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• W2030890665 abstract "The mechanism of dissociation reactions induced by calcium chelators has been studied for complexes of Drosophila calmodulin with target peptides, including four derived from the skeletal muscle myosin light chain kinase target sequence. Reactions were monitored by fluorescence stopped-flow techniques using a variety of intrinsic probes and the indicator Quin2. For most of the complexes, apparently biphasic kinetics were observed in several fluorescence parameters. The absence of any obvious relationship between dissociation rates and peptide affinities implies kinetic control of the dissociation pathway. A general mechanism for calcium and peptide dissociation was formulated and used in numerical simulation of the experimental data.Unexpectedly, the rate of the slowest step decreases with increasing [peptide]/[calmodulin] ratio. Numerical simulation shows this step could contain a substantial contribution from a reversible relaxation process (involving the species Ca2-calmodulin-peptide), convolved with the following step (loss of C-terminal calcium ions). The results indicate the potentially key kinetic role of the partially calcium-saturated intermediate species. They show that subtle changes in the peptide sequence can have significant effects on both the dissociation rates and also the dissociation pathway. Both effects could contribute to the variety of regulatory behavior shown by calmodulin with different target enzymes. The mechanism of dissociation reactions induced by calcium chelators has been studied for complexes of Drosophila calmodulin with target peptides, including four derived from the skeletal muscle myosin light chain kinase target sequence. Reactions were monitored by fluorescence stopped-flow techniques using a variety of intrinsic probes and the indicator Quin2. For most of the complexes, apparently biphasic kinetics were observed in several fluorescence parameters. The absence of any obvious relationship between dissociation rates and peptide affinities implies kinetic control of the dissociation pathway. A general mechanism for calcium and peptide dissociation was formulated and used in numerical simulation of the experimental data. Unexpectedly, the rate of the slowest step decreases with increasing [peptide]/[calmodulin] ratio. Numerical simulation shows this step could contain a substantial contribution from a reversible relaxation process (involving the species Ca2-calmodulin-peptide), convolved with the following step (loss of C-terminal calcium ions). The results indicate the potentially key kinetic role of the partially calcium-saturated intermediate species. They show that subtle changes in the peptide sequence can have significant effects on both the dissociation rates and also the dissociation pathway. Both effects could contribute to the variety of regulatory behavior shown by calmodulin with different target enzymes. INTRODUCTIONCalmodulin is involved in the regulation of a range of cellular functions, usually through its Ca2+-dependent activation of target proteins (1Klee C.B. Cohen P. Klee C.B. Calmodulin. Elsevier Science Publishers B.V., Amsterdam1988: 33-56Google Scholar). Ca4-CaM 1The abbreviations used are: CaMwild-type Drosophila calmodulinT26Wa SYNCAM calmodulin mutant with a T26W mutationsm- and sk-MLCKsmooth and skeletal muscle myosin light chain kinaseMasmastoparanMasXmastoparan XQuin28-amino-2-[(2-amino-5-methylphenoxy)-methyl]-6-methoxyquinoline-N,N,N′,N′-tetraacetic acid). binds to many target proteins with high affinity (Kd≈ nM) and binds peptides derived from the calmodulin binding regions of these proteins with similar affinities.The x-ray crystal structure of Ca4-CaM (2Babu Y.S. Sack J.S. Greenhough T.J. Bugg C.E. Means A.R. Cook W.J. Nature. 1985; 315: 37-40Crossref PubMed Scopus (796) Google Scholar, 2Babu Y.S. Sack J.S. Greenhough T.J. Bugg C.E. Means A.R. Cook W.J. Nature. 1985; 315: 37-40Crossref PubMed Scopus (796) Google Scholar, 3Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (967) Google Scholar, 4Chattopadhyaya R. Meador W.E. Means A.R. Quiocho F.A. J. Mol. Biol. 1992; 228: 1177-1192Crossref PubMed Scopus (614) Google Scholar) shows two globular domains with similar conformation, each containing two helix-loop-helix Ca2+ binding sites. Those in the C-domain have a higher affinity than those in the N-domain, and there is positive cooperativity between two sites within a domain (5Linse S. Helmersson A. Forsén S. J. Biol. Chem. 1991; 266: 8050-8054Abstract Full Text PDF PubMed Google Scholar). The crystal structure shows the two domains separated by an extended α-helix. In solution, this central helix contains a loop (residues 74-82) which allows the calmodulin domains to interact closely with the peptide (6Barbato G. Ikura M. Kay L.E. Pastor R.W. Bax A. Biochemistry. 1992; 31: 5269-5278Crossref PubMed Scopus (887) Google Scholar).Calcium binding to calmodulin induces a conformational change that exposes hydrophobic surfaces which comprise the binding site for target molecules. The solution structure of the complex of Ca4-CaM with M13, a 26-residue peptide derived from sk-MLCK, has been determined by NMR (7Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar). The M13 peptide is in an α-helical conformation, effectively enclosed by the N- and C-domains of the calmodulin. The N and C termini of the peptide interact primarily with the C- and N-domains of calmodulin, respectively, and the Trp-4 and Phe-17 residues of the peptide appear to play an important anchoring role. The structures of the complexes of Ca4-CaM with peptides derived from sm-MLCK and CaM kinase II have been determined by x-ray diffraction (8Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (939) Google Scholar, 9Meador W.E. Means A.R. Quiocho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Scopus (611) Google Scholar). The structures of the three complexes are rather similar, although there are significant differences in the positions of the peptides and the relative orientations of the calmodulin domains. In particular, the complex of Ca4-CaM with the CaM kinase II peptide has fewer peptide residues involved in helix formation and contact with the calmodulin, and the longer loop in the central helix region (residues 73-83) allows the domains to move closer together (9Meador W.E. Means A.R. Quiocho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Scopus (611) Google Scholar). The different structures show how calmodulin can adapt to bind peptides of different sequences, while maintaining high affinity in the interaction.In recent work we studied the peptide WFF, which corresponds to residues 1-18 of M13 and contains the major sites of interaction with CaM (7Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar). We have also permuted the sequence of WFF to include either Trp or Phe residues at positions 4, 8, and 17 (peptides FWF, FFW, and FFF: Table I). These peptides bind with high affinity to Ca4-CaM and retain the standard orientation with residues 4 and 17 interacting with CaM C- and N-domains, respectively (10Findlay W.A. Martin S.R. Beckingham K. Bayley P.M. Biochemistry. 1995; 34: 2087-2094Crossref PubMed Scopus (49) Google Scholar, 11Findlay W.A. Gradwell M.J. Bayley P.M. Protein Sci. 1995; 4: 2375-2382Crossref PubMed Scopus (18) Google Scholar). They are therefore well suited for investigating the effect of controlled structural modifications on the kinetics and equilibria of CaM-target peptide interactions (12Bayley P.M. Findlay W.A. Martin S.R. Biophys. J. 1996; 70Google Scholar, 13Bayley P.M. Findlay W.A. Martin S.R. Protein Sci. 1996; 7: 1215-1228Crossref Scopus (142) Google Scholar).Table IPeptide sequencesWFFKRRWKKNFIAVSAANRFKFWFKRRFKKNWIAVSAANRFFFWKRRFKKNFIAVSAANRWKFFFKRRFKKNFIAVSAANRFKMasXINWKGIAAMAKKLLMasINLKALAALAKKILCBP1LKLKKLLKLLKKLLKLG Open table in a new tab In the present work we have measured the dissociation kinetics of seven Ca4-CaM·peptide complexes (14Brown S.E. Bayley P.M. Martin S.R. Biophys. J. 1995; 68Abstract Full Text PDF Scopus (17) Google Scholar). The peptides studied are the four related to M13, the wasp venom peptides mastoparan and mastoparan X, and a synthetic peptide designed to form a basic amphiphilic α-helix (15DeGrado W.F. Adv. Protein Chem. 1988; 39: 51-124Crossref PubMed Scopus (323) Google Scholar). The effects of Ca2+ removal with a chelator were monitored using different fluorescent signals, including those of the chelator Quin2, peptide Trp, CaM Tyr (Tyr-138 in Drosophila CaM), and a Trp-containing calmodulin mutant, T26W (16Chabbert M. Lukas T.J. Watterson D.M. Axelsen P.H. Prendergast F.G. Biochemistry. 1991; 30: 7615-7630Crossref PubMed Scopus (31) Google Scholar, 17Kilhoffer M.-C. Kubina M. Travers F. Haiech J. Biochemistry. 1992; 31: 8098-8106Crossref PubMed Scopus (72) Google Scholar). Correlation of the kinetics observed with these different probes allows the deduction of a general mechanism for the dissociation of Ca4-CaM·peptide complexes. The results obtained are compared with other studies on the dissociation of CaM·peptide and CaM·protein complexes (18Johnson J.D. Holroyde M.J. Crouch T.H. Solaro R.J. Potter J.D. J. Biol. Chem. 1981; 256: 12194-12198Abstract Full Text PDF PubMed Google Scholar, 19Johnson J.D. Snyder C. J. Biol. Chem. Abstracts of the 11th IUPAB Congress. 1993; 28: 101Google Scholar, 20Kasturi R. Vasulka C. Johnson J.D. J. Biol. Chem. 1993; 268: 7958-7964Abstract Full Text PDF PubMed Google Scholar, 21Winder S.J. Walsh M.P. Vasulka C. Johnson J.D. Biochemistry. 1993; 32: 13327-13333Crossref PubMed Scopus (47) Google Scholar, 22Török K. Trentham D.R. Biochemistry. 1994; 33: 12807-12820Crossref PubMed Scopus (95) Google Scholar, 23Persechini A. White H.D. Gansz K.J. J. Biol. Chem. 1996; 271: 62-67Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 24Johnson J.D. Snyder C. Walsh M. Flynn M. J. Biol. Chem. 1996; 271: 761-767Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). We show that the predominant kinetic pathway is sensitive to changes in individual residues of the target peptides and deduce that the rate of the slowest step is determined by the contribution of a hitherto unsuspected kinetic relaxation mechanism involving the intermediate species Ca2-CaM·peptide, with two Ca2+ ions bound in the C-domain.RESULTSAssociation of Ca4CaM and PeptideFluorescence changes on mixing of WFF, FFW, or FWF (0.4 μM) with excess Ca4-CaM (at 20°C) were almost complete within the instrument deadtime, indicating a bimolecular association rate constant (kon) in excess of 8 × 108M−1 s−1. We therefore studied the association of FWF (0.4 μM) with Ca4-CaM (0.4 μM) at 9.5°C (Fig. 1D). The kon value obtained as described under “Materials and Methods” was 1 ± 0.5 × 109M−1 s−1. This is similar to a value of 9 × 108M−1 s−1 found for the reaction of a fluorescently labeled CaM with two peptides derived from sm-MLCK (22Török K. Trentham D.R. Biochemistry. 1994; 33: 12807-12820Crossref PubMed Scopus (95) Google Scholar) and to a value of 2 × 109M−1 s−1 deduced from NMR studies on a peptide derived from Bordetella pertussis adenylate cyclase (30Craescu C.T. Bouhss A. Mispelter J. Diesis E. Popescu A. Chiriac M. Bârzu O. J. Biol. Chem. 1995; 270: 7088-7096Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). These on-rates are close to the expected diffusion limit of 2 × 109M−1 s−1 (31Eigen M. Hammes G.G. Methods Enzymol. 1963; 25: 1-38Google Scholar, 32Rosenberry T.L. Neumann E. Biochemistry. 1977; 16: 3870-3878Crossref PubMed Scopus (40) Google Scholar). The high kon has important consequences for subsequent mechanistic arguments (see “The Kinetic Model”). Although slow processes attributed to conformational changes have been observed in studies of the association of fluorescently labeled CaMs with peptides and proteins (22Török K. Trentham D.R. Biochemistry. 1994; 33: 12807-12820Crossref PubMed Scopus (95) Google Scholar, 33Bowman B.F. Peterson J.A. Stull J.T. J. Biol. Chem. 1992; 267: 5346-5354Abstract Full Text PDF PubMed Google Scholar), no slow processes were observed in the association reactions studied here. Values of kon for the interaction of CaM with proteins tend to be lower (20Kasturi R. Vasulka C. Johnson J.D. J. Biol. Chem. 1993; 268: 7958-7964Abstract Full Text PDF PubMed Google Scholar, 21Winder S.J. Walsh M.P. Vasulka C. Johnson J.D. Biochemistry. 1993; 32: 13327-13333Crossref PubMed Scopus (47) Google Scholar), but a value of 5.3 × 108M−1·s−1 was measured for caldesmon (20Kasturi R. Vasulka C. Johnson J.D. J. Biol. Chem. 1993; 268: 7958-7964Abstract Full Text PDF PubMed Google Scholar).Calcium Dissociation from CalmodulinRates for the dissociation of the C-terminal Ca2+ ions from Ca4-CaM (Table II) agree well with previous values (34Martin S.R. Andersson Teleman A. Bayley P.M. Drakenberg T. Forsén S. Eur. J. Biochem. 1985; 151: 543-550Crossref PubMed Scopus (117) Google Scholar, 35Martin S.R. Maune J.F. Beckingham K. Bayley P.M. Eur. J. Biochem. 1992; 205: 1107-1114Crossref PubMed Scopus (64) Google Scholar, 36Suko J. Pidlich J. Bertel O. Eur. J. Biochem. 1985; 153: 451-457Crossref PubMed Scopus (27) Google Scholar). The rates observed for T26W SYNCAM are two to three times faster than those for wild-type Drosophila CaM (Table II) and are somewhat faster than the value of 14 s−1 reported for SYNCAM (37Haiech J. Kilhoffer M.C. Craig T.A. Lukas T.J. Wilson E. Guerra-Santos L. Watterson D.M. Adv. Exp. Med. Biol. 1990; 269: 43-56Crossref PubMed Google Scholar). We note, however, that the slow phase rate observed with T26W only corresponds to some 5% of the total amplitude; the major signal change reflects dissociation of the N-terminal Ca2+ ions. These small differences may be due to sequence differences between the proteins (38Roberts D.M. Crea R. Malecha M. Alvarado-Urbina G. Chiarello R.H. Watterson D.M. Biochemistry. 1985; 24: 5090-5098Crossref PubMed Scopus (112) Google Scholar).Table II.Observed rate constants (s−1) for the Quin2 and EGTA-induced dissociation of Ca2+ from Ca4-CaM and Ca4-CaM·peptide complexesSystemKdQuin2EGTA (Trp)EGTA (Tyr)EGTA (T26W Trp)nMCaM700 ± 200aThe relative amplitudes of the phases could not be determined for the Quin2 dissociations of CaM and T26W as most of the signal for the fast phase was lost in the deadtime of the instrument.NAbNot applicable.7.3 ± 0.58.5 ± 0.5T26W≥800NA bNot applicable.NA≥800 (≈95)21 ± 229 ± 5 (≈5)CaM·WFF0.12cS. R. Martin, W. A. Findlay, and P. M. Bayley, manuscript in preparation.10 ± 1 (30)12 ± 3 (10)NA6 ± 1 (85)1.4 ± 0.1 (70)1.5 ± 0.2 (90)0.7 ± 0.1 (15)CaM·FWF6.5cS. R. Martin, W. A. Findlay, and P. M. Bayley, manuscript in preparation.31 ± 4 (45)32 ± 4 (40)NA39 ± 5 (80)3.3 ± 0.4 (55)2.8 ± 0.4 (60)4.2 ± 0.5 (20)CaM·FFW1.6dAt 30°C, 25 mM Tris, 100 mM KCl, 1 mM CaCl2, pH 7.5 (10).2.4 ± 0.43.5 ± 0.2NA5.3 ± 0.5CaM·FFF1.0cS. R. Martin, W. A. Findlay, and P. M. Bayley, manuscript in preparation.1.0 ± 0.1fThe observed dissociations for the peptide FFF have a lag phase (Fig. 1B). The EGTA dissociation rates were determined using the equation for appearance of C in the reaction, A → B → C. The Quin2 signal was fitted to a single exponential (see “Materials and Methods”)..NA bNot applicable.1.4 ± 0.5fThe observed dissociations for the peptide FFF have a lag phase (Fig. 1B). The EGTA dissociation rates were determined using the equation for appearance of C in the reaction, A → B → C. The Quin2 signal was fitted to a single exponential (see “Materials and Methods”)..5.2 ± 0.5CaM·MasX0.9cS. R. Martin, W. A. Findlay, and P. M. Bayley, manuscript in preparation.65 ± 5 (70)91 ± 5gRelative amplitudes are not given for this system because the fast and slow phases had amplitudes with opposite sign.NA350 ± 501.3 ± 0.2 (30)0.65 ± 0.052.5 ± 0.4CaM·Mas0.3eAt 25°C in 45 mM MOPS, 200 mM KCl, 1 mM CaCl2, pH 7.3 (29).14 ± 1 (50)NA bNot applicable.1.4 ± 0.2140 ± 201.6 ± 0.2 (50)CaM·CBP1≈0.005cS. R. Martin, W. A. Findlay, and P. M. Bayley, manuscript in preparation.8 ± 1.5 (55)NA bNot applicable.1.1 ± 0.518 ± 40.7 ± 0.2 (45)a The relative amplitudes of the phases could not be determined for the Quin2 dissociations of CaM and T26W as most of the signal for the fast phase was lost in the deadtime of the instrument.b Not applicable.c S. R. Martin, W. A. Findlay, and P. M. Bayley, manuscript in preparation.d At 30°C, 25 mM Tris, 100 mM KCl, 1 mM CaCl2, pH 7.5 (10Findlay W.A. Martin S.R. Beckingham K. Bayley P.M. Biochemistry. 1995; 34: 2087-2094Crossref PubMed Scopus (49) Google Scholar).e At 25°C in 45 mM MOPS, 200 mM KCl, 1 mM CaCl2, pH 7.3 (29Malencik D.A. Anderson S.R. Biochem. Biophys. Res. Commun. 1986; 135: 1050-1057Crossref PubMed Scopus (11) Google Scholar).f The observed dissociations for the peptide FFF have a lag phase (Fig. 1B). The EGTA dissociation rates were determined using the equation for appearance of C in the reaction, A → B → C. The Quin2 signal was fitted to a single exponential (see “Materials and Methods”)..g Relative amplitudes are not given for this system because the fast and slow phases had amplitudes with opposite sign. Open table in a new tab Dissociation of Ca4-CaM·Peptide ComplexesFig. 1 shows typical traces for the dissociation of the CaM complexes of the peptides WFF, FFF, and MasX induced by excess chelator. A-C of Fig. 1 are representative of the range of kinetic behavior observed. The complexes clearly show diverse properties, when compared with one another for a given optical parameter or when compared for different optical parameters for the same complex. The results in Table II show rates and relative amplitudes for the complexes studied. Full discussion of these results requires a consideration of possible mechanisms (see “The Kinetic Model”). However, some generalizations can usefully be made here.The rates observed with the complexes and Quin2 (Table II) are frequently biphasic, with a fast rate of 5-100 s−1 and a slow rate of 0.5-5 s−1. The total amplitude corresponds to four Ca2+ ions. The rates are markedly slower than those observed with CaM alone, where fast and slow rates clearly correspond to dissociation from N- and C-domains, respectively (27Bayley P.M. Ahlström P. Martin S.R. Forsén S. Biochem. Biophys. Res. Commun. 1984; 120: 185-191Crossref PubMed Scopus (98) Google Scholar). The presence of peptide enhances the affinity of both N- and C-domain Ca2+ sites (see “The Kinetic Model”), and this assignment of the faster rate from the N-domain is evidently retained in the presence of peptide. By contrast, for FFW and FFF the Quin2 signals were monophasic, with a small lag phase in the case of Ca4-CaM·FFF (Fig. 1B). In this case it appears that a distinction between the N- and C-domain properties cannot be made.For those cases showing biphasic Quin2 signals, the EGTA-induced dissociation of the complexes with the Trp-containing peptides also shows biphasic character, with the greater amplitude in the slow phase. The existence of the fast phase in the Trp signal in the case of WFF and FWF is an important observation, with mechanistic significance (see “The Relaxation Step: Experimental Justification”). It raises questions of the fluorescence properties of intermediate species and, more fundamentally, of the effects of coupling individual kinetic steps with different optical parameters. The biphasic kinetics observed in the EGTA dissociation of CaM·MasX is clearly exceptional, since (Fig. 1C) there is an intermediate species of enhanced fluorescence and the two kinetic components are oppositely signed.The EGTA-induced dissociation of complexes of non-Trp-containing peptides with CaM shows a single phase, usually in the slow range, consistent with the origins of this signal at Tyr-138 in the C-domain of CaM. The EGTA-induced dissociation of Ca4-CaM·FFF shows a lag phase, evidencing the sequential nature of the process.The T26W mutant was used with all peptides, since it has a significant fluorescent change when Ca2+ dissociates from the N-domain. In general, biphasic and monophasic behavior follow the Quin2 signals for all complexes (except CaM·Mas and CaM·CBP1). The rates are generally similar although CaM·MasX again appears exceptional. The relative amplitudes of biphasic transients are however different, with the fast phase predominating, consistent with the involvement of the N-domain. The signal in the case of complexes of T26W with Trp-containing peptides is composite, with similar contributions from the loss of Ca2+ from the N-domain and the change due to complete dissociation of the peptide. These two contributions overlap and cannot readily be resolved.There is no obvious relationship between the observed rates and the affinities of the peptides for Ca4-CaM. Thus, for example, EGTA-induced dissociation rates for complexes with Mas or CBP1 are similar, even though the affinity of CBP1 for Ca4-CaM is 2 orders of magnitude higher than that for Mas. Clearly the reactions studied here involve the eventual dissociation of both Ca2+ and peptide from the initial complex, whereas the peptide affinities reflect only the association/dissociation reaction of the peptide from the complex with Ca4-CaM. Thus, the pathway for the chelator-induced dissociation of the peptide appears to be under kinetic, rather than thermodynamic, control. Taken together, the results indicate that the dissociation mechanism is a complex multi-step pathway, with observed rates resulting from the coupling of individual kinetic steps.The Kinetic ModelThe purpose of the kinetic model is to account for the reduction in the Ca2+ dissociation rates from CaM in the presence of peptides and to identify steps in the pathway where differences in the peptides produce significant kinetic effects. The complete kinetic scheme for the dissociation of Ca2+ and peptide from a Ca4-CaM·peptide complex becomes unduly complex if the four Ca2+ ions are considered to dissociate independently. It is reasonable, however, based on extensive experimental evidence for the dissociation kinetics of Ca4-CaM, to consider the Ca2+ ions as dissociating in pairs, one pair from each domain (34Martin S.R. Andersson Teleman A. Bayley P.M. Drakenberg T. Forsén S. Eur. J. Biochem. 1985; 151: 543-550Crossref PubMed Scopus (117) Google Scholar). This assumption results in the Ca2+ dissociation scheme shown in Fig. 2A, where C represents CaM and the subscripts P, N, and C represent peptide, and the N- and C-terminal Ca2+ pairs, respectively. Therefore, for example, CNCP represents the full Ca4-CaM·peptide complex, and C−CP represents the Ca2-CaM·peptide complex with 2 Ca2+ in the C-domain.Fig. 2Formal kinetic scheme for dissociation of a Ca4-CaM·peptide complex, assuming that the Ca2+ ions dissociate in pairs. C represents CaM and the subscripts P, N, and C represent peptide, N- and C-terminal Ca2+ pairs, respectively. a, full scheme; b, simplified scheme and rate constants used in the simulations. All steps involving Ca2+ dissociation are assumed to be irreversible (see “Materials and Methods”).View Large Image Figure ViewerDownload Hi-res image Download (PPT)This kinetic scheme can be simplified given knowledge of the properties of CaM itself. As shown in Fig. 2A, step 2 of the path involves peptide dissociation from CNCP to produce the species CNC− (i.e. Ca4-CaM). Dissociation of this species is known to proceed almost exclusively via steps 3 and 6, and not via steps 9 and 8, as the C-terminal Ca2+ ions dissociate about one hundred times slower from Ca4-CaM (CNC−) than the N-terminal Ca2+ ions (34Martin S.R. Andersson Teleman A. Bayley P.M. Drakenberg T. Forsén S. Eur. J. Biochem. 1985; 151: 543-550Crossref PubMed Scopus (117) Google Scholar). Correspondingly, we assume that dissociation of the C-terminal Ca2+ pair from CNCP is always slower than dissociation of the N-terminal pair and have therefore eliminated step 12 and the subsequent steps 10 and 11 This reduces the scheme to two competing pathways (Fig. 2B).In path A the N-terminal Ca2+ ions dissociate first (step 1), and in path B the peptide dissociates first (step 2). Path B is simply steps 2, 3, and 6 For path A, once the species C−CP forms it is possible for either the peptide to dissociate to form C−C− (step 4), or the C-terminal Ca2+ ions to dissociate to form C−−P (step 5). Step 4 is a reversible step and is a relaxation process in the mechanism, as opposed to a uni-directional irreversible step such as Ca2+ dissociation in the presence of a chelator. However, it is coupled to unidirectional steps 1 and 6 and is in parallel with step 5 It is necessary to know the relative rates of these processes to determine which path will predominate.The Relaxation Step: Theoretical JustificationThe values of k−4 and k−5 can be estimated, and dissociation via step 4 is found likely to predominate in certain cases. Constants k−1 and k−5, the N- and C-terminal Ca2+ dissociation rates, can be estimated for a typical Ca4-CaM·peptide complex as follows. The interaction of Ca4-CaM with a peptide may be characterized by Equation 1,Kd'/Kd=(K'/K)4 where Kd′ and Kd are the dissociation constants for the interaction of the peptide with apo-CaM and Ca4-CaM. K and K′ (average Ca2+ affinities) are equal to (K1K2K3K4)1/4 and (K1′K2′K3′K4′)1/4, respectively, where Ki and Ki′ are the stoichiometric Ca2+ association constants measured in the absence and presence of peptide (39Yazawa M. Ikura M. Hikichi K. Ying L. Yagi K. J. Biol. Chem. 1987; 262: 10951-10954Abstract Full Text PDF PubMed Google Scholar).The value of K for Drosophila CaM is 1.45 × 105M−1 (25Maune J.F. Klee C.B. Beckingham K. J. Biol. Chem. 1992; 267: 5286-5295Abstract Full Text PDF PubMed Google Scholar), and the values of Kd are known for each peptide (Table II). Values of Kd′ are less well established. Values of 620 and 25 mM have been reported for the peptides C28W and C20W from the CaM binding domain of the plasma membrane Ca2+ pump (40Yazawa M. Vorherr T. James P. Carafoli E. Yagi K. Biochemistry. 1992; 31: 3171-3176Crossref PubMed Scopus (52) Google Scholar), and values of 80 mM and 5.7 μM have been reported for bovine heart phosphodiesterase and troponin I (41Olwin B.B. Storm D.R. Biochemistry. 1985; 24: 8081-8086Crossref PubMed Scopus (122) Google Scholar). Since binding of the peptides WFF and FFW to apo-CaM cannot be detected by CD at peptide concentrations of 100 μM,2 it is reasonable to assume a Kd′ of more than 1 mM for the sk-MLCK peptides. For a typical peptide with a Kd of 1 nM, values of Kd′ in the range 1 to 100 mM would correspond to (K′/K)4 values in the range 106 to 108 and therefore to K′/K values in the range 30 to 100. Values in this range have been determined for other peptides (40Yazawa M. Vorherr T. James P. Carafoli E. Yagi K. Biochemistry. 1992; 31: 3171-3176Crossref PubMed Scopus (52) Google Scholar, 41Olwin B.B. Storm D.R. Biochemistry. 1985; 24: 8081-8086Crossref PubMed Scopus (122) Google Scholar).Stoichiometric Ca2+ association constants for CaM in the presence of the peptides used here show that both N- and C-domain Ca2+ affinities are enhanced, and consistent with this, peptide WFF (like other peptides) shows much lower affinity for Ca2-TR2C (Kd = 76 nM) than for Ca4-CaM (<0.2 nM) (13Bayley P.M. Findlay W.A. Martin S.R. Protein Sci. 1996; 7: 1215-1228Crossref Scopus (142) Google Scholar). Thus Ca2+ binding in both domains contributes to the enhanced peptide affinity, and the peptide affinity for a partially saturated Ca2-CaM will be less than for Ca4-CaM. The above calculation can be taken further, assuming that the difference between K and K′ is reflected in the Ca2+ dissociation rates. If the effect of the peptide on the N- and C-domain Ca2+ affinity is approximately equal, both the N- and C-terminal dissociation rates are expected to be decreased by the above factor of 30 to 100. Since the dissociation rates in the absence of peptide are ≈700 s−1 (N-terminal) and 8.5 s−1 (C-terminal), we predict k−1 values in the range 25-7 s−1 and k−5 values in the range 0.3-0.085 s−1. The rate of Ca2+ dissociation from C−CP (step 5) is therefore predicted to be much slower than 8.5 s−1 (Table II), the rate of Ca2+ dissociation from CaM in the absence of peptide (step 6).Calculations also show that k−4, the peptide dissociation rate from C−CP, is likely to be at least 10 s−1, owing to the lower affinity of the peptide for CaM after loss of the N-terminal Ca2+ ions, see above. By analogy with the kon for the association of FWF with Ca4-CaM, k4 is likely to be of the order of 109M−1 s−1, and hence the re-association reaction of the peptide becomes significant. Step 4 is reversible, i.e. it is a relaxation process. The rate of this step will be greater than k−4, and, since k−4 > k−5, it will exceed k−5. Hence, dissociation via step 4 should be significant. Even if the effect of the peptide is largely on the C-terminal Ca2+ sites, both k−4 and k−5 will be reduced, and the relaxation step 4 and the dissociation step 5 will remain in competition.The Relaxation Step: Experimental JustificationThe reversible nature of step 4 suggests that the observed rates should be affected by the [peptide]/[CaM] ratio. We therefore measured the dissociation reaction for Ca4-CaM·WFF as a function of the [WFF]/[CaM] ratio. At [WFF]/[CaM] = 1.05 the EGTA-induced dissociation of Ca4-CaM·WFF (monitored by peptide Trp fluorescence) is biphasic with a fast phase rate of 12" @default.
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• W2030890665 title "Kinetic Control of the Dissociation Pathway of Calmodulin-Peptide Complexes" @default.
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• W2030890665 doi "https://doi.org/10.1074/jbc.272.6.3389" @default.
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