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INTRODUCTION |
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 (
-conotoxins), voltage-sensitive Na+ channels
(
-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
-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
-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.
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EXPERIMENTAL PROCEDURES |
Synthesis of
N
-Fmoc-(
,
'-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
N
-Z-(
,
'-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
-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.
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RESULTS |
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
-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
NH-
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.
NH- CH fingerprint region of the NOESY
spectrum (mixing time, 250 ms) of con-T. The 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 ( 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.
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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
NH and
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
-carbons) nonexchangeable side chain protons (e.g.
CH2 of Lys,
CH3 of Leu,
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
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 ( CH, 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. (black), NH protons; (red), CH protons; (green),
side chain protons.
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Differences between the observed
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
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
-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
-helices. The
CH proton chemical shift
of Gla10, however, is opposite in sign from that expected
for a residue in an
-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
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
-helix of this peptide, we attribute the observation of the
positive chemical shift of the
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
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 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.
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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
-helical content of this
peptide is readily noted from the strong backbone
NH-
NH NOEs
(nn(i + 1)), the stronger
(nn(i + 1)) cross-peaks as compared with those of
CH-
NH (
n(i + 1)) connectivities, and the
presence of many (
n (i + 3)) and
(
n(i + 4)) NOEs. In addition, three-bond
CH-
NH coupling constants (3J
N) were
approximately 5 Hz for all residues with well resolved
CH-
NH
correlation peaks; these data are also strongly suggestive of the
presence of
-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.
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In addition to
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-
CH and Ala21-
CH3,
between Gln6-
CH and both Leu9-
CH and
Leu9-
CH3, and between Leu9-
CH
and Leu12-
CH3, among many others.
Furthermore, side chain-side chain NOE connectivities were found
between a number of residues, including Gln6-
CH2 and
Leu9-
CH3, Arg13-
NH and
Val17-
CH3, and
Glu16-
CH2 and
Asn20-
CH2. All of this information strongly
suggests a high structural order for con-T in complex with
Ca2+, consistent with an
-helical nature.
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 (
NH-
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
-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.
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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.
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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 -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.
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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
-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
-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
-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
-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
-helix was that of Gla4-Ala, where
approximately one-half of the
-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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Fig. 9.
Effects of Ca2+ on the
-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.
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The observation that the
-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
-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
-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. , wild-type con-T; , 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.
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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 ( ) or
con-T[Gla10-Ala/Gla14-Ala] ( ). The basal
binding level of [3H]MK-801 under these conditions in the
absence of spermine is approximately 500 cpm.
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DISCUSSION |
A variety of divalent cations, including Ca2+
and Mg2+, alter the CD properties of con-T (12) in a manner
suggesting that the relative
-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
-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
-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
-helical content. It is most likely that the population of molecules
in the
-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-
-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-
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
-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
-helix
was con-T[Gla4-Ala], which reduced the population of
-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
-helical character of this peptide, destabilization of the
-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
-helix macrodipole (14). However, the
population of con-T[Gla4-Ala] molecules in the
-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
-helical conformation and, in fact, somewhat decreased this
distribution. More quantitative comparisons of the C50
values for Ca2+ required to induce overall
-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
-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
-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
-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
-helical conformation of con-T. The Gla3
-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.