The binding isotherms of the divalent metal cations, Ca2+, Mg2+, and Zn2+, to the synthetic γ-carboxyglutamic acid-containing neuroactive peptides, conantokin-G (con-G) and conantokin-T (con-T), have been determined by isothermal titration calorimetry (ITC) at 25 °C and pH 6.5. We have previously shown by potentiometric measurements that con-G contains 2–3 equivalent Ca2+ sites with an average Kd value of 2800 μm. With Mg2+ as the ligand, two separate exothermic sites are obtained by ITC, one of Kd = 46 μmand another of Kd = 311 μm. Much tighter binding of Zn2+ is observed for these latter two sites (Kd values = 0.2 μm and 1.1 μm), and a third considerably weaker binding site is observed, characterized by a Kd value of 286 μm and an endothermic enthalpy of binding. con-T possesses a single exothermic tight binding site for Ca2+, Mg2+, and Zn2+, with Kdvalues of 428 μm, 10.2 μm, and 0.5 μm, respectively. Again, in the case of con-T, a weak (Kd = 410 μm) endothermic binding site is observed for Zn2+. The binding of these cations to con-G and con-T result in an increase in the α-helical content of the peptides. However, this helix is somewhat destabilized in both cases by binding of Zn2+ to its weakest site.Since the differences observed in binding affinities of these three cations to the peptides are substantially greater than their comparative Kd values to malonate, we conclude that the structure of the peptide and, most likely, the steric and geometric properties imposed on the cation site as a result of peptide folding greatly influence the strength of the interaction of cations with con-G and con-T. Further, since the Zn2+ concentrations released in the synaptic cleft during excitatory synaptic activity are sufficiently high relative to the Kd of Zn2+ for con-G and con-T, this cation along with Mg2+, are most likely the most significant metal ion ligands of these peptides in neuronal cells. The binding isotherms of the divalent metal cations, Ca2+, Mg2+, and Zn2+, to the synthetic γ-carboxyglutamic acid-containing neuroactive peptides, conantokin-G (con-G) and conantokin-T (con-T), have been determined by isothermal titration calorimetry (ITC) at 25 °C and pH 6.5. We have previously shown by potentiometric measurements that con-G contains 2–3 equivalent Ca2+ sites with an average Kd value of 2800 μm. With Mg2+ as the ligand, two separate exothermic sites are obtained by ITC, one of Kd = 46 μmand another of Kd = 311 μm. Much tighter binding of Zn2+ is observed for these latter two sites (Kd values = 0.2 μm and 1.1 μm), and a third considerably weaker binding site is observed, characterized by a Kd value of 286 μm and an endothermic enthalpy of binding. con-T possesses a single exothermic tight binding site for Ca2+, Mg2+, and Zn2+, with Kdvalues of 428 μm, 10.2 μm, and 0.5 μm, respectively. Again, in the case of con-T, a weak (Kd = 410 μm) endothermic binding site is observed for Zn2+. The binding of these cations to con-G and con-T result in an increase in the α-helical content of the peptides. However, this helix is somewhat destabilized in both cases by binding of Zn2+ to its weakest site. Since the differences observed in binding affinities of these three cations to the peptides are substantially greater than their comparative Kd values to malonate, we conclude that the structure of the peptide and, most likely, the steric and geometric properties imposed on the cation site as a result of peptide folding greatly influence the strength of the interaction of cations with con-G and con-T. Further, since the Zn2+ concentrations released in the synaptic cleft during excitatory synaptic activity are sufficiently high relative to the Kd of Zn2+ for con-G and con-T, this cation along with Mg2+, are most likely the most significant metal ion ligands of these peptides in neuronal cells. The N-methyl-d-aspartate (NMDA) 1The abbreviations used are: NMDA,N-methyl-d-aspartate; NMDAR, NMDA receptor; con-G, conantokin-G; con-T, conantokin-T; Gla, γ-carboxyglutamic acid; ITC, isothermal titration calorimetry; Mes, 4-morpholineethanesulfonic acid. subtype of glutamate receptor is a ligand-gated ion channel that displays high permeability for Ca2+. The marked excitotoxicity of glutamate is generally regarded as ascribable to its persistent interaction with the NMDA receptor (NMDAR), resulting in the establishment of neurodegenerative glutamatergic loops defined by uncontrolled elevations of intracellular Ca2+, followed by cell lysis and death. Because the Ca2+-mediated neuronal cell death, which is attendant to both acute (e.g. ischemia) and chronic (e.g. epilepsy, Parkinson's disease) neurodegenerative disorders, can be ameliorated by antagonists specific for the NMDAR (1Gotti B. Duverger D. Bertin J. Carter C. DuPont R. Frost J. Gaudilliere B. MacKenzie E.T. Rousseau J. Scatton B. J. Pharmacol. Exp. Ther. 1988; 247: 1211-1221PubMed Google Scholar, 2Chen H.S. Pellegrini J.W. Aggarwal S.K. Lei S.Z. Warach S. Jensen F.E. Lipton S.A. J. Neurosci. 1992; 12: 4427-4436Crossref PubMed Google Scholar, 3Meldrum B.S. Curr. Opin. Neurol. Neurosurg. 1992; 5: 508-513PubMed Google Scholar, 4Graham S.H. Chen J. Sharp F.R. Simon R.P. J. Cereb. Blood Flow Metab. 1993; 13: 88-97Crossref PubMed Scopus (143) Google Scholar, 5Biegon A. Joseph A.B. Neurol. Res. 1995; 17: 275-280Crossref PubMed Scopus (45) Google Scholar), extensive biochemical characterization of drug-receptor interactions focused on this receptor is a widely studied topic. Isolated from the venom ducts of predatory snails of the genusConus, the conantokins-G (con-G) and -T (con-T) are potent and selective inhibitors of NMDAR function (6Skolnick P. Boje K. Miller R. Pennington M. Maccecchini M.-L. J. Neurochem. 1992; 59: 1516-1521Crossref PubMed Scopus (56) Google Scholar, 7Chandler P. Pennington M. Maccecchini M.L. Nashed N.T. Skolnick P. J. Biol. Chem. 1993; 268: 17173-17178Abstract Full Text PDF PubMed Google Scholar, 8Zhou L.-M. Szendrei G.I. Fossom L.H. Maccecchini M.-L. Skolnick P. Otvos L. J. Neurochem. 1996; 66: 620-628Crossref PubMed Scopus (60) Google Scholar) and are the only peptide antagonists of this receptor subtype described to date. More specifically, this antagonism derives from a noncompetitive inhibitory effect of the polyamine agonist site of the receptor (6Skolnick P. Boje K. Miller R. Pennington M. Maccecchini M.-L. J. Neurochem. 1992; 59: 1516-1521Crossref PubMed Scopus (56) Google Scholar). The physiological responses elicited following intracranial injections in mice include a sleep-like state in neonatal mice and a hyperactive response in older animals (9Rivier J. Galyean R. Simon L. Cruz L.J. Olivera B.M. Gray W.R. Biochemistry. 1987; 28: 8508-8512Crossref Scopus (57) Google Scholar). An unusual feature of con-G and con-T is their high abundance of γ-carboxyglutamic acid (Gla) (10McIntosh J. Olivera B.M. Cruz L. Gray W. J. Biol. Chem. 1984; 259: 14343-14346Abstract Full Text PDF PubMed Google Scholar, 11Haack J.A. Rivier J. Parks T.N. Mena E.E. Cruz L.J. Olivera B.M. J. Biol. Chem. 1990; 265: 6025-6029Abstract Full Text PDF PubMed Google Scholar). Prior to its discovery in the conantokins, the presence of this amino acid in polypeptides and proteins had been noted only in certain bone proteins and in various blood coagulation factors. In these contexts, the ability of Gla to bind to Ca2+ plays an integral role in the adoption of functional protein conformers (12Nelsestuen G.L. J. Biol. Chem. 1976; 251: 5648-5656Abstract Full Text PDF PubMed Google Scholar). The interaction of Gla with Ca2+ has been established for the conantokins, in which cases it has been demonstrated that both con-G and con-T adopt a significant degree of α-helicity in the presence of this cation (13Prorok M. Warder S.E. Blandl T. Castellino F.J. Biochemistry. 1996; 35: 16528-16534Crossref PubMed Scopus (75) Google Scholar). This α-helical induction is particularly profound in the case of con-G, which is essentially structureless in its apo form but assumes a full end-to-end α-helical conformation when Ca2+ or Mg2+ is fully bound to the peptide (13Prorok M. Warder S.E. Blandl T. Castellino F.J. Biochemistry. 1996; 35: 16528-16534Crossref PubMed Scopus (75) Google Scholar, 14Blandl T. Zajicek J. Prorok M. Castellino F.J. Biochem. J. 1997; 328: 777-783Crossref PubMed Scopus (22) Google Scholar, 15Chen Z. Blandl T. Prorok M. Warder S.E. Li L. Zhu Y. Pedersen L.G. Ni F. Castellino F.J. J. Biol. Chem. 1998; 273: 16248-16258Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In contrast, con-T manifests appreciable α-helicity in the apo form which increases slightly in the divalent cation-bound state (13Prorok M. Warder S.E. Blandl T. Castellino F.J. Biochemistry. 1996; 35: 16528-16534Crossref PubMed Scopus (75) Google Scholar, 16Warder S.E. Chen Z.G. Zhu Y. Prorok M. Castellino F.J. Ni F. FEBS Lett. 1997; 411: 19-26Crossref PubMed Scopus (33) Google Scholar, 17Warder S.E. Prorok M. Chen Z. Li L. Zhu Y. Pedersen L.G. Ni F. Castellino F.J. J. Biol. Chem. 1998; 273: 7512-7522Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Because the smaller population of conformers associated with the more structurally rigid metal-bound forms of these peptides would be expected to afford more discriminate binding to the NMDAR site, metal complexation to these peptides may be necessary for full bioactivity. Of the many Conus peptides currently characterized, all of which appear to target neuronal or muscle cell receptors, only con-G and con-T lack intramolecular disulfide bridges. This observation lends support to the idea that conformational rigidity, either covalently or noncovalently imposed, is an important element of receptor recognition among members of the Conus-derived peptide family. In the case of the conantokins, metal ions other than Ca2+ have also been shown to effect α-helix induction. Of these, Ca2+, Mg2+, and Zn2+ are the most physiologically relevant metal cations in brain cells (14Blandl T. Zajicek J. Prorok M. Castellino F.J. Biochem. J. 1997; 328: 777-783Crossref PubMed Scopus (22) Google Scholar). Through CD-monitored titrations of con-G, we have found that the affinity of these divalent metal ions for the peptide is significantly greater for Mg2+ and Zn2+ than for Ca2+ (14Blandl T. Zajicek J. Prorok M. Castellino F.J. Biochem. J. 1997; 328: 777-783Crossref PubMed Scopus (22) Google Scholar). Although CD-monitored titrations represent a convenient approach for assessing relative metal ion affinities, their correspondingKd values cannot be accurately determined from this method since the estimated Kd values fall in the range of working peptide concentrations. This situation also complicates determination of the stoichiometry of metal binding. To gain a comprehensive quantitative assessment of these parameters, we have employed isothermal titration calorimetry (ITC) for monitoring the heat changes that accompany metal ion binding to the peptides. In addition to Kd and stoichiometry, values for ΔH and ΔS can also be extracted from an ITC profile. With these data in hand, the nature of metal ion binding to both con-G and con-T can be more rigorously The of the is to the properties of metal binding. The synthetic and for con-G = and con-T = have been described (13Prorok M. Warder S.E. Blandl T. Castellino F.J. Biochemistry. 1996; 35: 16528-16534Crossref PubMed Scopus (75) Google Scholar). of peptide determined by quantitative amino acid The binding isotherms of Ca2+, Mg2+, and Zn2+ to the and determined by ITC measurements of the heat changes titration of the metal ions of the relevant The titrations with an titration at 25 °C in a Mes, pH 6.5. in concentrations from in a of in the an of or in than the peptide in at The observed heat The observed heat for the heat of of by titrations in the of The resulting titration for the the for ITC by titrations of con-G and con-T as a function of metal ion on an at a cell at 25 The peptides in Mes, pH to a of 0.5 by a of for con-G and for con-T. The α-helical content determined from at the = Biochemistry. PubMed Scopus Google Scholar). a at °C in at at of and The used Mes, pH 6.5. The specific of con-T from its amino acid by the Gla of glutamate The of the determined to be data the single in the values to for all data represent the average of the from three separate titrations of con-G, con-T, and with various divalent metal cations at 25 pH 6.5. of the heat changes the binding of of Zn2+ to con-G and con-T are shown in the of The binding to a of as a function of the of is in Zn2+ titrations represent the more isotherms of the Ca2+, Mg2+, and Zn2+ data with and resulting from the complexation of Zn2+ with con-G and con-T, respectively. The of the data for con-G by a site to an = only Kd and to during the con-T, to be to The employed in the case of Mg2+ binding to con-G, an stoichiometry of titrations of all to during the obtained for to or This that of peptide of an apo the of the An to this observation in the case of the for con-T. these a to = = = = = = as these peptide of this as described In addition to the peptide binding sites that can be by Zn2+, the of Zn2+ binding to the also with Mg2+ and Ca2+ in that a endothermic in the titration of both peptides. This is significantly more in the case of con-T can be from the of the data in all other determined changes for both peptides are to be noted from the of site, with the of the associated with the weaker of the two Mg2+ sites in ion binding properties of con-G and con-T as determined by ITC data from The binding of Ca2+ to con-G is weak to to ITC since high concentrations of peptide would be necessary for an values represent the average of at two The of the for is data from M. Warder S.E. Blandl T. Castellino F.J. Biochemistry. 1996; 35: 16528-16534Crossref PubMed Scopus (75) Google The binding of Ca2+ to con-G is weak to to ITC since high concentrations of peptide would be necessary for an in a The values represent the average of at two The of the for is of the Kd values for Ca2+, Mg2+, and Zn2+ for con-G and con-T a increase in affinity for Zn2+ when with Mg2+ in to the peptides tighter than Ca2+. con-G, the of the Zn2+ sites (Kd = 0.2 μm) displays more binding than the of the Mg2+ sites (Kd = 46 μm) and a increase in affinity relative to the 2–3 Ca2+ con-T, this is somewhat with the strength of the interaction for the tight Zn2+ site and greater than that associated with the Mg2+ and respectively. In contrast, manifests only a for Zn2+ as with Mg2+ and Ca2+ and In the Kd values that the binding of these cations to range from to of than the Kd values associated with their complexation to the ΔH and ΔS values for metal ion binding to are The stoichiometry of determined for the of Zn2+ with that degree of may accompany binding of this metal ion to In an to the the endothermic observed for con-G and con-T, CD-monitored titrations of these peptides with Zn2+ that in the ITC can be for con-G an essentially increase in α-helicity the addition of Zn2+, to an at a of The of this with a of tight binding to of Zn2+, that all metal ion in the The of that a of a but defined in α-helicity is This noted at of Ca2+ and Mg2+ The CD-monitored titration of con-T with Zn2+ is in B. affinity binding of Zn2+, as with con-G, is in the of the a significant in α-helicity of the Again, this in α-helical content observed in Ca2+ and Mg2+ Because the in α-helical content with the endothermic of the ITC it that of the weakest Zn2+ site of both con-G and con-T a of the which is considerably more for the latter of the of con-T of a more of metal binding peptide peptide also for the in con-T and con-G α-helicity observed at To this of con-T in both the apo state and at various Zn2+ concentrations. shown in the of con-T increases from in its state to at a of and at a of Although these degree of the for the data the determined at and con-T in the presence of Zn2+ to be and to the expected the peptide The metal cation sites for con-G are with the cation sites from by a method the from the of the and peptides Z. Blandl T. Prorok M. Warder S.E. Li L. Zhu Y. Pedersen L.G. Ni F. Castellino F.J. J. Biol. Chem. 1998; 273: 16248-16258Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, Li L. Pedersen L.G. B. Biochemistry. 1997; PubMed Scopus Google Scholar). The structure of the form is for the of the of which are for metal ion This would be in the of a of and a site. The of these as metal binding by the of Mg2+ to the structure the method Z. Blandl T. Prorok M. Warder S.E. Li L. Zhu Y. Pedersen L.G. Ni F. Castellino F.J. J. Biol. Chem. 1998; 273: 16248-16258Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This approach also as another likely site for ITC to this third which may bind Mg2+ to be to this However, a weak site, during the Zn2+ titration of con-G and may binding at this site. peptide Gla to support a the tight Mg2+ site is by and with the weaker site of and Z. Blandl T. Prorok M. Warder S.E. Li L. Zhu Y. Pedersen L.G. Ni F. Castellino F.J. J. Biol. Chem. 1998; 273: 16248-16258Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The of and as determined from the structure of con-T is to that found in con-G and, by can be a likely binding for metal ions S.E. Chen Z.G. Zhu Y. Prorok M. Castellino F.J. Ni F. FEBS Lett. 1997; 411: 19-26Crossref PubMed Scopus (33) Google Scholar). of Ca2+ this structure the a structure the of and as site site by the two of and the of binding site for Ca2+ and Mg2+, but observed with Zn2+, a more with and that the high affinity site of con-T these have demonstrated that are Ca2+ of either con-G or con-T (13Prorok M. Warder S.E. Blandl T. Castellino F.J. Biochemistry. 1996; 35: 16528-16534Crossref PubMed Scopus (75) Google Scholar). In the presence of concentrations of Mg2+, the of both peptides has also been However, with Zn2+, the titration of con-T data for which a with peptide be In from the titration one of the unusual of con-T with Zn2+ include peptide which to helix To the of con-T the employed in both the calorimetry and we for of the apo and peptide of the of the to the single for and con-T the of of as the of is a of the data are with of con-T. However, when with the of the of this peptide determined from is of the increase in in the presence of Zn2+ also to be with the of We that these can be in of an high specific of the peptide that employed in the Because the value of for Gla has been we to it the glutamate value of in the of con-T. This is an of the for this when that the presence of an in Gla the associated with this by of its and for In in the presence of Zn2+ the and interaction of the metal ion with the Gla may the of the high of Gla in con-T, its to the value of the peptide is The observation that an increase in peptide at high Zn2+ concentrations is in support of the of con-T. The associated with binding in the expected with the degree of conformational by metal binding. In the case of Mg2+ complexation to con-G, the peptide a in α-helicity Mg2+ binding (14Blandl T. Zajicek J. Prorok M. Castellino F.J. Biochem. J. 1997; 328: 777-783Crossref PubMed Scopus (22) Google Scholar), the in the ΔH for binding of of is The for con-T, which a smaller increase in α-helicity of its metal ion sites with Mg2+, is to a greater in the con-G than in the con-T that to these ΔH values since metal ion binding to malonate, which possesses an ΔH to the binding The of that of metal ions to the Gla of the peptide may to The that accompany the exothermic are somewhat the significant degree of that metal ion binding on these peptides. with as of the metal ion from its and of and are likely in the peptide the values for metal complexation to malonate, it is that the of of The ΔH values observed for by Zn2+ of the weak sites of both con-G and con-T with a degree of α-helix as from CD-monitored titrations The values to weak site Zn2+ binding for both peptides is also with the of The nature of this in con-G is to state with the of Mg2+ to con-G Z. Blandl T. Prorok M. Warder S.E. Li L. Zhu Y. Pedersen L.G. Ni F. Castellino F.J. J. Biol. Chem. 1998; 273: 16248-16258Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) as a third binding Because all Gla of con-G on the of the helix in the structure Z. Blandl T. Prorok M. Warder S.E. Li L. Zhu Y. Pedersen L.G. Ni F. Castellino F.J. J. Biol. Chem. 1998; 273: 16248-16258Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), of an divalent cation would be expected to to the We that ITC titrations with various Gla to of con-G the nature of this third site and these are currently in con-T, we that the greater degree of α-helix that Zn2+ can be in of the of The of both and con-T that the of is an in both This is by data on the which displays α-helical content in both its and forms S.E. Prorok M. Chen Z. Li L. Zhu Y. Pedersen L.G. Ni F. Castellino F.J. J. Biol. Chem. 1998; 273: 7512-7522Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In the presence of high Zn2+ we that is from its to interaction role to with in the of a weak site, the α-helix in the the important the of the metal ion that in the conformation of these peptides. has been established in with bone and blood proteins that Ca2+ their functional In the case of and protein which and Gla in their for Mg2+ Ca2+ has been noted from titration G.L. M. J. Biol. Chem. 1976; 251: Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, G.L. Biochemistry. PubMed Scopus Google Scholar, M. S. B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). a is observed for Ca2+ in of their Kd values for the smaller for Mg2+ as with Ca2+ this to to appreciable differences in these However, for con-G, the Mg2+ tight site and weak site affinities in with Ca2+ are and respectively. con-T, Mg2+ displays a increase in binding strength with Ca2+. con-G and con-T Kd values for Zn2+ than for either Ca2+ or greater affinity for Zn2+ Ca2+ has been for a which a J. Chem. 1998; Scopus Google Scholar). that the greater of interactions Zn2+, ascribable to its as as for this are the to this binding. However, the of these is by which that only a increase in affinity for with Ca2+. This would that the binding sites of peptides, which bind Zn2+, are particularly to this smaller metal ion their binding site cation are the strength of binding in these may more on steric and geometric than on The that Zn2+ is the metal ion of function is by that that high concentrations of μm) can be in the synaptic cleft during excitatory synaptic activity S.H. 1984; PubMed Scopus Google Scholar, L. Y. Res. 1987; PubMed Scopus Google Scholar). injections of the mice have been shown to a sleep-like state in these at an estimated brain of μm (13Prorok M. Warder S.E. Blandl T. Castellino F.J. Biochemistry. 1996; 35: 16528-16534Crossref PubMed Scopus (75) Google Scholar). a Zn2+ of μm, this would result in essentially of the two tight Zn2+ sites in con-G and of the weak site. In the case of con-T, of the high affinity Zn2+ site and of the affinity Zn2+ site would be at these peptide and concentrations. a Mg2+ of tight and weak site of con-G would be and respectively. con-T, essentially of the peptide would be with a Ca2+ of Ca2+ would be considerably than Mg2+ and Zn2+, with and of the metal sites in con-G and con-T, respectively. We that Mg2+, as as Zn2+, are more for the role of in metal ions for the However, it be noted that the high and by Ca2+ proteins the ability to other This feature of may the of the conantokins, the binding by Ca2+ with Mg2+ and Zn2+. the of these metal ions in the with to the NMDAR is by their in receptor the associated with the receptor Mg2+ of the receptor M.L. G.L. 1984; PubMed Scopus Google Scholar, L. P. P. A. A. 1984; PubMed Scopus Google Scholar), and and by Zn2+ G.L. M.L. 1987; Scopus Google Scholar, J. Neurosci. 1990; PubMed Google Scholar). the role of these metal ions in of antagonism of the receptor is for a full of the these peptides and one of the associated with in this
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