Thermodynamics of Binding of Calcium, Magnesium, and Zinc to the N-Methyl-D-aspartate Receptor Ion Channel Peptidic Inhibitors, Conantokin-G and Conantokin-T*

Mary Prorok and Francis J. CastellinoDagger

From the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The binding isotherms of the divalent metal cations, Ca2+, Mg2+, and Zn2+, to the synthetic gamma -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 µM and 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 Kd values 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 alpha -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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The N-methyl-D-aspartate (NMDA)1 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 (1-5), 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 genus Conus, the conantokins-G (con-G) and -T (con-T) are potent and selective inhibitors of NMDAR function (6-8) 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 (6). The physiological responses elicited following intracranial injections in mice include a sleep-like state in neonatal mice and a hyperactive response in older animals (9).

An unusual feature of con-G and con-T is their high abundance of gamma -carboxyglutamic acid (Gla) (10, 11). 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 (12). 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 alpha -helicity in the presence of this cation (13). This alpha -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 alpha -helical conformation when Ca2+ or Mg2+ is fully bound to the peptide (13-15). In contrast, con-T manifests appreciable alpha -helicity in the apo form which increases slightly in the divalent cation-bound state (13, 16, 17). 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 alpha -helix induction. Of these, Ca2+, Mg2+, and Zn2+ are the most physiologically relevant metal cations in brain cells (14). 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+ (14). Although CD-monitored titrations represent a convenient approach for assessing relative metal ion affinities, their corresponding Kd 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 Delta H and Delta S 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 analyzed. The purpose of the current communication is to elaborate the thermodynamic properties of metal ion-conantokin binding.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Peptide Synthesis-- The materials, synthetic protocols, and purification procedures for obtaining con-G (G-E-gamma -gamma -L-Q-gamma -N-Q-gamma -L-I-R-gamma -K-S-N-CONH2, gamma  = Gla) and con-T (G-E-gamma -gamma -Y-Q-K-M-L-gamma -N-L-R-gamma -A-E-V-K-K-N-A-CONH2, gamma  = Gla) have been described earlier (13). Concentrations of peptide stock solutions were determined by quantitative amino acid analysis.

Isothermal Calorimetry-- The binding isotherms of Ca2+, Mg2+, and Zn2+ to the conantokins and malonate were determined by ITC measurements of the heat changes accompanying titration of the metal ions into solutions of the relevant sample. The titrations were performed with an OMEGA titration calorimeter (Microcal, Inc., Northhampton, MA) at 25 °C in a buffer containing 10 mM Mes, 100 mM NaCl, pH 6.5. Peptide samples ranging in concentrations from 0.23-1.0 mM in a total volume of 1.4 ml were placed in the reaction cell. After equilibration, an appropriate concentration of CaCl2, MgCl2, or Zn(OAc)2 (typically 30-50× higher in concentration than the peptide solution) in matching buffer was delivered at discrete intervals. The observed heat was measured after each injection. The total observed heat effects were corrected for the heat of dilution of ligand by performing control titrations in the absence of peptide. The resulting titration curves were deconvoluted for the best-fit model using the ORIGIN for ITC software package supplied by Microcal.

Circular Dichroism-- CD titrations of con-G and con-T as a function of metal ion concentration were performed on an AVIV model 62DS spectrometer at 222 nm using a 0.1-cm path length cell thermostatted at 25 °C. The peptides were dissolved in 10 mM Mes, 100 mM NaCl, pH 6.5, to a final concentration of 0.5 mM. Mean residue ellipticities were calculated by using a mean residue molecular mass of 133 Da for con-G and 128 Da for con-T. The fractional alpha -helical content was determined from mean residue ellipticities at 222 nm using the empirical relationship f(alpha -helix) = (-[theta ]222 - 2340)/30300 (18).

Sedimentation Equilibrium-- Experiments were conducted using a Beckman XL-I analytical ultracentrifuge operating at 20 °C in absorbance mode at 280 nm at rotor speeds of 45,000 and 52,000 rpm. The buffer used was 10 mM Mes, 100 mM NaCl, pH 6.5. The partial specific volume of con-T (0.72 ml/g) was calculated from its amino acid composition by assigning the Gla residue the <A><AC>v</AC><AC>&cjs1171;</AC></A> of glutamate (0.66 ml/g). The density of the buffer was determined to be 1.005 g/ml. Sedimentation data were analyzed using the single ideal species model included in the Beckman XL-I software. Baseline offset values were constrained to zero for all data sets. Calculated molecular weights represent the average of the results from three separate scans.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Calorimetric titrations of con-G, con-T, and malonate with various divalent metal cations were performed at 25 °C, pH 6.5. Examples of the heat changes accompanying the binding of incremental additions of Zn2+ to con-G and con-T are shown in the top panels of Fig. 1. The binding isotherm corresponding to a plot of integrated heats as a function of the molar ratio of Zn2+/peptide is displayed in the lower panels. These Zn2+ titrations represent the more complicated isotherms of the Ca2+, Mg2+, and Zn2+ data sets with 3 and 2 enthalpic transitions resulting from the complexation of Zn2+ with con-G and con-T, respectively. The deconvolution of the data for con-G was achieved by fixing a multiple site model to an n = 3, thereby allowing only Kd and Delta H values to float during the iterative nonlinear least squares minimization. For con-T, n1 was constrained to be equal to n2. The same constraint was employed in the case of Mg2+ binding to con-G, wherein an apparent stoichiometry of 2 was obtained. For those titrations involving an n of approximately 1, all parameters (n, Kd, Delta H) were allowed to float during the minimization process. Excellent fits were obtained for stoichiometries corresponding to integer or near-integer values. This strongly suggests that neither metal-induced aggregation of peptide monomer nor metal-induced dissociation of an apo aggregate occurred under the conditions of the calorimetric titrations. An exception to this general observation occurred in the case of the Zn2+ titration for con-T. For these data, a second acceptable model was generated corresponding to Kd1 = 60 nM, n1 = 0.45; Kd2 = 760 nM, n2 = 0.53; Kd3 = 440 µM, n3 = 0.95. Insofar as these parameters might indicate Zn2+-induced peptide aggregation, further exploration of this possibility was pursued, as described below.


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Fig. 1.   Calorimetric titrations of the conantokins with Zn(OAc)2. The ligand (5-µl injections) were added to the peptide solutions at 200-s intervals. A, con-G (0.3 mM) titrated with 14 mM Zn(OAc)2. B, con-T (0.5 mM) titrated with 12 mM Zn(OAc)2. The titrations were performed at 25 °C in a buffer containing 10 mM Mes/100 mM NaCl, pH 6.5.

In addition to the additional peptide binding sites that can be occupied by Zn2+, the thermodynamic signature of Zn2+ binding to the conantokins was also unique compared with Mg2+ and Ca2+ in that a late endothermic transition occurs in the titration of both peptides. This is significantly more pronounced in the case of con-T (Fig. 1, Table I). As can be seen from the summary of the data in Table I, all other experimentally determined enthalpic changes for both peptides are exothermic. Also to be noted from Table I are the positive entropies attending occupation of each titratable site, with the exception of the entropy associated with the weaker of the two Mg2+ sites in con-G.

                              
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Table I
Metal ion binding properties of con-G and con-T as determined by ITC titrations
The values represent the average of at least two experiments. The error estimate of the parameters for replicate experiments is <10%.

Comparison of the Kd values for Ca2+, Mg2+, and Zn2+ for con-G and con-T shows a dramatic increase in affinity for Zn2+ when compared with Mg2+ which, in turn, binds to the peptides much tighter than Ca2+. For con-G, the tightest of the 3 Zn2+ sites (Kd = 0.2 µM) displays 200-fold more avid binding than the tightest of the 2 Mg2+ sites (Kd = 46 µM) and a 12,000-fold increase in affinity relative to the 2-3 eq Ca2+ sites. For con-T, this discrimination is somewhat less pronounced, with the strength of the interaction for the tight Zn2+ site being approximately 40- and 1000-fold greater than that associated with the Mg2+ and Ca2+ sites, respectively. In contrast, malonate manifests only a very modest selectivity for Zn2+ as compared with Mg2+ and Ca2+ (2.3- and 6.5-fold, respectively). In addition, the absolute Kd values that characterize the binding of these cations to malonate range from 1 to 4 orders of magnitude higher than the same Kd values associated with their complexation to the conantokins (Table I). Both Delta H and Delta S values for metal ion binding to malonate are positive. The stoichiometry of approximately 0.6 determined for the association of Zn2+ with malonate suggests that some degree of dimerization (or possibly higher order aggregation) may accompany binding of this particular metal ion to malonate.

In an attempt to address the phenomena underlying the endothermic transitions observed for con-G and con-T, CD-monitored titrations of these peptides with Zn2+ were performed under conditions that paralleled those implemented in the ITC experiments. As can be seen for con-G (Fig. 2A), an essentially linear increase in alpha -helicity attends the addition of Zn2+, up to an apparent plateau occurring at a metal/peptide ratio of 2:1 (m:m). The linear phase of this titration, with a midpoint of 1:1 (m:m), reflects extremely tight binding up to 2 eq of Zn2+, such that virtually all added metal ion exists in the peptide-bound form. The inset of Fig. 2A reveals that above a Zn2+/peptide molar ratio of 2, a small but defined decrease in alpha -helicity is induced. This phenomenon was not noted at similar metal/peptide ratios of Ca2+ and Mg2+ (data not shown). The CD-monitored titration of con-T with Zn2+ is illustrated in Fig. 2B. High affinity binding of Zn2+, as with con-G, is seen in the early portion of the profile, while a significant decrease in alpha -helicity accompanies occupation of the second Zn2+ site. Again, this decrease in alpha -helical content was not observed in similarly conducted Ca2+ and Mg2+ titrations. Because the CD-titratable diminutions in alpha -helical content correlate closely with the endothermic phases of the ITC profiles, it appears that occupancy of the weakest Zn2+ site of both con-G and con-T effects a partial unwinding of the alpha -helix, which is considerably more pronounced for the latter peptide.


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Fig. 2.   Circular dichroism-monitored Zn(OAc)2 titrations of the conantokins. A, con-G (0.5 mM). The inset is an expansion of latter portion of the titration. B, con-T (0.5 mM). The titrations were performed at 222 nm in a buffer containing 10 mM Mes, 100 mM NACL, PH 6.5, 25 °C.

As indicated above, calorimetric analysis of the Zn2+ titration profile of con-T was suggestive of a more complex model of metal binding involving peptide association. Zn2+-induced peptide dimerization (or higher order aggregation) could also account for the decrease in con-T and con-G alpha -helicity observed at higher Zn2+/peptide molar ratios. To address this issue, sedimentation equilibrium analysis of con-T was performed in both the apo state and at various Zn2+ concentrations. As shown in Fig. 3, the apparent molecular weight of con-T increases from 3160 in its uncomplexed state to 3390 at a Zn2+/peptide ratio of 1:1 (m:m), and 3650 at a cation/peptide ration of 5.6:1 (m:m). Although these results imply some degree of self-association, the residuals for the fitted data do not display any systematic deviation (Fig. 3). Furthermore, the molecular weights determined at 0.65 mM and 0.9 mM con-T in the presence of 5 mM Zn2+ were calculated to be 3710 and 3510, respectively, opposite to the trend expected if the peptide were undergoing self-association.


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Fig. 3.   Representative sedimentation equilibrium scans and calculated fits for apparent molecular weight for con-T (bottom panels). Experiments were performed at a rotor speed of 45,000 rpm at a final peptide concentration of 0.5 mM. Distribution of residuals for the indicated fits is shown in the top panels. A, con-T in the absence of metal cations. B, con-T at 0.5 mM Zn(OAc)2. C, con-T at 2.8 mM Zn(OAc)2. The buffer was 10 mM Mes, 100 mM NaCl, pH 6.5, 20 °C.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The multiple metal cation sites detected for con-G (Table I) are consistent with the multiple cation sites proposed from modeling by a genetic algorithm/molecular dynamics simulation method using the coordinates provided from the NMR-derived structures of the Ca2+- and Mg2+-bound peptides (15, 19). The NMR structure of the Mg2+-complexed form is notable for the spatial proximity of the side chain carboxylates of Gla10 and Gla14, which are optimally positioned for metal ion coordination. This proximity would be unlikely in the absence of a stabilizing cationic bridge. A cluster comprised of Gla3, Gla4, and Gla7 represents a second potential coordination site. The viability of these side chains as metal binding loci was supported by the simulated docking of Mg2+ to the Mg2+-loaded NMR-derived structure using the genetic algorithm/molecular dynamics simulation method (15). This approach also identified Gla7 as another likely site for Mg2+ in con-G. ITC experiments failed to detect this putative third locale which may bind Mg2+ too weakly to be amenable to this calorimetric approach. However, a third, albeit relatively weak site, was detected during the Zn2+ titration of con-G and may reflect binding at this proposed Gla7 site. Synthetic peptide variants containing individual Gla to Ala substitutions strongly support a model wherein the tight Mg2+ site is maintained by Gla10 and Gla14, with the weaker site being comprised of Gla3, Gla4, and Gla7 (15). The orientation of Gla10 and Gla14 as determined from the Ca2+-bound NMR solution structure of con-T is strikingly similar to that found in con-G and, by analogy, can be considered a likely binding locus for metal ions (16). Docking of Ca2+ into this structure using the aforementioned genetic algorithm/molecular dynamics simulation yielded a lowest energy structure showing the carboxylates of Gla10 and Gla14 as coordination site donors. A second site appears maintained by the two carboxylates of Gla3 and the side chain amide carbonyl of Gln6. A second binding site was not experimentally detected for Ca2+ and Mg2+, but was observed with Zn2+, a more avid ligand. Titrations with Zn2+ of individual [Gla10Ala]con-T and [Gla14Ala]con-T variants suggest that the high affinity site of con-T includes these residues.2

Previous studies have demonstrated that no detectable higher order species are present upon Ca2+ loading of either con-G or con-T (13). In the presence of saturating concentrations of Mg2+, the monomeric molecularity of both peptides has also been established.3 However, with Zn2+, the calorimetric titration of con-T yielded data for which a fit consistent with peptide dimerization could be generated. In addition, from the CD titration data, one interpretation of the unusual trend of decreasing con-T helicity coinciding with increasing Zn2+ concentration could include Zn2+-induced peptide aggregation upon weak-site occupancy, which leads to helix unwinding. To test the possibility of Zn2+-induced con-T aggregation under the conditions employed in both the calorimetry and CD experiments, we opted for sedimentation equilibrium analysis of the apo and Zn2+-complexed peptide states (Fig. 3). Inspection of the residuals of the fits to the single ideal species model for uncomplexed and Zn2+-chelated con-T reveals the random distribution of points diagnostic of ideal solute behavior. Insofar as the distribution of residuals is considered a rigorous indicator of analyte ideality, the data are consistent with strictly monomeric solution behavior of con-T. However, when compared with the calculated sequence-based molecular weight of 2680, the higher apparent weight-average molecular weight of this peptide determined from sedimentation equilibrium (3160) is suggestive of association. Furthermore, the increase in apparent molecular weight in the presence of Zn2+ also appears to be inconsistent with the contention of solute ideality. We contend that these disparate results can be addressed in terms of an artificially high partial specific volume <A><AC>&ngr;</AC><AC>&cjs1171;</AC></A> of the peptide (0.72 ml/mg) that was employed in the molecular weight determination. Because the value of <A><AC>&ngr;</AC><AC>&cjs1171;</AC></A> for Gla has not been determined, we assigned to it the glutamate value of 0.66 ml/mg in calculating the <A><AC>&ngr;</AC><AC>&cjs1171;</AC></A> of con-T. This is almost certainly an overestimate of the <A><AC>&ngr;</AC><AC>&cjs1171;</AC></A> for this residue when considering that the presence of an additional carboxylate in Gla should effectively lower the <A><AC>&ngr;</AC><AC>&cjs1171;</AC></A> associated with this residue by virtue of its increased hydrophilicity and capacity for hydration. In addition, in the presence of Zn2+ the intimate and preferential interaction of the metal ion with the Gla side chains may profoundly perturb the nu  of Gla. When considering the relatively high weight percentage of Gla in con-T, its contribution to the nu  value of the peptide is significant. The observation that an increase in molecular weight does not attend increased peptide concentration at high Zn2+ concentrations is additional compelling evidence in support of the nonassociative behavior of con-T.

The exotherms associated with peptide-metal binding correlate in the expected fashion with the degree of conformational change effected by metal binding. In the case of Mg2+ complexation to con-G, wherein the peptide undergoes a dramatic change in alpha -helicity upon Mg2+ binding (14), the total change in the Delta H for binding of 2 eq of ligand is -8.7 kcal/mol. The enthalpic change for con-T, which undergoes a distinctly smaller increase in alpha -helicity upon saturation of its metal ion sites with Mg2+, is -4.4 kcal/mol. Clearly, bond formation occurs to a greater extent in the con-G metal-induced transition than in the con-T system. It seems unlikely that electrostatic components contribute to these favorable Delta H values since metal ion binding to malonate, which occurs strictly through electrostatic interactions, possesses an unfavorable Delta H (Table I), leaving entropic forces to drive the binding event. The effective neutralization of clustered, destabilizing negative charge that occurs upon chelation of metal ions to the Gla head groups of the peptide may simply allow favorable intrapeptide hydrogen bonds to prevail.

The positive, albeit small, entropies that accompany the exothermic transitions are somewhat surprising considering the significant degree of order that metal ion binding imposes on these peptides. Factors with favorable entropies, such as release of the metal ion from its coordination sphere and release of structured water surrounding polar and non-polar residues, are likely contributing in the peptide systems. From the positive TDelta S values for metal complexation to malonate, it is probable that such contributions take entropic precedence, despite the absence of some elements of rotational bond freedom.

The positive Delta H values observed for occupancy by Zn2+ of the weak sites of both con-G and con-T correlate with a small degree of alpha -helix unwinding as implied from parallel CD-monitored titrations (Fig. 2). The relatively large positive entropy values linked to weak site Zn2+ binding for both peptides is also consistent with the notion of increased structural disorder. The nature of this destabilization in con-G is difficult to state with certainty because the results of simulated Mg2+ docking to con-G (15) point toward Gla7 as a third binding locus. Because all five Gla residues of con-G exist on the same face of the helix in the metal-loaded structure (15), inclusion of an additional divalent cation would be expected to impart increased stabilization to the peptide. We expect that ITC titrations with Zn2+ employing various Gla to Ala variants of con-G will address the nature of this third site and these studies are currently in progress. For con-T, we contend that the greater degree of alpha -helix unwinding that occurs upon Zn2+ loading can be explained in terms of the capping potential of Gla4. The NMR solution structures of both apo- and Ca2+-loaded con-T indicate that the side chain of Gla4 is an apparent capping residue in both structures. This contention is supported by CD data on the [Gla4Ala]con-T variant which displays compromised alpha -helical content in both its apo- and Ca2+-loaded forms (17). In the presence of high Zn2+ concentrations, we suggest that Gla4 is recruited from its side-chain to main-chain interaction role to share with Gla3 in the maintenance of a weak metal-binding site, thus destabilizing the alpha -helix in the N-terminal vicinity.

Finally, the current study raises important issues concerning the identity of the metal ion that predominates in the bioactive conformation of these peptides. It has been established in numerous studies with Gla-containing bone and blood proteins that Ca2+ mediates their functional aspects. In the case of prothrombin, factor IX, and protein C, which contain 10, 12, and 9 Gla residues, respectively, in their N-terminal domains, no major preference for Mg2+ versus Ca2+ has been noted from intrinsic fluorescence titration experiments (20-23). Only a slight (2.5-fold) preference is observed for Mg2+ over Ca2+ in terms of their Kd values for malonate (Table I). Despite the smaller ionic radius for Mg2+ (86 pm) as compared with Ca2+ (114 pm), this disparate charge to radius ratio fails to impart appreciable selectivity differences in these cases. However, for con-G, the Mg2+ tight site and weak site affinities in comparison with Ca2+ are 175- and 9-fold, greater, respectively. For con-T, Mg2+ displays a 42-fold increase in binding strength compared with Ca2+. Both con-G and con-T display distinctly lower Kd values for Zn2+ than for either Ca2+ or Mg2+. A pronounced (ca. 10,000-fold) greater affinity for Zn2+ over Ca2+ has recently been reported for a Gla-containing de novo designed peptide, which undergoes a metal-induced helical transition (24). These investigators maintain that the greater covalent character of electrostatic interactions involving Zn2+, ascribable to its filled 3d orbitals as well as higher charge density for this cation, are the major contributors to this preferential binding. However, the primacy of these factors is not borne out by our malonate data, which indicate that Zn2+ manifests only a modest (6.5-fold) increase in affinity for malonate compared with Ca2+. This would suggest that the binding sites of those peptides, which avidly bind Zn2+, are particularly amenable to this smaller metal ion simply because their induced binding site cation pockets are intrinsically small. Hence, the strength of metal-ion binding in these molecules may rely more on proper steric and geometric fit than on purely electrostatic considerations.

The consideration that Zn2+ is the dominant metal ion effector of conantokin function is supported by studies that indicate that transiently high local concentrations of Zn2+ (ca. 300 µM) can be attained in the synaptic cleft during excitatory synaptic activity (25, 26). Intracerebral injections of the conantokins into pre-2-week-old mice have been shown to induce a prolonged sleep-like state in these subjects at an estimated final brain concentration of 15 µM (13). Assuming a free Zn2+ concentration of 300 µM, this would result in essentially complete occupancy of the two tight Zn2+ sites in con-G and approximately 50% occupancy of the weak site. In the case of con-T, saturation of the high affinity Zn2+ site and 40% occupancy of the low affinity Zn2+ site would be attained at these peptide and ligand concentrations. At a cerebrospinal fluid Mg2+ concentration of 1.5 mM, tight and weak site loading of con-G would be 97% and 80%, respectively. For con-T, essentially 100% of the peptide molecules would be complexed with Mg2+. At a cerebrospinal fluid Ca2+ concentration of 1.5 mM, Ca2+ loading would be considerably less than Mg2+ and Zn2+, with 30% and 77% occupancy of the metal sites in con-G and con-T, respectively. We therefore propose that Mg2+, as well as Zn2+, are more plausible candidates for the role of in vivo metal ions for the conantokins. However, it should be noted that the high coordination numbers and irregular coordination geometry displayed by Ca2+ confer upon Ca2+-bound proteins the ability to bridge phospholipid membranes and/or other proteins. This particular feature of Ca2+-complexed biomolecules may mediate the functionality of the conantokins, despite the compromised binding capacity displayed by Ca2+ compared with Mg2+ and Zn2+. Dissecting the roles of these metal ions in the conantokins with respect to the NMDAR is complicated by their involvement in receptor function, namely the Ca2+ permeability associated with the receptor (see above), voltage-dependent Mg2+ blockage of the receptor (27, 28), and allosteric voltage-insensitive and -sensitive blocks by Zn2+ (29, 30). Elucidating the precise role of these metal ions in terms of conantokin antagonism of the receptor is essential for a full pharmacological profile of the these peptides and represents one of the major investigative challenges associated with future work in this area.

    FOOTNOTES

* This work was supported by Grant HL-19982 from the National Institutes of Health (to F. J. C.) and the Kleiderer-Pezold family endowed professorship (to F. J. C.).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.

Dagger To whom correspondence should be addressed. Tel.: 219-631-6456; Fax: 219-631-8017; E-mail: castellino.1{at}nd.edu.

1 The abbreviations used are: NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; con-G, conantokin-G; con-T, conantokin-T; Gla, gamma -carboxyglutamic acid; ITC, isothermal titration calorimetry; Mes, 4-morpholineethanesulfonic acid.

2 S. E. Warder, unpublished data.

3 M. Prorok, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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