Sequence Requirements for the N-Methyl-D-aspartate Receptor Antagonist Activity of Conantokin-R*

Tamas Blandl, Jaroslav Zajicek, Mary Prorok, and Francis J. CastellinoDagger

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

Received for publication, July 25, 2000, and in revised form, November 26, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Conantokin-R (con-R), a gamma -carboxyglutamate-containing 27-residue peptide, is a natural peptide inhibitor of the N-methyl-D-aspartate (NMDA) subtype glutamate receptor. Synthetic analogs of con-R were generated to evaluate the importance of the individual structural elements of this peptide in its NMDA receptor antagonist activity, measured by inhibition of the spermine-enhanced binding of the NMDA receptor-specific channel blocker, [3H]MK-801, to rat brain membranes. Progressive C-terminal truncations of the 27-residue peptide revealed stages of severe activity loss. These occurred at con-R[1-11] and con-R[1-7], corresponding to the deletions of Leu12-Pro27 and Met8-Pro27 respectively. A second set of analogs featured single Ala substitutions in the fully active con-R[1-17] fragment. The replacement of Met8 and Leu12 by Ala resulted in approximate 20- and 55-fold decreases of inhibitor potency, respectively. In addition to these two residues, the only other positions where a single Ala substitution led to substantial losses (from 11-fold to >1000-fold) of activity were those of the first five N-terminal amino acids. Based on the above findings, the binding epitope of con-R was localized to the N-terminal turn of the helix and other residues on one face along two subsequent turns. This contribution pattern of the side chains in activity closely resembles the results obtained with another member of this peptide family, conantokin-T. The secondary structure and metal ion binding properties of the con-R variants were also evaluated using circular dichroism spectroscopy. Divalent cation-dependent increases of alpha -helix content were observed in most analogs. However, analogs with replacement of Gla11 and Gla15, as well as truncation fragments shorter than 15 residues, lost the ability to be stabilized by metal ions. These results confirmed the location of the primary divalent cation binding locus at Gla11 and Gla15. Additional interactions were indicated by the reduced alpha -helix stability in the Ala analogs of Gla4, Lys7, and Arg14.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The conantokins are a family of neuroactive peptides isolated from venoms of various species of marine snails of the genus Conus. The three well characterized members of this group include con-G,1 from Conus geographus (1), con-R, from Conus radiatus (2), and con-T, from Conus tulipa (3). The conantokins posses a high content of Gla, a post-translational modification that has also been observed in several other Conus-derived venom peptides (4-9). The presence of Gla was first reported in vertebrate blood and bone proteins (10, 11), where it arises from the gamma -carboxylation of glutamic acid by a vitamin K-dependent process (12). The responsible enzyme, a vitamin K-dependent gamma -carboxylase, has been purified from bovine liver (13), and its cDNA has been cloned, sequenced, and expressed (14-16). A similar vitamin K-dependent enzyme activity was found in the microsomal fraction of C. radiatus venom ducts (17). The cloning of the cDNA for con-G allowed examination of the sequence requirements of the Conus-derived carboxylase, which showed substantial differences between the carboxylation signal sequence specificities of mammalian and Conus carboxylases, despite the similar cofactor requirements (18).

con-R contains 27 amino acids, exceeding by 10 and 6 residues the sizes of con-G and con-T, respectively. Other features that distinguish this peptide from con-G and con-T include a single amino acid insertion at Ala10 and a five-member disulfide loop near the C terminus. con-R also possesses a free alpha -carboxylate at its C terminus, unlike con-G and con-T, both of which feature amidated C termini. Several amino acids are rigidly conserved among the conantokins. These include the N-terminal Gly-Glu sequence, four Gla residues, and an Arg residue preceding the Gla closest to the C terminus of the peptides. In addition, Lys7, Met8, Leu12, and Lys19 are present in both con-R and con-T. It is also relevant that a recently obtained clone of the con-G cDNA coded for Val5, which is the same residue as in con-R (18).

In addition to the occurrence of the post-translationally modified Gla, an additional remarkable structural feature of the conantokins is their stable end-to-end alpha -helical secondary structure (19-21). In con-G, the alpha -helical conformation is dependent on the presence of divalent metal ions (22, 23), whereas con-T is alpha -helical in the absence of cations (24). The primary metal ion binding site has been localized to Gla10 and Gla14 in both con-G and con-T (23, 25, 26). Metal ion chelation by these residues leads to a strong stabilization of the alpha -helical fold. The examination of the secondary structure and cation binding properties of con-R revealed that residues 1-20 form a moderately stable alpha -helix, whereas the C-terminal disulfide loop segment is in a nonhelical conformation (27). Additionally, full metal ion binding capacity of this peptide was retained by a 17-residue N-terminal fragment (27).

The common receptor target of these peptides is the NMDAR (3, 28). Evidence in support of NMDAR antagonism by the conantokins was obtained by both electrophysiological (2, 29, 30) and pharmacological (31) approaches. Structure-activity relationships of con-G (32-34) and con-T (25, 35) have been examined, and critical residues were identified in each peptide. In this communication, a similar analysis of con-R is reported. Chemical modifications were utilized to evaluate the functional importance of structural elements in con-R. Several functionally important residues were identified, and this information was compared with the results that are available on the other two peptides.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peptide Synthesis, Purification, and Characterization-- Syntheses of con-R (NH2-GEgamma gamma V5AKMAA10gamma LARgamma 15NIAKG20CKVNC25YP-COOH) and its analogs were performed by automated solid phase peptide synthesis on an Applied Biosystems (Foster City, CA) model 433A peptide synthesizer (19). The Fmoc protection strategy was used with the following side chain protecting groups: tert-butyl ester for Gla, Glu, Tyr; trityl for Asn, Cys; 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl for Arg; and butyloxycarbonyl for Lys. The derivatized Gla was synthesized as described previously (36). con-R, containing a C-terminal free acid, was synthesized on a preloaded H-Pro-2-chlorotrityl resin support (NovaBiochem, San Diego, CA), as described (27). All of the truncated variants were synthesized as the peptide amides, using 5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)-valeric acid-p-methylbenzylhydrylamine resin support (PerSeptive Biosystems, Framingham, MA). Peptides were cleaved with 88% trifluoroacetic acid in the presence of the radical scavengers dithiothreitol and triisopropylsilane. Peptide purification was typically achieved by anion exchange chromatography on an fast protein liquid chromatography Bioscale DEAE20 (Bio-Rad) column, followed by desalting on a Sephadex G-15 column (1.5 × 100 cm) that was equilibrated and eluted with 0.1% NH4OH. The peptides were characterized by reverse-phase high performance liquid chromatography and matrix-assisted laser desorption ionization mass spectrometry, as described (19). Concentrations of peptide stock solutions were determined by quantitative amino acid analysis.

[3H]MK-801 Binding Assays-- Adult Harlan Sprague-Dawley rats were sacrificed by decapitation, and forebrains were removed and processed (31). 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 glutamate and glycine, with varying concentrations of peptide (25). The final concentrations of [3H]MK-801 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. Incubations were carried out for 2 h at room temperature. The assays were terminated by rapid filtration over Whatman GF/B filters pretreated with 0.03% polyethyleneimine, using a 24-well cell harvester (Brandel, Gaithersburg, MD). 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 were 60-300% over basal values, depending on the membrane preparation. 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.

In Vivo Mouse Injection Assays-- Two age groups of mice were injected with peptide or buffer intracranially as previously described (19). Mice (10-days and 30-days of age; n = 4) received 30 µl each of 150 µM or 450 µM peptide solution, respectively, in sterile saline (150 mM NaCl, 1 mM Ca2Cl, 10 mM Tris-Cl, pH 7.40). Behavioral observations were made in a blinded fashion for 8 h post-injection and at 24 h post-injection. con-T solutions at the same concentrations were used as positive controls.

Circular Dichroism Spectroscopy-- CD spectra of con-R analogs were collected at 25 °C in 10 mM sodium borate, 100 mM NaCl, pH 6.5, on an Aviv (Lakewood, NJ) model 200SF spectrometer using a 1-cm-pathlength cell. The peptide solutions and buffers used were treated with Chelex-100 resin prior to the experiments to ensure that they contained no multivalent metal ions. CD spectra of the peptides were acquired in 100% aqueous buffer with the above composition or with the addition of 20 mM CaCl2, 1.5 mM MgCl2, or 1.5 mM ZnCl2. The peptide concentration was 35 µM. The alpha -helical content was determined from mean residue ellipticities at 222 nm using the empirical relationship, falpha  = (-[Theta ]222-2340)/30,300 (37).

NMR Studies-- Peptide samples (~2 mM) were dissolved in a solution containing 10 mM sodium borate, 100 mM NaCl, 20 mM MgCl2, pH 6.5, 10% 2H2O. Two-dimensional total correlation spectroscopy (spin-lock interval, 60 ms) and nuclear Overhauser enhancement spectroscopy (mixing time, 150 ms) spectra were recorded at 5 °C at a spectral window of 6200 Hz on a three channel Varian UnityPlus spectrometer (599.89 MHz, 1H) employing standard pulse sequences (38, 39) using the hypercomplex phase sensitive method (39). The H2O signal was suppressed by presaturation. Relaxation delays of 1.6 and 2.8 s (including presaturation) were used in the total correlation spectroscopy and nuclear Overhauser enhancement spectroscopy experiments, respectively. Time domain data (t2 and t1) were recorded as 2048 × 256 complex matrices with 16 and 56 scans per t1 increment for the total correlation spectroscopy and nuclear Overhauser enhancement spectroscopy spectra, respectively.

Linear prediction to the 512 complex data points was applied to the fids in the t1 domain, and zero filling in both t2 and t1 domains was used (40). Final 4K × 2K complex time domain data sets were Fourier transformed with Gaussian apodization functions applied in both dimensions. A first order base-line correction was applied in F2 domain. All spectra were processed identically using Varian software that accompanied the spectrometer. The spectra were referenced relative to the signal of DSS (delta  = 0 ppm).

Isothermal Titration Calorimetry Analyses-- The binding isotherms of Mg2+ to the con-R variants were determined by 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 Na-Mes, 100 mM NaCl, pH 6.5. The exact protocols used as well as the methods for data analyses have been published previously.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To elucidate the detailed structure-activity relationships in the con-R sequence, a number of analogs were synthesized. Progressive C-terminal truncations were introduced in a series of variants. In addition, in a second set of con-R variants, individual side chain residues were replaced with Ala. Members of this latter group were 17 amino acids in length, based on the fully active con-R[1-17] sequence. The NMDAR antagonist activity of each analog was determined by the [3H]MK-801 binding assay (Fig. 1) and compared with the potency of wild-type con-R.



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Fig. 1.   Inhibition of the polyamine enhanced [3H]MK-801 binding by N-terminal fragments of con-R. The assay results are presented in a normalized form. The 100% value represents bound ligand in the presence of spermine (50 µM); 0% refers to basal binding (no added spermine). The parameters were fit by nonlinear regression using Microcal Origin software. , con-R[1-14]; black-square, con-R[1-12]; open circle , con-R[1-8]. These experiments were performed in triplicate.

The results with the C-terminal truncation series are shown in Table I. The potencies of con-R[1-20], con-R[1-17], and con-R[1-15] are comparable with the full-length peptide. Truncations through residues resulted in potency decreases of less than 1 order of magnitude. Sequences between con-R[1-11] and con-R[1-8] suffered substantial (120-150-fold) decreases of activity. con-R[1-7] and con-R[1-6] displayed IC50 values that were >100 µM, which represented a diminution of potency of at least 1000-fold.


                              
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Table I
Effects of C-terminal truncations on the NMDAR antagonist and conformational properties of con-R

The data obtained from substitutions of Ala at individual side chains are summarized in Table II. These analogs were generated as 17-amino acid N-terminal fragments, because con-R[1-17] displayed the same potency as full-length con-R. Alterations to any of the N-terminal four amino acids decreased the potency of the peptides. These same residues were similarly important to the activities of con-G (34). Glu2 and Gla4 are required for activity, because their removal results in a minimum of a 1000-fold decrease in peptide potency. Ala substitutions of Gly1 and Gla3 reduced activity by 11- and 160-fold, respectively. Three additional analogs, con-R17[V5A], con-R17[M8A], and con-R17[L12A] displayed 20-60-fold reductions in activity. Alterations at Lys7 and Arg14 did not result in significant effects on activity. The simultaneous replacement of Gla11 and Gla15 had a small adverse effect, viz. a 9- or 4-fold potency decrease for the double-Ala or double-Glu analog, respectively. The sequence positions of these residues were altered by deleting Ala10 of con-R[1-17] in con-R16[Delta A10]. This shifted the tight metal ion site at Gla11 and Gla15 to identical locations of these residues because they are found in con-G and con-T (Gla10 and Gla14). The potency of this analog decreased by 28-fold.


                              
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Table II
Effects of the side chains on the NMDAR antagonist and conformational properties of con-R

The in vivo effects of con-R variations were also tested by intracranial injections of mice with peptide solutions. The 15-µg dose of full-length con-R used for the 10-day of age group resulted in a sleep-like inactive state within 15 min post-injection, which lasted 6-8 h. Similar effects were seen with the positive control group, which was injected with the same concentration of con-T. To a second, 30-day of age group, a quantity of 45 µg of con-R was administered. These mice also exhibited a sleep-like condition for 2-6 h, but unlike the age 10-day group, these animals awakened periodically and showed considerable disorientation, impaired movement, e.g., dragging hind legs, and vocalized repeatedly. At 6-8 h post-injection, members of this group showed some signs of hyperactivity, such as constant walking and cage climbing. The 30-day-old positive control group injected with con-T underwent a shorter inactive sleep period (<1 h) and exhibited disoriented/hyperactive behavior 2-8 h after injection. Animals from both age groups appeared normal in behavior after 24 h. The mouse intracranial injection experiments were also performed with con-R[1-17]. The 10-day-old mice receiving the same dose of con-R[1-17] as wild-type con-R and con-T slept for 2.5-4 h, a significantly shorter period than the 6-8 h observed for the other two peptides. The 30-day-old animals exhibited an impaired state for up to 2 h, after which normal activity was resumed, with no signs of disorientation or hyperactivity. Again, in this case, although similar syndromes were observed as in the groups receiving an equivalent dose of con-R or con-T, the duration of the effects was shorter.

The in vivo results with con-R17 were compared with point variants of this peptide. Three peptides were chosen, viz. con-R17[E2A], con-R17[K7A], and con-R17[M8A]. con-R17[E2A], which did not display NMDAR activity in the MK-801 binding assay (Table I), also did not show any in vivo activity after intracranial injections in mice. Similarly, con-R17[K7A], which showed full potency in the in vitro MK-801 binding assay, induced a sleep-like state, similar to wild-type con-R[1-17] in the in vivo mouse assay. Lastly, con-R17[M8A], which showed an approximate 50-fold reduction in activity compared with con-R[1-17] in the MK-801 binding assay, displayed a qualitatively intermediate effect in the mouse in vivo assay. This variant induced a very sluggish behavioral state in mice, but sleep did not occur.

The overall conformation of each analog was examined by CD spectroscopy in the absence of multivalent cations and in the presence of Ca2+ or Mg2+, respectively. These secondary structure observations revealed that con-R-derived peptides of 15 amino acids or longer displayed the cation-dependent increase of alpha -helical character. Truncation analogs shorter than this length had progressively less alpha -helical content in the apo-form and a slight decrease of alpha -helicity upon loading with high concentrations of either Ca2+ or Mg2+. Fig. 2 illustrates the CD spectra of con-R[1-15] and con-R[1-14], in both the absence of metal ions and in the presence of Mg2+. The above described differences in cation-dependent conformational transitions are clearly observed in these two analogs. Additionally, the alpha -helicity of the apo-peptide is higher for the shorter analog. This can be explained by the deletion of Gla15 and, therefore, the elimination of charge repulsion between Gla11 and Gla15 in the absence of complex forming cations. In the Ala replacement series, some peptides showed altered conformational properties. Analogs of Gla4, Lys7, and Arg14 had lower alpha -helicity, both in the apo- and the metal ion-bound states. Similarly, the Lys7 right-arrow Ala modification resulted in reduced overall alpha -helicity. The double replacement of Gla11 and Gla15 increased the apo alpha -helix content, with Ala being more alpha -helix promoting than Glu. However, metal ion binding resulted in a decrease rather than an increase of alpha -helical conformation for these analogs.



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Fig. 2.   CD spectra of con-R N-terminal fragments. , apo-con-R[1-15]; triangle , con-R[1-15]/Mg2+; black-diamond , apo-con-R[1-14]; , con- R[1-14]/Mg2+.

To further investigate whether possible conformational alterations influenced the activity of these variant peptides, 1H NMR analyses were performed on three variants of con-R[1-17] in their Mg2+-bound forms, viz. con-R17[E2A], con-R17[K7A], and con-R17[M8A], that showed a nearly full loss of NMDAR inhibitory activity, no loss in activity, and partial loss of activity, respectively. The alpha H chemical shift index values of these three variants, relative to those of wild-type con-R[1-17], are illustrated in Fig. 3. Only minor relative changes are noted for these peptides up to residue Gla11, which for con-R17[E2A] and con-R17[K7A] displays relatively large differences from wild-type con-R[1-17]. However, this residue shows anomalous conformational behavior in con-G (23), con-R (26), and con-T (24) and appears to be greatly influenced by metal binding. In any case, the largest changes at this location, which are also in parallel, are in the two variants, con-R17[E2A] and con-R17[K7A]. Because opposite effects were observed on the activity of these variants, this alteration does not appear to be meaningful in terms of NMDAR inhibitory activity. To determine whether the changes in chemical shift index indices at Gla11 for these latter two variants indeed did not reflect macroscopic alterations in metal binding to the variant peptides, isothermal titration calorimetry analysis of the binding of Mg2+ to both the Glu2 right-arrow Ala and Lys7 right-arrow Ala variants were conducted. The Kd values obtained were 14 and 11 µM, respectively (data not shown). These values are comparable with the Kd of 18 µM previously obtained for Mg2+/con-R17 binding (27), and indicate that the Mg2+ binding affinities are similar for the three peptides. Similarly, other deviations in the chemical shift index values at residues that are near the C terminus of the peptide occur to the same degree with these same two peptides. This also indicates that such changes are not important with regard to the NMDAR activity of these peptides.



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Fig. 3.   Residue-specific alpha H chemical shift index (CSI) differences of con-R variant 17-mer peptides versus con-R[1-17]. The deviations of the alpha H chemical shifts of Mg2+-bound con-R residues from their random coil values of the variant peptides are subtracted from similar values of wild-type con-R[1-17]. Gray bars, con-R17[M8A]; black bars, con-R17[E2A]; white bars, con-R17[K7A]. The values indicated are in ppm.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

con-R is the most recently isolated member of the conantokin class of neuroactive conopeptides, and it is perhaps the most interesting example of these peptides, based on the modest amount of information that is available on this peptide at present. con-R is at least 4-fold more potent in the in vitro [3H]MK-801 binding NMDAR assay than either con-G or con-T. It is also significant that this peptide shows some selectivity to NMDAR containing the NR2B and NR2A subunits (2). In addition to the subunit selectivity and perhaps as a consequence, con-R also shows high potency, as well as a favorable protective index against behavioral toxicity in anticonvulsant models when compared with NMDA inhibitors with different mechanisms, e.g. ifenprodil and MK-801 (2).

The primary structure of con-R also possesses features that are unique in its class, such as the C-terminal extension that contains a single disulfide loop. A conformational analysis of full-length con-R, along with two variants lacking the disulfide region, revealed that this latter segment is a major factor in governing the conformational behavior of the peptide (27). Although the receptor antagonist and metal ion binding properties of the analogs lacking the disulfide bridge were comparable with the full-length peptide, the secondary structure of the variants was shown to consist of a more stable alpha -helix. This is due to the elimination of alpha -helix breaker sequence elements, such as Gly20 and the (i, i+4) disulfide linkage. In full-length con-R, this structure perpetuates the secondary structure disruption to the N-terminal region, which otherwise possesses a higher intrinsic alpha -helix propensity (27).

In the present study, a number of con-R analogs were generated to examine their NMDAR antagonist structure-activity relationships. con-R[1-15] possesses wild-type like potency in the [3H]MK-801 binding assay. Therefore, it can be assumed that this segment of con-R contains all of the structural elements needed for receptor inhibition. The progressive truncation series showed two major drops in activity, which occurred at con-R[1-11] and con-R[1-7]. These boundaries correspond to the elimination of Leu12 and Met8. The replacement of these two side chains by Ala also resulted in major decreases of potency.

This contribution of the side chains has some similarities to the results obtained with con-G and con-T variants. The N-terminal turn of the alpha -helix and residues on one face along two subsequent turns were found to be important contributors to the receptor antagonist activity of the latter peptides. In con-R, replacement of Gly1, Glu2, Gla3, or Gla4 by Ala resulted in a 10-fold to >1000-fold diminution in their activities. This segment of the sequence is 100% conserved among the three conantokins. Analyses of the structure-activity relationships of both con-G (32-34) and con-T (25, 35) showed that the N-terminal four amino acids are major contributors to the NMDAR inhibitory activity of both peptides. These four residues are equally crucial in con-R. Three additional analogs, Val5 right-arrow Ala, Met8 right-arrow Ala, and Leu12 right-arrow Ala, suffered 20-60-fold reductions in inhibitor potencies. Some of these side chains probably contribute the indicated binding energies to ligand-receptor interactions. Others may be involved in effecting a productive binding by inducing a conformational change in the receptor that is necessary for inhibition.

Residue 5 is functionally important in both con-G and con-T, although not interchangeable (34, 35). Whereas the Val5 differs from both of the other conantokins, a Val residue was encoded in a cDNA clone of con-G (18). Although this side chain is less bulky than either the Tyr or Leu found in the peptide sequences of con-T or con-G, respectively, it still has sufficient volume to be involved in putative van der Waal's-type interactions with the NMDAR protein surface. The replacement of Val5 with Ala resulted in a 60-fold diminution in potency. Ala6 is in a homologous position to Gln6 of con-G and con-T. Although this side chain is conserved between the latter two peptides, its replacement by Ala had no effect on the activity of either. Therefore, Ala6 of con-R probably occupies a noncritical position in the sequence.

Lys7 is identical to the amino acid found in con-T at this position. In contrast, con-G has a Gla in the analogous position. The Lys7 right-arrow Gla modification of con-T resulted in a peptide with diminished apo alpha -helicity (26). This showed that the radically different side chains in the two homologous sequences confer significantly disparate conformational behavior. This residue does not seem to be involved in interactions, because both Ala and Lys variants retain full activity in con-G, as does the con-R17[K7A] analog of con-R. Therefore, it is expected that, as in con-T, Lys7 in con-R serves to stabilize the alpha -helical conformation of the peptide. Met8 of con-R is again identical to this residue of con-T. An Ala replacement of the latter caused a 16-fold activity decrease.2 In contrast, the corresponding analog of con-R had an approximate 57-fold potency loss. Despite this difference, Met8 seems to play an important role in the function of both. Residue 9 is not conserved among the three conantokins. Gln9 is required for the NMDAR inhibitory activity of con-G, whereas Leu9 of con-T can be replaced by Ala without loss of potency. Ala9 in con-R most likely occupies a nonimportant position in the sequence.

Ala10 constitutes a single amino acid insertion, considering that Gla11 and Gla15 correspond to Gla10 and Gla14 in the other two conantokins. Gla11 can be replaced by Ala in all three of the conantokins without consequence to NMDAR antagonism. However, the tight metal ion binding site is compromised after this alteration. Ile12 in con-G was found to be required for the NMDAR antagonist activity of that peptide (34). Leu12 of con-T also contributes in this regard, although to a lesser extent.2 Leu12 is similarly involved in the inhibition of the NMDAR by con-R, despite the altered side chain and the metal ion binding site compared with the other two peptides. Because of the insertion at Ala10, Ala13 has no obvious counterpart in the other two sequences and is probably not important for activity.

Arg14 is analogous to Arg13 found in both con-G and con-T. An Ala replacement of this residue had a minor effect on NMDAR inhibition in con-G and was without consequence in con-T. Likewise, no adverse effect of that modification resulted in con-R. This conserved side chain plays a role in the secondary structure stabilization in each molecule, because Ala substitutions decrease the alpha -helix content of each. Because the fragment con-R[1-15] displays full receptor antagonist potency, further modifications beyond residue 15 were not made. Upstream of Gla15, con-R has no homology with con-G and con-T, except for Lys19, also found in con-T.

The deletion variant con-R16[Delta A10] displayed a 28-fold decrease in inhibitory activity. The modification in this peptide caused the metal ion binding site formed by Gla11 and Gla15 to shift upstream by one position. This is identical to the positions of these residues in both con-G and con-T. The metal ion-induced conformational change in this variant is similar to that in con-R[1-17], which indicates that the cation site is still functional. The likely reason for the reduced activity is the shifting of Leu12, an important contributor to activity, to sequence position 11. The position change of this side chain results in a spatial displacement and also a change in orientation, viz. a 100° turn in relation to the alpha -helix register, which may prevent a positive interaction between the peptide and the receptor.

There are several Ala residues in the sequence of con-R, and their locations are of interest in light of the Ala scan results of the three peptides. Ala6, Ala9, Ala10, and Ala13 appear to be positioned where the different residues found in one or both of the other conantokin sequences could be substituted by Ala without functional consequences.

The structure-activity relationships of con-R are more similar to those of con-T than to con-G (Fig. 4). In fact, there is just one amino acid of importance in con-R and con-T that is not identical. Even this residue occupies the same position, viz. sequence number 5, which is a Val in con-R and a Tyr in con-T. The shortest segment of con-R with measurable activity was con-R[1-8]. In con-G, residue segment (1-13) is the shortest fragment that displays detectable activity (34). con-T is more similar to con-R in this respect as well, because its residue (1-8) fragment is still active, but the (1-6) fragment is inactive.2 These functional similarities are underscored by the extensive primary structure homology that exists between con-T and the first 20 residues of con-R. There is 50% identity between these two sequences, after accounting for the insertion at Ala10 in con-R.



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Fig. 4.   Contributions of side chains in the NMDAR antagonist activity of con-G, con-T, and con-R. Amino acids are color coded according to importance in NMDAR inhibition. Red, when replaced by Ala, the potency of the resulting peptide decreases by at least 100-fold. Green, when replaced by Ala, the potency of the resulting peptide decreases by 10-60-fold. Black, when replaced by Ala, the potency of the resulting peptide does not substantially change.

The results of the in vivo mouse injection experiments reveal distinct differences for con-R[1-17] compared with full-length con-R. Both the 10-day-old and the age 30-day-old groups exhibited comparable syndromes for the two peptides, nevertheless the effects lasted for 2-4 h with con-R[1-17] and 6-8 h with con-R. Considering that the in vitro NMDAR antagonist potencies of the two compounds are the same, their different bioavailability/turnover is the likely reason for these differences. Although the C-terminal disulfide loop segment is without apparent secondary structure (27), this motif may convey properties that increase the half-life of the peptide in vivo.

In light of structural studies of con-G and con-T (20, 21, 23-25), the secondary structure results for con-R analogs show that a commonality exists. The divalent cation-dependent increases of alpha -helix content were observed in all of the analogs that contained both Gla11 and Gla15. The analogs with double replacement of these residues, as well as the truncation fragments that were shorter than 15 residues, did not display this phenomenon. This is the direct consequence of removing the helix stabilizing (i, i+4) metal ion binding site. Additional interactions were indicated by the helix stability changes in the Ala analogs of Lys7 and Arg14. Both of these peptides had a decreased secondary structure. These positive residues are placed (i, i+3) from Gla residues, which can provide electrostatic stabilization between those side chains. The Ala analog of Gla4 was also destabilized significantly. The NMR-derived solution structure of con-T (24) indicated that the side chain of Gla4 is involved in N-terminal capping interactions with both the terminal amine and the backbone amide nitrogens in the first residues of the alpha -helix. It is very likely that Gla4 in con-R fulfills a similar function.

In conclusion, the results presented herein on the structure-activity relationships of con-R provided additional insight into the NMDAR antagonism of this peptide. The data also allowed comparisons to be made between con-R and the other two well characterized conantokins. The three peptides have similar mechanisms of action at the NMDAR, although subtle differences are also noted. con-R is more closely related to con-T than to con-G, with regard to both the secondary structure properties and the receptor antagonism.


    FOOTNOTES

* This work was supported by Grant HL-19982 from the National Institutes of Health (to F. J. C.), a Kleiderer-Pezold family endowed professorship (to F. J. C.), an American Heart Association Scientist Development Grant (to M. P.), and a predoctoral fellowship from the American Heart Association, Indiana Affiliate (to T. B.).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: Dept. of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556. Tel.: 219-631-6456; Fax: 219-631-8017; E-mail: castellino.1@nd.edu.

Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M006648200

2 S. E. Warder, T. Blandl, R. C. Klein, F. J. Castellino, and M. Porok, J. Neurochem., in press.


    ABBREVIATIONS

The abbreviations used are: con-G, conantokin-G; con-R, conantokin-R; con-T, conantokin-T; Gla, gamma -carboxyglutamic acid; Fmoc, 9-fluorenylmethyloxycarbonyl; NMDAR, N-methyl-D-aspartate receptor; Mes, 2-(N-morpholino)ethanesulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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