Acidic/IQ Motif Regulator of Calmodulin*

The small IQ motif proteins PEP-19 (62 amino acids) and RC3 (78 amino acids) greatly accelerate the rates of Ca2+ binding to sites III and IV in the C-domain of calmodulin (CaM). We show here that PEP-19 decreases the degree of cooperativity of Ca2+ binding to sites III and IV, and we present a model showing that this could increase Ca2+ binding rate constants. Comparative sequence analysis showed that residues 28 to 58 from PEP-19 are conserved in other proteins. This region includes the IQ motif (amino acids 39-62), and an adjacent acidic cluster of amino acids (amino acids 28-40). A synthetic peptide spanning residues 28-62 faithfully mimics intact PEP-19 with respect to increasing the rates of Ca2+ association and dissociation, as well as binding preferentially to the C-domain of CaM. In contrast, a peptide encoding only the core IQ motif does not modulate Ca2+ binding, and binds to multiple sites on CaM. A peptide that includes only the acidic region does not bind to CaM. These results show that PEP-19 has a novel acidic/IQ CaM regulatory motif in which the IQ sequence provides a targeting function that allows binding of PEP-19 to CaM, whereas the acidic residues modify the nature of this interaction, and are essential for modulating Ca2+ binding to the C-domain of CaM.

Calmodulin (CaM) 2 is a 17-kDa Ca 2ϩ receptor found in all eukaryotic cells, where it regulates activities ranging from neural transmission to growth and differentiation (for a condensed review see Ref. 1). This daunting task requires that CaM interact with a large number of proteins and that it properly sense Ca 2ϩ signals that vary greatly in frequency and amplitude (for review see Refs. 2 and 3). To effectively fulfill its diverse roles, mechanisms have evolved to regulate, or fine-tune CaM activity over short time frames. For example, phosphorylation of a small protein called ARPP-21 promotes high affinity binding to Ca 2ϩ -CaM, (neurogranin or Ng) thereby competitively inhibiting activation of other CaM-dependent enzymes (4).
PEP-19 (Purkinje cell protein 4 or pcp4) and RC3 (neurogranin or Ng) are small proteins expressed primarily in neuronal tissues, but with no known activity other than binding to CaM in the presence or absence of Ca 2ϩ . We found that both PEP-19 and RC3 have profound effects on the rate-limiting kinetics of Ca 2ϩ binding to the C-domain of CaM. Specifically, PEP-19 accelerates the rates of both association and dissociation of Ca 2ϩ without greatly affecting the overall K Ca of the C-domain (5). RC3 accelerates the rate of Ca 2ϩ dissociation from CaM, but has a lesser effect on the association rate, thereby decreasing the affinity of binding Ca 2ϩ to the C-domain of CaM (6). Importantly, both PEP-19 and RC3 exert these effects even when CaM is bound to CaM-dependent protein kinase II (CKII␣) (5,6).
These results suggest that PEP-19 and RC3 could have broad extrinsic effects on CaM-related signaling pathways by modulating the Ca 2ϩ binding properties of free or enzyme-bound CaM. This is consistent with the phenotype of RC3 knock-out mice, which show defects in synaptic plasticity (7), attenuated phosphorylation of hippocampal protein kinase A and C substrates (8), and altered Ca 2ϩ dynamics in cortical neurons (9).
Both PEP-19 and RC3 contain an IQ motif. This rather loose consensus sequence (IQXXXRGXXXR) was first identified as the light chain binding site in conventional myosins, but was subsequently recognized as a CaM binding sequence in numerous other proteins (10). IQ motif proteins exhibit diverse modes of interaction with CaM that include Ca 2ϩ -dependent or independent binding (10), binding to both or only one domain of CaM (5,(11)(12)(13), binding multiple CaMs to multiple IQ motifs (14), and exchange of CaM between the IQ motif and other sites in the same protein (15,16).
These intriguing structure-function relationships of IQ motifs led us to identify amino acids in PEP-19 that are required to modulate Ca 2ϩ binding to CaM. We show here that the consensus IQ CaM binding motif is necessary, but not sufficient to mimic the effect of intact PEP-19 on CaM. An adjacent highly acidic amino acid sequence acts in synergy with the IQ motif to modulate Ca 2ϩ binding to the C-domain of CaM. We propose that this acidic/IQ sequence constitutes a new CaM regulatory motif.
from Dr. Madeline Shea (University of Iowa). Synthetic peptides purchased from Sigma Genosys had greater than 90% purity, and were further purified as necessary by C4 reverse phase HPLC using 0 -60% acetonitrile gradient in water, 0.1% trifluoroacetic acid.
NMR Methodology-NMR spectra of isotope-labeled CaM and PEP-19 were generated using Varian Inova 800 MHz and Bruker DRX800 MHz spectrometers with room temperature triple resonance probes, as well as a Bruker DRX 600 MHz spectrometer equipped with 5-mm TXI CryoProbe. Backbone assignments for Ca 2ϩ -CaM in the absence and presence of PEP-19 were reported previously (5). Titration of [ 15 (39 -62), which allowed assignments to be made by following chemical shift changes from the free to bound forms of CaM. All titrations were conducted at a CaM concentration of 50 M. To eliminate dilution and pH effects, titrations were made from a stock solution of concentrated unlabeled PEP-19 or peptides containing 50 M Ca 2ϩ -CaM in the appropriate buffer. At each titration point a measured amount of sample was removed from the NMR tube and replaced with the identical volume from the stock solution. Titrations were carried out until a 10-fold excess of ligand was added. The data were processed using the Autoscreen module in FELIX 2002 software (19). The average amide chemical shift change was calculated using the following formula: where ⌬␦H ϭ change in 1 H chemical shift and ⌬␦n ϭ change in 15 N chemical shift.
Chemical shift changes for CaM backbone amide 1 H and 15 N nuclei were analyzed separately to derive K d values for binding PEP-19 to CaM because the relative contribution of change in each dimension can vary significantly. In general, backbone 15 N chemical shifts experienced the greatest change relative to the spectral window.
Generation of Fluorescently Labeled Proteins-Labeling of CaM(K75C) or CaM(T110C) with either acylodan or IAEDANS was previously reported (18,20). To obtain the fluorescent CaM used in the FRET study, a double Cys CaM mutant, CaM(T34C,T110C), was labeled with IAEDANS (donor) and DDPM (accepter) to generate CaMD/A. CaM(T34C,T110C) was first reacted with 0.4 mol of IAEDANS/mol of protein in 20 mM Tris-HCl at pH 7.5 and 100 mM KCl for 2 h at 20°C in the dark. Free IAEDANS was removed using a Bio-Gel P-6DG (Bio-Rad) desalting column. The IAEDANS-labeled CaM averaged 0.3 mol of IAEDANS/mol of protein. A portion of this partially labeled protein was saved as the donor-alone protein (CaMD), whereas the rest was labeled with excess DDPM to give CaMD/A. Free DDPM was removed by desalting. PEP-19 and PEP(39 -62) with C-terminal Gly-Cys extensions were labeled with DDPM as described for CaM, but free DDPM was removed using a semi-prep C4 reverse phase HPLC column with a 0 -60% acetonitrile gradient. Protein and peptide concentrations were determined using the Pierce BCA protein assay with a bovine serum albumin standard and color developed at 60°C.
Equilibrium Binding of PEP-19 to CaM-Solutions of fluorescently labeled CaM derivatives were prepared in a buffer of 20 mM MOPS, pH 7.5, 100 mM KCl, and 1 mM dithiothreitol. Concentrated stock solutions of PEP-19, PEP(39 -62), or their DDPM-labeled derivatives, were prepared by dissolving lyophilized protein or peptide in the labeled CaM solution to eliminate dilution of CaM during titration. The increase in volume was less than 10%. We assessed potential nonspecific FRET effects using DDPM coupled to free Cys, and found a linear, 5% decrease in fluorescence from donor-labeled CaM per increment of 25 M Cys-DDPM. The FRET effect between donorlabeled CaM and acceptor-labeled PEP-19 or PEP(39 -62) was corrected for this nonspecific effect, and the upper concentration of DDPM-labeled ligands was limited to 50 M.
Dissociation constants (K d ) were derived from fluorescence or NMR data by fitting titration curves to the following equation, which does not require measurement of free ligand concentrations, Where S ϭ fluorescence or NMR signal at a given titration point; S i ϭ initial signal in the absence of ligand; S f ϭ final signal in the presence of excess ligand; L ϭ total ligand added at a given titration point; C t ϭ total CaM concentration; and K d ϭ dissociation constant. The equation was used to fit plots of S versus L with S i , S f , and K d as fitted variables.
Equilibrium Ca 2ϩ Binding-Macroscopic equilibrium Ca 2ϩ binding constants were determined using the competitive binding assay described by Linse et al. (21). Calcium was removed from buffers by passage over a Calcium-Sponge column (Molecular Probes). Residual Ca 2ϩ detected using Indo-1 was typically Ͻ10 Ϫ7 M. CaM was decalcified by adding 1-5 mM BAPTA followed by desalting into Ca 2ϩ -free buffers. This effectively removed greater than 95% of Ca 2ϩ from CaM as determined by Tyr fluorescence. All pipette tips, cuvettes, and other labware were rinsed with 0. were performed by addition of 2-, 3-, 5-, or 10-l aliquots of the Ca 2ϩ stock, to an initial sample volume of 0.7 ml. The decrease in absorbance of Br 2 BAPTA at 263 nm was monitored using a Cary/Varian 100 spectrophotometer. The number and volume of aliquots was adjusted to achieve an even distribution of data points on the binding isotherm. The total Ca 2ϩ concentration was then calculated based on the initial volume and total added volume at each titration point. Macroscopic calcium binding constants were calculated essentially as described by Linse et al. (21) using the following equations,  Calcium k off rates were determined using 2 M CaM, 20 M Ca 2ϩ , and 300 M Quin-2. Fluorescence from Quin-2 was detected using an excitation wavelength of 334.5 nm and Oriel emission cut-off filter 51282. A stopped-flow experiment to measure Ca 2ϩ k on rates was devised using the Ca 2ϩ -sensitive chromophore Br 2 BAPTA as a buffer to maintain free Ca 2ϩ levels in the range of 0.5 to 5 M, and as a chromophore to monitor Ca 2ϩ binding. The kinetics of binding Ca 2ϩ to Br 2 BAPTA and the N-domain of CaM are very fast and thus, the increase in absorbance observed upon mixing apo-CaM with Ca 2ϩ / Br 2 BAPTA solutions is due to the release of Ca 2ϩ from Ca 2ϩ / Br 2 BAPTA as Ca 2ϩ binds to the C-domain of CaM. The observed change in absorbance was fitted to a single exponential equation. All buffers contained 20 mM MOPS, pH 7.5, and 100 mM KCl. The assay was performed at 20°C. Typically, Buffer A contained 5 M BAPTA with or without 2 to 4 M decalcified CaM or EGTA, whereas Buffer B contained 250 M Br 2 BAPTA and sufficient Ca 2ϩ to achieve a desired free Ca 2ϩ upon mixing equal volumes of Buffers A and B. This allows less than a 3% change in the concentrations of Br 2 BAPTA and Ca 2ϩ -Br 2 BAPTA as Ca 2ϩ binds to CaM, thereby maintaining a reasonably constant level of free Ca 2ϩ .
Free Ca 2ϩ levels of Ca 2ϩ /Br 2 BAPTA solutions were calculated from the absorbance at 263 nm (Abs obs ) versus controls in the absence (Abs min ) or presence (Abs max ) of excess Ca 2ϩ using the following equation.

Ca 2ϩ
Free ϭ 1.59 M ϫ ͑Abs obs Ϫ Abs min ͒ ͑Abs max Ϫ Abs obs ͒ (Eq. 5) Free Ca 2ϩ levels in the optical chamber of the stopped-flow instrument at 20°C and pH 7.5 were determined from the observed rates of binding Ca 2ϩ to EGTA present in solution . Free Ca 2ϩ levels were in close agreement when calculated based on Br 2 BAPTA absorbance or observed rates of binding Ca 2ϩ to EGTA.

Microscopic Kinetic Model for Ca 2ϩ
Binding to the C-domain of CaM-Linked differential equations for the forward and reverse microscopic binding events illustrated in Fig. 6A were incorporated into a computational model using Berkeley Madonna software. Initial parameter values were taken from the current study and literature reports. The overall average apparent dissociation constant (K d or K Ca50 ) of 2.3 Ϯ 0.3 M was derived from Table 2 and other reports using a variety of techniques at pH 7.4 to 7.5 with 90 to 100 mM KCl (5,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). The macroscopic dissociation constants, K d1 and K d2 , correspond to sequential binding of the first and second Ca 2ϩ ions, regardless of which site is filled first. Starting values for K d1 and K d2 given in Table 2 are very close to those reported with others (21,22). The microscopic dissociation constants K dIII and K dIV correspond to binding the first Ca 2ϩ to site III or IV, respectively, whereas K dIII/IV and K dIV/III correspond to binding the second Ca 2ϩ when the other site is already occupied. The model includes algebraic relationships that relate macroscopic and microscopic association constants (K a ; K a ϭ 1/K d ) as follows, where c is the coupling factor (31).
These relationships allow for the calculation of microscopic equilibrium binding constants if K 1 , K 2 , and the magnitude of difference between microscopic binding constants is known. K 1 and K 2 are reported in Table 2, and Evenas et al. (32) who used Ca 2ϩ binding mutants to show that the relative Ca 2ϩ binding affinity of site IV is ϳ6.3-fold greater than site III in both the 0-Ca 2ϩ and 1-Ca 2ϩ states of the C-domain.
Microscopic rate constants were constrained by the results of Malmendal et al. (32) who used NMR relaxation methods to conclude that the first Ca 2ϩ ion binds preferentially to site IV, and that the k off of site IV (k offIV ) was 5100 s Ϫ1 . If the 6.3-fold difference in affinity of binding Ca 2ϩ to sites IV and III were due exclusively to k off rates, then k offIII would be ϭ 32,000 s Ϫ1 , which is consistent with an exchange rate of 27,000 s Ϫ1 determined for transition between the open and closed conformation of the C-domain in which site IV was mutated (33). These k off values would correspond to k onIV and k onIII rates of around 300 M Ϫ1 s Ϫ1 , which is consistent with a diffusion-limited event, and similar to the k on for Ca 2ϩ binding to the N-domain of CaM. Microscopic rate constants for binding the second Ca 2ϩ ion were constrained by the fact that the observed k off (k off,Obs ) for dissociation of both Ca 2ϩ ions from the C-domain measured using stopped-flow experiments described as above and best fits a single exponential rate between 8.5 s Ϫ1 and 12.6 s Ϫ1 (5,25,34,35). Because NMR relaxation data show that the rate of dissociation of Ca 2ϩ from the 1-Ca 2ϩ state is very fast, then k off,Obs reflects the rate-limiting dissociation of the first Ca 2ϩ from either site III or IV of the 2-Ca 2ϩ state, followed by very rapid release of the second Ca 2ϩ . This means that k off,Obs ϭ k off,IV/III ϩ k off,III/IV , and it allows constraint of microscopic rate constants using the following relationships.
Thus, k dIII/IV and k dIV/III are calculated if k off,Obs is defined within an experimentally observed range, and k off,IV/III is varied between 0 and the defined k off,Obs . The model includes a Ca 2ϩ buffer based on Br 2 BAPTA with K d ϭ 1.59 M, a diffusion limited k on ϭ 500 M Ϫ1 s Ϫ1 and k off ϭ 795 s Ϫ1 . This allowed simulation of stopped-flow experiments described above to measure the rate of association of Ca 2ϩ with the C-domain of CaM, and to use the built-in curve fit function of Berkeley Madonna to optimize parameter sets against experimental data.
Global parameter optimization and error analysis were also performed in the MATLAB computing environment (The MathWorks). Although the computational model ( Fig. 1) has 8 kinetic parameter values, the experimental data and algebraic constraints described above and under "Results " reduced the unknown parameters to k on,III and k off,IV/III . However, to take into account the different reported values of k off,Obs , we also treated k off,III/IV as an additional unknown parameter for the global optimization. We set the maximum values of k on,III , k off,IV/III , and k off,III/IV (500 M Ϫ1 s Ϫ1 , 50 s Ϫ1 , and 50 s Ϫ1 , respectively) and divided the entire parameter space into 200 ϫ 500 ϫ 500 ϭ 5 ϫ 10 7 grid points. The error (root mean square difference) was calculated for each of these 5 ϫ 10 7 grid points by comparing the simulated Ca 2ϩ association and dissociation rates with the experimental data. This systematic parameter optimization revealed a single distinct region of the parameter space in which the computational model best fit the data. The estimate of k on,III resulted in a unique set of parameter values of the model, which was again reconfirmed by the lsqnonlin function of the MATLAB Optimization Toolbox.

Identifying Sequences in PEP-19 That Bind to Ca 2ϩ -CaM-A
BLAST protein similarity search identified PEP-19 orthologs with high degrees of identity throughout their primary sequences. Other proteins of diverse size and from diverse species had sequence similarity to the C-terminal portion of PEP-19 that includes the IQ motif and cluster of adjacent acidic residues (see Fig. 1A). RC3 also has an acidic cluster but with a sequence that differs from PEP-19 (see "Discussion " for more details). The corresponding sequences from myosin V and the voltage-gated Ca 2ϩ channel Ca v 1.2 are shown in Fig. 1A to emphasize the absence of acidic clusters in these IQ motif proteins.
The sequence comparisons in Fig. 1A led us to hypothesize that both the IQ motif and the adjacent acidic region in PEP-19 are necessary to modulate the Ca 2ϩ binding of CaM. The peptides shown in Fig. 1B were synthesized to directly test this hypothesis. PEP(28 -62) spans the acidic and IQ regions. PEP(39 -62) includes the core IQ motif (10), and is the minimal region in PEP-19 shown to have CaM antagonist activity (36). PEP (28 -45) Fig. 2A shows that the greatest effects of PEP-19 are localized to the C-domain of Ca 2ϩ -CaM, primarily in helix F, the linker between helices F and G, and in helix H. These data indicate a single major binding site for PEP-19 in the C-domain of Ca 2ϩ -CaM.   (20), was used as a positive control because it causes CaM to adopt a compact shape with the N-and C-domains in close proximity (38). Fig. 3 shows that binding CKII-(293-312) to Ca 2ϩ -CaMD/A causes a large decrease in fluorescence due to a FRET effect. PEP-19 and PEP(28 -62) cause much smaller decreases in fluorescence, indicating they do not induce CaM to adopt a highly compact shape. CaM remains extended when bound to PEP(39 -62) because fluorescence from CaMD/A is essentially unaffected by the peptide. Fig. 4A plots Ca 2ϩ -CaM amide chemical shifts as a function of increasing concentrations of PEP-19 or PEP(28 -62). Both data sets fit well to a single-site binding model with K d values of 29 and 24 M for PEP-19 and PEP(28 -62), respectively (see Table  1). Because PEP(39 -62) affects amides in the N-and C-domains of Ca 2ϩ -CaM, the average responses for selected residues in these domains were analyzed separately as shown in Fig.  4B. Chemical shift changes reached a maximum, but the chemical shift response curve for residues in the N-domain was right shifted relative to residues in the C-domain, suggesting that Nand C-domains sense different binding events. The lines in Fig.  4B show a fit to a single-site binding model, but the fits were poor relative to those shown in Fig. 4A. This was not surprising given the potential complexity of chemical shift changes in response to multiple ligands, and the high concentration of  Table 1). Interestingly, even though Figs. 2C and 3 indicate multiple binding sites for PEP(39 -62) on Ca 2ϩ -CaM, the response of Ca 2ϩ -CaM ACR to this peptide fit a single-site model with an apparent K d of 0.24 M. Thus, the affinity of binding PEP (39 -62) to one site in Ca 2ϩ -CaM is about 70-fold greater relative to binding intact PEP-19. Fig. 4D shows results of a FRET assay using derivatives of PEP-19 and PEP(39 -62) with C-terminal Cys residues labeled with the FRET acceptor DDPM, and CaM(T110C) labeled with the FRET donor IAEDANS. A large decrease in fluorescence of up to 70% due to FRET quenching was observed upon binding acceptor-labeled peptides to donor-labeled Ca 2ϩ -CaM. Changes in fluorescence upon binding acceptor-labeled PEP-19 to donor-labeled Ca 2ϩ -CaM fit a single-site model with a K d of 20 M, which is comparable with the values derived from both NMR and CaM ACR (see Table 1). Interestingly, changes in fluorescence upon binding PEP(39 -62) indicate at least two classes of binding sites. A K d of 0.16 M for the higher affinity site is comparable with that derived using CaM ACR , whereas the K d of 27 M for the low affinity site is consistent with the K d for binding intact PEP-19 to Ca 2ϩ -CaM (see Table 1).

Relative Affinities of Binding PEP-19 Polypeptides to CaM-
Together, the data in Figs. 2-4 show that PEP-19 and PEP(28 -62) bind predominately to a single site in the C-domain of CaM. In contrast, there are at least two binding sites for PEP(39 -62) on CaM, located in the N-and C-domains.
Comparative Effects of PEP-19 Peptides on Equilibrium Ca 2ϩ Binding- Fig. 5A and Table 2 compare the macroscopic Ca 2ϩ dissociation constants, K d1 through K d4 , for CaM in the absence or presence of PEP-19 derivatives. Linse et al. (21) assigned K d1 /K d2 and K d3 /K d4 to Ca 2ϩ binding sites in the C-and N-domains of free CaM, respectively. We have adopted these assignments because the binding constants in Table 2 for free CaM are in agreement with those reported by other groups using the same technique under similar conditions (21,22). Similar to previous reports using a variety of techniques (21,(27)(28)(29), Table 2 demonstrates strong positive cooperativity of Ca 2ϩ binding to the C-domain of CaM because K d2 Ͻ K d1 /4, with a lower limit for the free energy of cooperativity (⌬⌬G c ) of Ϫ3.4 kcal/mol. Table 2 shows that intact PEP-19 and PEP(28 -62) have little effect on K d3 or K d4 , but have significant effects on K d1 and K d2 . Because both PEP-19 and PEP(28 -62) have relatively small effects on amide chemical shifts in the N-domain (see Fig. 2), we have assigned K d3 and K d4 as macroscopic  binding constants for the N-domain, and K d1 to K d2 to the C-domain of CaM bound to either PEP-19 or PEP(28 -62). Both PEP-19 and PEP(28 -62) cause a significant decrease in the cooperativity of Ca 2ϩ binding to the C-domain of CaM from a ⌬⌬G c of Ϫ3.4 to Ϫ1.3 kcal/mol. PEP(39 -62) has little effect on K d2 , K d3 , or K d4 , but increases the affinity of binding the first Ca 2ϩ (K d1 ) from 17 M with free CaM to 1.4 M in the presence of the peptide. This results in a lower degree of cooperativity of Ca 2ϩ binding to the putative C-domain of CaM, but a 3-fold increase in overall affinity.

Comparative Effects of PEP-19 Peptides on Ca 2ϩ Binding Kinetics-
The rate of dissociation of Ca 2ϩ from the N-domain of CaM is very fast and occurs within the deadtime of the stopped-flow fluorimeter at room temperature (1.7 ms). Thus, only the slower release of two Ca 2ϩ ions from the C-domain of CaM can be readily detected. Fig. 5B and Table 3 show that both intact PEP-19 and PEP(28 -62) greatly increase the rate of dissociation of Ca 2ϩ from CaM. PEP-19 has a similar effect on a recombinant CaM C-terminal fragment, CaM-(76 -148), which further supports its domain-specific effect, and assignment of K d1 and K d2 in Table 2 to the C-domain of CaM. In contrast, the shorter PEP(39 -62) has the opposite effect of decreasing the observed rate of Ca 2ϩ dissociation by about 3-fold. The magnitude of this slow phase is consistent with the release of 2 Ca 2ϩ ions from the C-domain, and the 3-fold decrease in rate would account for the higher affinity of Ca 2ϩ binding to the C-domain in the presence of PEP(36 -62) shown in Table 2.
"Experimental Procedures" describes an assay to measure Ca 2ϩ k on rates at free Ca 2ϩ levels maintained between 0.5 and 5 M using Br 2 BAPTA as both a Ca 2ϩ buffer and a chromophore to monitor Ca 2ϩ binding to CaM. EGTA was used in control experiments, because its high affinity for Ca 2ϩ at pH 7.5 (K d ϭ 0.038 M) ensures saturation at all levels of free Ca 2ϩ used in the assay, but the Ca 2ϩ association rate for EGTA is easily measured using stopped-flow techniques. Fig. 5C shows the expected increase in rate of association of Ca 2ϩ with EGTA at increasing free Ca 2ϩ levels. best fit an exponential decay with two rate constants. For these data points, a weighted average rate was determined from (R 1 ϫ A 1 ϩ R 2 ϫ A 2 )/(A 1 ϩ A 2 ). Panel C shows the rate of change in absorbance when a solution of EGTA (2 M) was rapidly mixed with Br 2 BAPTA solutions of increasing free Ca 2ϩ levels as described under "Experimental Procedures." All data sets best fit a single exponential equation. The observed rate was used to calculate the free Ca 2ϩ levels in the optical chamber (see   5D shows the rate of Ca 2ϩ binding to CaM at free Ca 2ϩ levels ranging from about 0.5 to 5 M. The data best fit a single exponential rate at all Ca 2ϩ levels. The increased magnitude of change at higher free Ca 2ϩ levels is consistent with an increased percent saturation of the C-domain with Ca 2ϩ , which has an overall K d of 2.3 M. We observed no effect of either PEP (39 -62) or PEP(29 -45) on the rate of Ca 2ϩ binding to CaM, however, Fig. 5E shows that both intact PEP-19 and PEP (28 -62) greatly increased the observed Ca 2ϩ k on .

DISCUSSION
The primary goal of the current study was to define residues in PEP-19 that modulate Ca 2ϩ binding to CaM. During the course of these experiments we also showed that PEP-19 attenuates the degree of positive cooperativity of Ca 2ϩ binding to sites III and IV. Positive cooperativity simply means that binding the first Ca 2ϩ ion increases the affinity of binding the second Ca 2ϩ . With respect to the macroscopic binding constants K d1 and K d2 shown in Fig. 6A, positive cooperativity is implied if K d2 Ͻ K d1 /4. Because K d ϭ k off /k on , this criteria can be satisfied by a wide range of rate constants. It is therefore not immediately apparent from macroscopic Ca 2ϩ binding constants how changes in cooperativity could account for the large effects of PEP-19 on the rates of Ca 2ϩ binding.
Little is known about the microscopic equilibrium Ca 2ϩ binding and rate constants for CaM, but it is these parameters that would provide the greatest insight into the mechanism of action of PEP-19. Thus, we developed a kinetic model for cooperative Ca 2ϩ binding to CaM that is based on experimental data and algebraic expressions that relate microscopic and macro-scopic binding and rate constants (see "Experimental Procedures" for details). The model was used to derive and optimize the microscopic rate constants shown in Fig. 6A for transition of the C-domain of CaM from the 0-Ca 2ϩ to 2-Ca 2ϩ states. The models were tested by comparing experimental data with a simulation using rate constants derived from the model to predict the pseudo first-order rate of association of Ca 2ϩ with the C-domain of CaM. Fig. 6B shows that the simulation closely approximates the experimental data, with both data sets showing a non-linear relationship between free Ca 2ϩ and the rate of Ca 2ϩ binding.
A key feature of the kinetic model described in Fig. 6 is that binding the first Ca 2ϩ ion to either site III or IV is characterized by fast rate constants, whereas binding the second Ca 2ϩ occurs with much slower rates. In essence, binding the first Ca 2ϩ to either site III or IV increases the affinity of binding the second Ca 2ϩ , which defines positive cooperativity, but it also drastically slows the rates of this second binding event. This immediately implies that the observed attenuation of cooperativity by PEP-19 could accelerate Ca 2ϩ rate constants by allowing greater expression of rapid rates associated with independent binding of Ca 2ϩ to sites III and IV.
Our results demonstrate that the core IQ sequence (amino acids 39 -62) is necessary to promote binding of PEP-19 to CaM, but that it does not mimic other properties of PEP-19. The core IQ motif binds to at least two sites on CaM. One site has a K d similar to that of binding intact PEP-19, whereas another site binds the IQ motif with higher affinity. This must be considered when evaluating data that utilize IQ peptides taken out of context of the intact protein. For example, our results are consistent with a previous report showing that a synthetic peptide called camstatin, which spans the IQ motif in PEP-19 (residues 36 -60), was a more effective inhibitor of nNOS than intact PEP-19 (36). It is likely that the properties of camstatin are similar to those of PEP (39 -62). Although intact PEP-19 binds to Ca 2ϩ -CaM with relatively low affinity (K d of 20 to 30 M), it is present at high concentrations in brain (39), and the inset to Fig. 5 shows that PEP-19 at a concentration of 5 M significantly increases the rate of dissociation of Ca 2ϩ from CaM.
Our data show that coupling the acidic-rich amino acids 28 -40 to the core IQ motif is necessary to mimic intact PEP-19 with respect to preferential binding to the C-domain of CaM,

), and PEP(39 -62) on calcium binding affinity and cooperativity
The macroscopic dissociation constants were derived as described in Material and Methods. All values are the average mean Ϯ S.E. of (n) independent titrations. The square root of the product of macroscopic dissociation constants for each domain is equivalent to the K Ca50 for binding to each domain. The precision of this value is greater than the individual dissociation constants. The overall decrease in free energy from binding two Ca 2ϩ ions to the N-or C-domains (⌬G tot ) was calculated as ⌬G tot ϭ ϪRT ln(K 1 ϫ K 2 ). The upper limit for the change in free energy due to cooperative Ca 2ϩ binding (⌬⌬G c ) was calculated as ⌬⌬G c ϭ ϪRT ln(4 ϫ K 2 /K 1 ). Free energy values are in kcal/mole.  Fig. 1) implies a functional significance that extends beyond PEP-19. Of particular interest are large proteins that may have intrinsic activity, such as the sea urchin protein in Fig. 1A that also encodes fibronectin type II and multiple PLAT/LH2 domains. The acidic/IQ motif may mediate direct CaM-dependent regulation of larger proteins, with modulation of Ca 2ϩ binding to CaM as an integral feature of this regulation. A critical role for acidic residues in modulating Ca 2ϩ binding to CaM implies mechanisms involving interactions between Ca 2ϩ and PEP-19. The acidic region may function as a negatively charged antenna that electrostatically "steers" Ca 2ϩ ions to and from sites III and IV. A more specific interaction with Ca 2ϩ is suggested by the primary sequence in Fig. 1A. With the exception of Pro-37, residues Glu-29 to Glu-40 in PEP-19 conform well to the consensus sequence of an EF-hand Ca 2ϩ binding loop, with oxygen-containing side chains at coordination positions X, Y, Z, ϪY, and ϪZ. Interestingly, the acidic region of RC3 does not have a similar distribution of acidic residues. The role of this putative Ca 2ϩ binding loop is currently being studied.
Synergy between the core IQ sequence and adjacent residues to achieve unique functionality is a paradigm that may apply to other IQ motif proteins. For example, residues N-terminal to the IQ motif of Ca V 1.2 are not highly acidic. Instead, this sequence includes a Phe residue (see open arrow in Fig. 1A) that anchors Ca V 1.2 to the N-domain of CaM (see open arrow in Fig.  1A) (11,12). This region of Ca V 1.2 may play an important regulatory role because channel facilitation and inactivation is thought to be mediated by dynamic differential binding of the N-and C-domains of CaM to the IQ region (15). A corresponding Phe residue is not present in PEP-19. Thus, different modules extending N-terminal to the IQ motifs of PEP-19 versus Ca V 1.2 appear to confer unique functionalities to these CaM binding proteins.
In summary, this study reports a comprehensive new kinetic model that can account for cooperative Ca 2ϩ binding to the C-domain of CaM and provides a mechanistic model for the effects of PEP-19 at the level of attenuating cooperativity. We also show that the effects of PEP-19 on CaM rely on the synergy between the core IQ motif that targets PEP-19 to CaM, and an adjacent acidic cluster that modulates Ca 2ϩ binding. We propose that this acidic/IQ motif is a regulator of CaM signaling found in diverse proteins and species.