The Roles of Individual gamma -Carboxyglutamate Residues in the Solution Structure and Cation-dependent Properties of Conantokin-T*

Scott E. WarderDagger , Mary ProrokDagger , Zhigang Chen§, Leping Li, Yi Zhu§, Lee G. Pedersenpar , Feng Ni§, and Francis J. CastellinoDagger **

From the Dagger  Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, the § Montreal Joint Center for Structural Biology, Biotechnology Research Institute, Biomolecular NMR Laboratory, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada, the  National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, and the par  Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The solution structure of the Ca2+-loaded conantokin-T (con-T), a gamma -carboxyglutamate (Gla)-containing 21-residue peptide (NH2-G1Egamma gamma Y5QKMLgamma 10NLRgamma A15EVKKN20A-CONH2,gamma  = Gla), has been elucidated by use of distance geometry calculations with experimental distances derived from two-dimensional 1H NMR spectroscopy. An end-to-end alpha -helix was the dominant conformation in solution, similar to that of apo-con-T, except that reorientation of several side chains occurred in the Ca2+-coordinated complex. The most notable examples of this were those of Gla10 and Gla14, which were more optimally positioned for complexation with Ca2+. In addition to the stabilization offered to the alpha -helix by Ca2+ binding, hydrophobic clustering of the side chains of Tyr5, Met8, Leu9, and Leu12, and ionic interactions between Lys7 and Gla3/Gla10 and between Arg13 and Gla14, along with hydrogen bonding between Gln6 and Gla10, were among the side chain interactions likely playing a significant role in maintenance of the alpha -helical conformation. Docking of Ca2+ in the con-T structure was accomplished using genetic algorithm-molecular dynamics simulation approaches. The results showed that one Ca2+ ion is most likely coordinated by four side chain oxygen atoms, two each from Gla10 and Gla14. Another bound Ca2+ ion has as its donor sites three oxygen atoms, two from Gla3 and one from Gln6. To examine the functional roles of the individual Gla residues, a series of variant peptides have been synthesized with Ala substituted for each Gla residue, and several properties of the resulting variants have been examined. The data obtained demonstrated the importance of Gla10 and Gla14 in stabilizing binding of the highest affinity Ca2+ site and in governing the conformational change induced by Ca2+. The critical nature of Gla3 and Gla4 in inhibition of the spermine-induced potentiation of the binding of MK-801 to open ion channels of the N-methyl-D-aspartate receptor was established, as well as the role of Gla4 in stabilizing the apo-con-T alpha -helical conformation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

con-T1 is a neuroactive peptide found in the venom of Conus tulipa (1). It is among a vast number of small peptides, typically 10-30 amino acids in length, employed by these and other species of cone snails to immobilize their prey and their predators (2, 3). The targets of this array of peptides are neuroreceptors and skeletal muscle receptors, and the remarkable selectivity shown by this general class of peptides has also encouraged their use as laboratory reagents to block specific classes of these receptors (4, 5). Specifically, a general class of Conus peptides, the conotoxins, which contain a relatively high number of disulfide bonds, interact with nicotinic acetylcholine receptors (alpha -conotoxins), voltage-sensitive Na+ channels (omega -conotoxins), and voltage-sensitive Ca2+ channels (µ-conotoxins) (6). Conantokins, such as the 17-amino acid residue peptide con-G (7) and the homologous 21-residue con-T (1), are Gla-containing peptides without disulfide bonds. They have been shown to function as noncompetitive inhibitors of the enhancement by spermine and spermidine of Ca2+ flow into neurons, by targeted action on glutamate/glycine receptors of the NMDA subclass (8-11).

Certain structural features of these small peptides are relevant to their functions. con-G and con-T contain five and four residues of Gla/mol of peptide, respectively (1, 7). Because of this feature, both peptides interact with divalent cations that are important to the functions of neuronal cells, such as Ca2+ and Mg2+ (3, 12), as well as other metal ions (12, 13). The binding of Ca2+ and similar cations induces a large conformational change in con-G (12) from an essentially random structure to an alpha -helix. On the other hand, con-T, although it also interacts with these cations, does not undergo as dramatic a conformational change, because apo-con-T is already highly organized in an alpha -helical conformation (12, 14).

The conantokins possess the potential to serve as agents that inhibit the flow of Ca2+ into neurons via the NMDA receptor route and thus eliminate the harmful effects of entry of this cation into neuronal cells. However, these peptides cannot readily be directly employed as pharmaceutical agents for this purpose in humans because they do not cross the blood-brain barrier. Thus, as part of a rational drug design program, elucidation of the structure-function relationships of these peptides is essential. A recent emphasis on this topic is witnessed by publications on elaboration of the NMR-derived, three-dimensional structures of apo-con-G (13, 15), Ca2+·con-G (13), and apo-con-T (13, 14, 16). Most of these investigations have focused on backbone conformations, but one has rigorously defined side chain orientations of apo con-T (14). In the present phase of our efforts in this area, we have defined the three-dimensional conformation of the Ca2+·con-T complex and have employed mutant con-T peptides to attempt to understand the role of individual Gla residues in defining its metal binding properties and its bioactivity. The results of this study are summarized in this report.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Synthesis of Nalpha -Fmoc-(gamma ,gamma '-di-O-tBu)-L-Gla-OH-- The derivatized Gla was synthesized by previously described methodology (17), except for the resolution of the D- and L-isomers of Nalpha -Z-(gamma ,gamma '-di-O-tBu)-Gla-OMe, which was accomplished by the method involving enzymatic hydrolysis of the L-isomer using papain (18).

Peptide Synthesis, Purification, and Characterization-- The peptides were synthesized on a 0.1 mmol scale on a PAL resin support (PerSeptive Biosystems, Framingham, MA) using an Applied Biosystems (Foster City, CA) model 433A peptide synthesizer, as described previously (12). Purification of con-T and mutant con-T peptides was accomplished by fast protein liquid chromatography on a Bioscale DEAE-20 column (Bio-Rad) equilibrated with 10 mM NaBO3, pH 8.0. A 500-ml linear gradient of NaCl, from 10 mM NaBO3, pH 8.0 (start solution), to 10 mM NaBO3, 500 mM NaCl, pH 8.0 (limit solution), was employed. The target material was pooled, lyophilized, and then desalted on a Sephadex G-15 (Pharmacia Biotech Inc.) column that was equilibrated and eluted with 0.1% NH4OH. The peptides were characterized by reverse-phase high performance liquid chromatography and delayed extraction-matrix-assisted laser desorption ionization-time of flight mass spectrometry, as described in an earlier communication (12).

Calcium Binding-- The Ca2+ binding isotherms for each peptide were determined by potentiometry at 25 °C using a semi-micro Ca2+-selective electrode (Orion, Los Angeles, CA). A fused double-walled titration vessel with inlet and outlet ports was attached to a circulating water bath for precise temperature control in these titrations. Chelex-100 (Bio-Rad)-treated peptides (1-2 mM peptide in 10 mM NaBO3, 100 mM NaCl, pH 6.5) were titrated with CaCl2. Data analyses were conducted as described previously (19).

Circular Dichroism-- CD spectra were recorded between 190 and 260 nm on an Aviv model 62DS spectrometer. Peptides were dissolved in 10 mM NaBO3, 100 mM NaCl, pH 6.5, to a final concentration of 35 µM. A 1-cm-path-length cell was employed. Spectra representing the average of five scans were collected at a 1.0-nm bandwidth at 1.0-nm intervals. The alpha -helical contents at various Ca2+ concentrations were calculated according to a previously published method (20).

Two-dimensional 1H NMR-- The peptide was dissolved in 10 mM NaBO3, 100 mM NaCl, pH 6.5, to a final concentration of approximately 2 mM. A volume of 50 µl of 2H2O was added to 450 µl of the peptide solution to provide the NMR deuterium lock signal. The internal reference was 4,4-dimethyl-4-silapentane-L-sulfonate. Dilute HCl to was added to adjust the pH of the sample to 6.5 to minimize the loss of NH signals that would occur at higher pH values. One- and two-dimensional NMR experiments were carried out on a Bruker AMX-500 spectrometer. The data were collected, processed, and analyzed as described previously for structure determination of apo-con-T (14). Briefly, sequence-specific assignments of the proton resonances for con-T in the presence of Ca2+ were achieved by combining the procedures of spin system identification using TOCSY and DQF-COSY, followed by sequential assignments through NOE connectivities (21). Assigned NOE cross-peaks were characterized as strong, medium, or weak as determined from the number of contours and converted to distance upper bounds of 2.7, 3.7, and 5.0 Å, respectively. Spectral processing was carried out using FELIX (Biosym Technologies, San Diego, CA) and an in-house program, nmrDSP, on Silicon Graphics workstations. Spectral contrast-enhancement methods were applied to resolve severely overlapped proton resonances (22, 23). The Sybyl software package (Tripos, Inc., St. Louis, MO) was used for spectral visualization.

Peptide conformations, free from steric overlaps and consistent with the NMR data, were generated by DG calculations using the fixed bond lengths and bond angles provided in the ECEPP/3 data base (24). All side chain-side chain or side chain-main chain medium range (i + 2 and i + 4) constraints were set to an upper bound of 5 Å and a lower bound of 2.0 Å. In the absence of metal coordination sites, we chose not to employ energy minimization as a structural refinement because of the potential bias that this could introduce in the side chain orientations. Because the major backbone NOE contacts in apo-con-T and in Ca2+·con-T were essentially the same, our approach to modeling the Ca2+·con-T NMR-derived structure was to start with the 10 energy-minimized convergent structures of apo-con-T (14) as templates for the DG calculations, with the view that the generated structures that satisfied the distance constraints would be the best approximations of metal-loaded, energy-minimized structures. Initial structures that were generated from well resolved NOEs allowed several degenerate resonances to be assigned. Remaining NOEs were inputted as ambiguous constraints that take into account all of the possible interactions in an unbiased manner.

Identification of the Positions of the Metal Ions in con-T-- Docking of the metal ions in con-T and further energy minimization-based refinement of the metal-bound structure was accomplished by the genetic algorithm-molecular dynamics simulation approach described previously (25). The initial coordinates for con-T were those determined by NMR in this study for the Ca2+·con-T complex. The genetic algorithm was employed to determine the initial positions of the Ca2+ ions by searching through the O-O midpoints that were within 6 Å of each other, using all oxygen atoms in the midpoint calculation. A total of 150 midpoints was found. The lowest Amber (26) energy structure, verified also by a systematic search, was subjected to a Particle Mesh Ewald (27) molecular dynamics simulation. For this simulation, the peptide was solvated in a 9.0-Å box of TIP3P water. The H2O, Na+, and Ca2+ were then energy-minimized at constant volume as described (25). Next, a Particle Mesh Ewald molecular dynamics simulation was performed on Na+, Ca2+, and H2O for 100 ps, followed by energy minimization. Another Particle Mesh Ewald molecular dynamics simulation was then performed for 150 ps on side chain residues, the ions, and H2O for 150 ps, with fixed backbone, followed by data collection over 300 ps.

[3H]MK-801 Binding Assays-- Adult Sprague-Dawley rats were sacrificed by decapitation after being administered isoflurane vapor. Forebrains were removed and processed as described previously (9). Control experiments showed that use of this anesthetic did not affect the con-T inhibition isotherms.

Inhibition assays were performed in triplicate in a total volume of 500 µl in 5 mM Na+-Hepes, 4.5 mM Tris-Cl, pH 7.4, in the nominal absence of glycine and glutamate, with varying concentrations of peptide. The final concentrations of [3H]MK-801 (New England Nuclear, 23.9 Ci/mmol) and spermine were 5 nM and 50 µM, respectively. Binding was initiated by the addition of 300 µl of membrane suspension containing 100-200 µg of protein, and incubation was carried out at room temperature for 2 h. Assays were terminated by rapid filtration over Whatman GF/B filter strips (presoaked in assay buffer containing 0.03% polyethyleneimine) using a Brandel (Gaithersburg, MD) 24-well cell harvester. Two 5-ml washes with cold buffer followed. Basal [3H]MK-801 binding was defined as the amount of radioligand bound in the nominal absence of spermine. Observed enhancements in [3H]MK-801 binding at 50 µM spermine versus basal levels varied among different membrane preparations and were in the range of 60-300%. However, these differences in basal [3H]MK-801 binding had no bearing on the absolute IC50 value for con-T in different experiments. Nonspecific binding was determined in the presence of 50 µM MK-801 and represented 5-10% of total ligand bound in the presence of 50 µM spermine.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

NMR of Ca2+·con-T-- In a previous study, it was demonstrated that the CD spectrum of con-T underwent a small change to a more alpha -helical conformation consequent to the binding of Ca2+ to this peptide (12). In the present study, the nature of this conformational alteration was further explored, with the ultimate goal of elucidating the Ca2+-bound structure of con-T. The one-dimensional 1H NMR spectrum of Ca2+·con-T showed well resolved resonances, similar to those observed earlier for apo-con-T (14). This allowed ready assignment of all of its proton resonances using a combination of TOCSY, DFQ-COSY, and NOESY experiments. An example of the NOESY spectrum of the alpha NH-alpha CH fingerprint region of Ca2+·con-T illustrating some of the sequential assignments is provided in Fig. 1. The complete proton resonance assignments of Ca2+-loaded con-T, obtained by use of this combination of methods, are listed in Table I.


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Fig. 1.   alpha NH-alpha CH fingerprint region of the NOESY spectrum (mixing time, 250 ms) of con-T. The alpha NH resonances of Glu2 and Gla3 are present at 9.16 and 9.12 ppm, respectively, and are not shown in the figure. Connectivities are only illustrated for (alpha n, i + 1) interactions. The buffer was 10 mM NaBO3, 100 mM NaCl, 40 mM Ca2+, 10% 2H2O, pH 6.5, at 5 °C. The final con-T concentration was 2 mM.

                              
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Table I
Proton resonance assignments of conantokin-T in the presence of Ca2+ at 5 °C
The chemical shifts were determined in 10 mM NaBO3, 100 mM NaCl, pH 6.5, at a peptide concentration of 2 mM and a Ca2+ concentration of 40 mM. The chemical shifts cited are relative to 4,4-dimethyl-4-silapentane-L-sulfonate, which was set to 0 ppm.

Several of the proton resonances of con-T in the presence of Ca2+ were shifted from those previously obtained for apo-con-T, and this is revealing in terms of possible differences in the structures of these peptides. A graphical summary of the chemical shift differences between Ca2+·con-T and apo-con-T of the alpha NH and alpha CH backbone protons for each of the residues of this peptide are illustrated in Fig. 2. Also present in this figure are representative differences in the terminal (farthest sequentially removed from the beta -carbons) nonexchangeable side chain protons (e.g. epsilon CH2 of Lys, delta CH3 of Leu, delta CH2 of Arg, and so forth) between these two peptides. The largest chemical shift changes observed in the backbone protons occur in the residue 10-18 region for the amide protons. Smaller differences are present in the alpha CH protons, except for that of Gla10, which undergoes a particularly large alteration. Of the terminal side chain protons, Gln6 and Arg13 show the most dramatic shifts.


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Fig. 2.   Proton chemical shift (alpha CH, alpha NH, and side chain) differences between apo-con-T and Ca2+-loaded con-T. The chemical shifts plotted were obtained by subtraction of the apo-con-T spectrum from that of Ca2+·con-T for each of the proton groups illustrated. The particular side chain protons chosen were those that were at terminal and nonexchangeable locations. The buffer was 10 mM NaBO3, 100 mM NaCl, pH 6.5, in the absence of Ca2+ or in the presence of 40 mM Ca2+ at 5 °C. The final con-T concentration was 2 mM. square  (black), alpha NH protons; diamond  (red), alpha CH protons; open circle  (green), side chain protons.

Differences between the observed alpha CH chemical shifts and their random coil values provide valuable secondary structural information (28). Specifically, upfield shifts of these proton spins of >= 0.1 ppm from their random coil values indicate the presence of helical structure, if these values occur uninterrupted over four or more consecutive residues. The random coil values of each con-T residue were determined experimentally by obtaining the spectrum in 6 M urea. The differences between the alpha CH proton chemical shifts for Ca2+·con-T from their same values in 6 M urea are illustrated in Fig. 3. Also plotted in Fig. 3, for comparison, are the values similarly obtained for apo-con-T (14). In the case of Ca2+·con-T, as with apo-con-T, residues 2-21 possess relative chemical shifts >0.1 ppm, indicating a high content of alpha -helical structure. This is especially evident in the region of residues 5-17, where chemical shift differences >0.6 ppm are found, indicating the presence of a significant population of alpha -helices. The alpha CH proton chemical shift of Gla10, however, is opposite in sign from that expected for a residue in an alpha -helical conformation. This was also observed in a previous work (13), in which it was attributed to the unknown random coil chemical shift value of the alpha CH proton of Gla10. However, our data, which show that this chemical shift is not anomalous, allow this interpretation to be abandoned. Because neither CD nor NOESY data demonstrate any serious disruption in the continuum of alpha -helix of this peptide, we attribute the observation of the positive chemical shift of the alpha CH proton of Gla10 to the previously unknown consequence of direct Ca2+ coordination to this residue. This is also a very likely explanation for the slightly smaller relative alpha CH proton chemical shifts for several other Ca2+·con-T residues as compared with those of apo-con-T (Fig. 3).


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Fig. 3.   Chemical shift index for the alpha CH-proton resonances of Ca2+·con-T. The chemical shift differences were calculated by subtraction of the values obtained in 6 M urea from those displayed in a buffer containing 10 mM NaBO3, 100 mM NaCl, 10% 2H2O, pH 6.5, ± 40 mM Ca2+ at 5 °C. The final con-T concentration was 2 mM. Black bars, apo-con-T; white bars, Ca2+·con-T.

Structure of Ca2+·con-T-- The structure of con-T in complex with Ca2+ was primarily derived from DG calculations utilizing NOE connectivities present in this peptide. A summary of the NOE connectivities for intraresidue sequential (i + 1) and medium (i + 2, i + 3, and i + 4) NOEs used in the DG calculations are shown in Fig. 4. The high alpha -helical content of this peptide is readily noted from the strong backbone alpha NH-alpha NH NOEs (nn(i + 1)), the stronger (nn(i + 1)) cross-peaks as compared with those of alpha CH-alpha NH (alpha n(i + 1)) connectivities, and the presence of many (alpha n (i + 3)) and (alpha n(i + 4)) NOEs. In addition, three-bond alpha CH-alpha NH coupling constants (3Jalpha N) were approximately 5 Hz for all residues with well resolved alpha CH-alpha NH correlation peaks; these data are also strongly suggestive of the presence of alpha -helical structure.


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Fig. 4.   The sequential and interresidue backbone NOEs observed for Ca2+·con-T. The thickness of the bars are a qualitative measure of the strengths of the NOE cross-peaks. An asterisk (*) indicates that the assignment of the cross-peak was ambiguous due to resonance overlaps. The buffer was 10 mM NaBO3, 100 mM NaCl, pH 6.5, at 5 °C. The final con-T concentration was 2 mM.

In addition to alpha n(i + 3) NOE contacts observed for most pairs of residues, backbone-side chain NOE connectivities were found between many proton pairs (Table II), such as those present between Lys18-alpha CH and Ala21-beta CH3, between Gln6-alpha CH and both Leu9-gamma CH and Leu9-delta CH3, and between Leu9-alpha CH and Leu12-delta CH3, among many others. Furthermore, side chain-side chain NOE connectivities were found between a number of residues, including Gln6-gamma CH2 and Leu9-delta CH3, Arg13-eta NH and Val17-gamma CH3, and Glu16-gamma CH2 and Asn20-delta CH2. All of this information strongly suggests a high structural order for con-T in complex with Ca2+, consistent with an alpha -helical nature.

                              
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Table II
Distance constraints used for Ca2+ · con-T structure calculations

The Ca2+-loaded structure of con-T was generated from 160 distance constraints, including 4 intraresidue, 70 sequential, 86 of medium range, and 5 group NOEs (Table II). The convergent structures from the apo-con-T study (14) were used as starting templates for the DG calculations to generate the 10 metal-loaded structures with the lowest distance violations (Fig. 5) with their backbone (alpha NH-alpha CH) atoms superimposed. This cluster of structures possess RMSD values to the mean structure of 1.0 Å for backbone atoms and 1.5 Å for all heavy atoms. The average structure is provided in Fig. 6. Analysis of this structure indicates that additional stabilization of the alpha -helix can originate from an extensive electrostatic network on one face of the helix, involving Gla3, Lys7, Gla10, Arg13, Gla14, and Lys18, and an appropriately spaced hydrophobic cluster of side chains, comprising Tyr5-Met8-Leu9-Leu12, on the opposite face. This situation also occurs in the apo-con-T structure (14).


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Fig. 5.   Stereo view of a cluster of 10 low energy conformations of Ca2+·con-T derived from distance geometry calculations. Individual structures have been superimposed on their backbones. The Gla residues are labeled and colored red.


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Fig. 6.   Stereo view of the average conformation of Ca2+·con-T from Fig. 5. This conformation was generated from the average of the 10 conformations shown in Fig. 5.

The structure of Ca2+·con-T is compared with the apo-con-T (14) in Fig. 7. The structures are very similar in backbone conformations, and changes were observed in only a few of the backbone residues, viz, Gln6, Gla10, Arg13, and Gla14. These changes are noted in NOE data of Table II and from similar data for apo-con-T (14). For example, in apo-con-T, there are no side chain-side chain NOE connectivities between Gla10 and Gla14 (14), whereas in Ca2+·con-T, NOEs are seen between Gla10HG and Gla14HG and between Gla10HG and Gla14HN.


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Fig. 7.   Comparison of the average apo-con-T and Ca2+·con-T conformations. The backbones of the peptides have been superimposed. The structure of apo-con-T was taken from Ref. 14.

Locating the Ca2+ Ions in con-T-- With the establishment of the Ca2+-loaded con-T structure, it became of interest to attempt to locate Ca2+ ions in their binding site(s). For this purpose, a genetic algorithm-molecular dynamics simulation procedure was employed as described (25), using a 2Ca2+·con-T binding model with 1 Na+ ion as the counterion to balance charge. An average structure was generated from the resulting coordinates and is illustrated in Fig. 8.


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Fig. 8.   The Ca2+-bound refined structure of con-T. A total of two Ca2+ ions (black) were docked in this structure using the genetic algorithm, beginning with the NMR-derived coordinates of con-T loaded with Ca2+. The structure was then refined by molecular dynamics simulation. In this illustration, the side chains (from the beta -carbons) of all amino acid side chains of con-T are illustrated, and certain key residues are labeled. All side chain carbon atoms are colored orange, nitrogen atoms are blue, oxygen atoms are red, and the sulfur atom of Met8 is green. A, space-filling representation. B, relationships between side chain residues implicated in binding to Ca2+ and the Ca2+ ions. This view also includes Gla4, which does not appear to be involved in Ca2+ binding. The orientation is as in A. The representations in A and B are not on the same scale.

This structure shows Ca2+ ions located at one site, containing the side chains of Gla10 and Gla14, with another at Gla3 and Gln6. Gla4 is not suitably located to provide such a donor site for Ca2+. In this model, two oxygen atoms from both Gla10 and Gla14 coordinate one Ca2+ ion, and two oxygen atoms from the gamma -carboxylate of Gla3 and one from the side chain carbonyl atom from Gln6 are donor groups for binding of the other Ca2+. During the course of these calculations, energy minimization was conducted to derive the final structure with Ca2+ ions in their appropriate locations. In comparing the two models, viz, the NMR-derived structure (Fig. 7) and that further refined through Ca2+ docking and energy minimizations (Fig. 8), it is clear that no major differences in backbone or side chain conformations exit. The most notable changes occur in the ring orientation of Tyr5 and the side chain positioning of Lys7, along with other minor differences. None of these, however, alter the face of the helix on which these residues reside, and none change the conclusions regarding the alpha -helix stabilizing forces. On the whole, the differences observed in the peptide structures revealed by the two models are surprisingly small. These are not further elaborated upon herein because the uncertainties in deriving the models themselves from these disparate approaches are likely as large as the small differences in positions of flexible side chains between them. Instead, it is stressed that the two models are very similar and provide a very good set of models with which to explore structure-function relationships of this peptide.

con-T Variants-- To test the importance of side chains found to be critical in stabilization of Ca2+ binding to con-T, several variant peptides were constructed, and their properties were assessed. This group consisted of changes of all Gla residues and one other residue, Gln6, that was identified as a possible contributor to stabilization of Ca2+ in the molecular dynamics simulation. In all cases, Ala was the amino acid that was substituted, because Ala would favor alpha -helix stability (29) but would not provide side chain atoms that could coordinate Ca2+.

The effects of these substitutions on the stability of the apo-con-T helix and that of the Ca2+-loaded peptide, as well as the quantitative ability of Ca2+ (C50 values) to induce the alpha -helical conformation in the variant peptides, are summarized in Table III. Representative CD titrations with Ca2+ of con-T, con-T[Gla3-Ala] and con-T[Gla14-Ala] are provided in Fig. 9. The only variation in this series that resulted in significant destabilization of the apo-con-T alpha -helix was that of Gla4-Ala, where approximately one-half of the alpha -helical content was observed as compared with the wild-type peptide (Table III). However, the C50 values characterizing the Ca2+-induced conformational transition in this peptide were similar for all peptides, except for those of con-T[Gla10-Ala], con-T[Gla14-Ala], and con-T[Gla10-Ala/Gla14-Ala], where substantially higher concentrations of Ca2+ were required to induce their Ca2+-dependent conformations. Furthermore, in these latter cases, the conformational change appeared to be very small and of marginal significance, and it was opposite in character to that induced in the other mutant peptides, the final extents of which were similar to those of con-T and con-T[Gla3-Ala] (Fig. 9).

                              
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Table III
Calcium affinities and bioactivities of con-T and mutant con-T peptides


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Fig. 9.   Effects of Ca2+ on the alpha -helical contents of apo-con-T, con-T[Gla3Ala], and con-T[Gla14Ala]. The molar ellipticities at 222 nm are plotted against the free Ca2+ concentrations. The buffer was 10 mM NaBO3, 100 mM NaCl, pH 6.5, at 25 °C.

The observation that the alpha -helicity of con-T[Gla10-Ala], con-T[Gla14-Ala], and con-T[Gla10-Ala/Gla14-Ala] decreases as the peptide is titrated with Ca2+ can be due to two possible factors: 1) electrostatic screening of favorable side chain-side chain or backbone-side chain interactions, and/or 2) the coordination of metal to individual Gla residues that leads to a specific disruption of critical charge-charge interactions. These possibilities were examined by investigating the ionic strength dependence on alpha -helicity for the peptides con-T[Gla10-Ala], con-T[Gla14-Ala], and con-T[Gla10-Ala/Gla14-Ala] using NaCl. The results revealed that nonspecific ionic strength changes alone cannot account for the decrease in alpha -helicity for these peptides. Therefore, the Ca2+-induced decreases are primarily due to specific coordination with the remaining Gla residues.

The binding isotherms of Ca2+ to each of the con-T mutant peptides were established by Ca2+-specific electrode titrations. Examples of the titration data obtained are illustrated in Fig. 10. In most cases, the binding data were fit to a model with two independent sites, one strong site with a Kd within the range of 0.2-0.5 mM and one weaker site of Kd approximately 10-fold higher. Exceptions have been observed in the cases of con-T[Gla10-Ala], con-T[Gla14-Ala], and con-T[Gla10-Ala/Gla14-Ala], wherein the data could be fit several different models of very weak Ca2+ binding. For these peptides, the binding characteristics were determined through Michaelis-Menten fits, which showed that the amount of free Ca2+ required to reduce the binding to 50% was approximately 1-4 mM. These results indicate that only the strong Ca2+ sites have been eliminated by mutations at Gla10 and Gla14 and/or that these two mutations led to peptides that required different Ca2+ binding models to satisfy the experimental data.


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Fig. 10.   Binding isotherms of Ca2+ to con-T mutants. Modified Scatchard plots are used to display the data. black-square, wild-type con-T; open circle , con-T[Gla4-Ala]. The experimental line is fit to wild-type con-T using an independent two-site model with Kd values 0.25 mM and 2.5 mM.

Lastly, the effects of the mutations in con-T on the ability of the resulting peptides to inhibit MK-801 binding to open rat brain NMDA receptor channels in the presence of exogenous spermine has been examined. The neuronal membranes were washed and thus contained only low levels of glutamate and glycine, but these ligands were present at sufficient concentrations to lead to significantly increased ion channel opening upon addition of spermine. The amount of [3]MK-801 binding to the membranes, as a function of the concentration of con-T-derived peptides, is shown for con-T and for con-T[Gla10-Ala/Gla14-Ala] in Fig. 11. Values of the IC50 for peptide inhibition have been calculated from the concentration midpoints of the differences between the basal level of MK-801 binding to the peptide and that induced by spermine at a peptide concentration of zero. The values obtained are summarized in Table III. Very large increases in the IC50 value were seen for con-T[Gla3-Ala] and con-T[Gla4-Ala], and a more modest increase was observed for con-T[Gla10-Ala] and con-T[Gla10-Ala/Gla14-Ala]. Thus, major binding determinants for the peptide to its site on the membrane are provided by Gla3 and Gla4 of con-T.


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Fig. 11.   Effects of con-T and con-T[Gla14Ala] on the binding of [3H]MK-801 to washed rat neuronal cells. The concentration of spermine used was held constant at 50 µM under conditions of endogenous levels of glutamate and glycine in washed membranes. [3H]MK-801 binding was then measured as a function of the concentrations of con-T (bullet ) or con-T[Gla10-Ala/Gla14-Ala] (open circle ). The basal binding level of [3H]MK-801 under these conditions in the absence of spermine is approximately 500 cpm.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

A variety of divalent cations, including Ca2+ and Mg2+, alter the CD properties of con-T (12) in a manner suggesting that the relative alpha -helical content of the peptide is increased. The Gla residues of this peptide, which serve as Ca2+ binding loci when present in blood coagulation proteins and in proteins and peptides present in bone, are the strongest candidate residues for metal ion coordination in con-T. Titrations by ion-specific electrode methods reveal that con-T has one strong Ca2+ binding site, and possibly a second weaker site of interaction (12). A previous study has detailed the backbone and side chain conformations of con-T and concluded that a significant population of molecules were end-to-end alpha -helices (14). Despite this, changes occurred in both the NMR and in the CD spectra of con-T as a result of addition of Ca2+ that indicated that a higher content of alpha -helix resulted from this interaction (Figs. 2-4 and 9 and Table II). As shown in Fig. 2, these changes did not appear to be in backbone conformations but rather in side chain orientations, and such types of changes should not have such dramatic influence on the alpha -helical content. It is most likely that the population of molecules in the alpha -helical conformation increases in the presence of Ca2+, and the side chain reorientations reflect the optimization of Ca2+ binding sites.

The NMR-derived solution conformation of Ca2+-loaded con-T has been determined by methods similar to those published for the apo-con-T structure (14). However, in this case, only DG calculations of backbone-backbone NOEs and of backbone-side chain and side chain-side chain NOE constraints were employed to calculate the structure at this stage. Because the exact position of the bound Ca2+ ion(s) was unknown, full energy minimization was not performed in these structure calculations. This is because the exclusion of the bound ions from the energy minimizations would have resulted in nonoptimal definition of metal-induced conformation. However, the major differences in side chain conformation that result from Ca2+ binding to con-T are centered at Gln6, Gla10, Arg13, and Gla14. Such changes are consistent with the binding of Ca2+ to Gla10 and Gla14. In apo-con-T, the guanidino group of Arg13 appears to structurally mimic the role of Ca2+ in the metal-loaded form, reducing electrostatic repulsion between the Gla10 and Gla14 side chains. This model is predicted from our previous structural work (14) and is consistent with the observation that con-T[Arg13-Ala] reduces the apo-alpha -helicity by >50% (data not shown). Therefore, binding of a metal ion to the tight site concomitant with displacement of the Arg13 side chain is consistent with a significant chemical shift in this residue.

Gla3 and that of Gln6 are in spatial proximity in the metal-peptide complex, either as the result of hydrogen bonding or because they are directly coordinated with the metal. The chemical shift changes of the Gla6-epsilon NH2 side chain protons in the Ca2+·con-T complex can be perhaps explained by the amide carbonyl serving as a ligand for Ca2+. In addition, disruption of the Gln6/Gla10 apo-con-T hydrogen bond may also contribute to the chemical shift changes.

The roles of the individual Gla residues of con-T as donors for binding of Ca2+ and their abilities to function in promotion of the Ca2+-induced conformational change in con-T have been directly assessed through study of variant con-T-based peptides. The Ca2+ binding isotherm displays one clear, tight binding site of approximately 0.25 mM (12), and additional evidence suggests that at least one additional, weaker site is present, with a Kd >10-fold higher than the strong site (Table III). Alteration of Gla3 or Gln6 to Ala residues preserves the binding at the tight Ca2+ site, but diminishes Ca2+ binding at the weaker site (Table III). This result is in concert with the model of the Ca2+-docked con-T (Fig. 8), which shows that these two residues are capable of coordinating Ca2+. Alteration of Gla4 to Ala did not affect Ca2+ binding, again in agreement with this model, which predicts that Gla4 would not coordinate Ca2+. Both this model and that derived from NMR measurements (Fig. 7) place Gla4 on the opposite side of the face of the alpha -helix that appears to house the Ca2+ binding loci. On the other hand, alteration of Gla10 and/or Gla14 to an Ala residue resulted in Ca2+ binding that was so weak that a unique binding model could not be fit. Therefore, for these peptides, an average binding constant was estimated from a Michaelis-Menten fit of the binding data (Table III). The value obtained was at least 15-fold larger than that displayed for the strong Ca2+ binding site of native con-T. Thus, we propose that Gla10 and Gla14 serve as coordination sites for the tightly bound cation.

The only mutant that affected the stability of the apo-con-T alpha -helix was con-T[Gla4-Ala], which reduced the population of alpha -helical molecules to one-half of their values in wild-type con-T and the other mutants tested (Table III). Thus, despite the change of Gla4 to a residue that in itself should not be disruptive to the alpha -helical character of this peptide, destabilization of the alpha -helix nonetheless occurred. This conclusion is in agreement with our analysis of the apo-con-T structure, in which Gla4 was predicted to be an important capping residue at the amino terminus and to interact favorably with the alpha -helix macrodipole (14). However, the population of con-T[Gla4-Ala] molecules in the alpha -helical conformation substantially increased upon addition of Ca2+, as was the case for wild-type con-T and most of the other mutants. The notable exceptions to this were con-T[Gla10-Ala], con-T[Gla14-Ala], and con-T[Gla10-Ala/Gla14-Ala], wherein addition of Ca2+ did not increase the population of molecules in the alpha -helical conformation and, in fact, somewhat decreased this distribution. More quantitative comparisons of the C50 values for Ca2+ required to induce overall alpha -helix transitions were similar for wild-type con-T, con-T[Gla3-Ala], con-T[Gla4-Ala], and con-T[Gln6-Ala]. On the other hand, the same values for alpha -helix destabilization in con-T[Gla10-Ala], con-T[Gla14-Ala], and con-T[Gla10-Ala/Gla14-Ala] were approximately 10-fold higher. Thus, the Ca2+ site localized at Gla10 and Gla14 is essential for stabilization of the alpha -helix in con-T.

Finally, the effects of these mutations on the bioactivity of con-T have been examined through the effects of these peptides on the spermine-induced [3H]MK-801 binding to washed rat neuronal membranes. The efficacy data of Table III are in general agreement with previous results using con-G mutants (11) in that mutations at Gla3 and Gla4 greatly reduced the ability of these peptides to inhibit the spermine-induced potentiation of MK-801 binding to open membrane channels. Mutations at Gla10 and Gla14 in con-T, as was the case with con-G, did not affect this property to the same extent, although the effect of the Gla10 mutation was significant (Table III). Thus, it appears that the nature of the amino acid residues at the amino terminus of these peptides may be a critical property in its bioactivity, with Ca2+ binding at other loci perhaps stabilizing critical conformations. This could explain the reasons for the tight homology of Gly1-Glu-Gla-Gla at the amino terminus of the currently known conantokin structures. Alternatively, the cation-induced alpha -helices might simply be a reflection that such structures can be formed from other peptide-ligand interactions, such as might occur when these peptides interact with groups at or on the membrane surface.

In conclusion, we have provided experimentally based models of the solution structure of Ca2+-loaded con-T and identified some groups most likely involved in coordination of the metal cations. The major Ca2+ binding site is coordinated by side chain carboxylate oxygen atoms from Gla10 and Gla14, and Ca2+ binding at this location serves to dramatically stabilize the alpha -helical conformation of con-T. The Gla3 gamma -carboxylate group in cooperation with the Gln6 side chain carbonyl moiety likely coordinate a second weakly bound Ca2+ site. Gla4 does not appear to function in this regard. The major roles of Gla3 and Gla4 appear to reside in their abilities to serve as major determinants of the bioactivity of con-T on NMDA receptor ion channels. These results have provided major insights into structure-function relationships of this neuroactive naturally occurring polypeptide and will serve as a focal point in design of new con-T-based active molecules.

    FOOTNOTES

* This work was supported by Grants HL-19982 (to F. J. C.) and HL-06530 (to L. G. P.) from the National Institutes of Health, a grant from the National Research Council of Canada (Publication No. 41403), Grant MT-12566 from the Medical Research Council of Canada (to F. N.), a grant from Ciba-Geigy Canada Ltd. (to F. N.), the Kleiderer-Pezold family endowed professorship (to F. J. C.), and postdoctoral (to M. P.) and predoctoral (to S. E. W.) fellowships from the American Heart Association, Indiana Affiliate.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

1 The abbreviations used are: con-T, conantokin-T; con-G, conantokin-G; NMDA, N-methyl-D-aspartic acid; CD, circular dichroism; DG, distance geometry; NOE, nuclear Overhauser effect.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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