75)ACR, acrylodan-labeled CaM(C75); acrylodan, 6-acryloyl-2-dimethylaminonapthalene; BSA, bovine serum albumin; CaM-kinase II, Ca2+/calmodulin-dependent protein kinase II; MOPS, 4-morpholinepropanesulfonic acid.

2 All amino acid designations in this study refer to the alpha  isoform of rat CaM-kinase II (GenBankTM accession no. P11275).

3 All CaM-kinase II synthetic peptides used in this study are designated CKII with the corresponding primary amino acid sequence number given in parentheses.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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