(Received for publication, June 18, 1996, and in revised form, September 5, 1996)
From The Centre for Drug Design and Development, The University of Queensland, Brisbane, Qld 4072, Australia
Conantokin-G and conantokin-T are two paralytic
polypeptide toxins originally isolated from the venom of the
fish-hunting cone snails of the genus Conus. Conantokin-G
and conantokin-T are the only naturally occurring peptidic compounds
which possess N-methyl-D-aspartate receptor
antagonist activity, produced by a selective non-competitive antagonism
of polyamine responses. They are also structurally unusual in that they
contain a disproportionately large number of acid labile
post-translational -carboxyglutamic acid (Gla) residues. Although no
precise structural information has previously been published for these
peptides, early spectroscopic measurements have indicated that both
conantokin-G and conantokin-T form
-helical structures, although
there is some debate whether the presence of calcium ions is required
for these peptides to adopt this fold. We now report a detailed
structural study of synthetic conantokin-G and conantokin-T in a range
of solution conditions using CD and 1H NMR spectroscopy.
The three-dimensional structures of conantokin-T and conantokin-G were
calculated from 1H NMR-derived distance and dihedral
restraints. Both conantokins were found to contain a mixture of
-
and 310 helix, that give rise to curved and straight
helical conformers. Conantokin-G requires the presence of divalent
cations (Zn2+, Ca2+, Cu2+, or
Mg2+) to form a stable
-helix, while conantokin-T adopts
a stable
-helical structure in aqueous conditions, in the presence
or absence of divalent cations (Zn2+, Ca2+,
Cu2+, or Mg2+).
Conantokin-G (con-G)1 and conantokin-T
(con-T) are highly conserved polypeptides originally isolated from the
venoms of piscivorous cone snails Conus geographus (1) and
Conus tulipa (2), respectively. These polypeptides are just
two components in the very complex venom that these snails have
developed for rapidly immobilizing their fast moving prey (3). Evidence
indicates that both con-G and con-T possess
N-methyl-D-aspartate receptor (NMDA receptor) antagonist activity, produced by a selective non-competitive antagonism of polyamine enhancement of NMDA receptor agonist activity (4-7). To
date, these are the only naturally occurring peptides known to have
this property. Con-G and con-T are also structurally unusual, in that
they lack the disulfide motifs commonly found in conotoxin peptides and
instead contain a high proportion of the base-stable but acid-labile
-carboxyglutamic acid (Gla) residue (8, 9). Gla residues were
originally identified in the vitamin K-dependent blood-clotting factors including prothrombin. Subsequently they were
found in other vertebrate proteins such as osteocalcin (calcium-binding protein) (10, 11) and have been implicated in Ca2+ binding
(12, 13). The discovery of Gla residues in several Conus
peptides has established that this post-translational modification has
a much wider phylogenetic distribution than previously thought (14).
Although no precise structural information has yet been published for
these peptides using either high field NMR or x-ray techniques,
previous CD spectroscopic measurements have indicated that con-G and
con-T are folded in a stable -helical conformation (7, 9). However,
there is some dispute over whether these structures are stabilized by
Ca2+ (4, 7) and furthermore, whether Ca2+ is
essential to the biological function of the conantokin peptides (7). To
investigate this intriguing problem we have synthesized con-G and con-T
using Boc chemistry and investigated their solution structures in
detail by CD and 1H NMR spectroscopy. The effects of the
solution environment including pH, monovalent cation (K+),
divalent cations (Zn2+, Ca2+, Cu2+,
and Mg2+), EDTA, and TFE on the structures of con-G and
con-T were monitored by CD spectroscopy. Both peptides were also
examined under several conditions by 1H NMR spectroscopy
and their three-dimensional structures were calculated based on the
NMR-derived restraints.
Chemicals and Reagents
All coded Boc-L-amino acids and reagents used during chain assembly were peptide synthesis grade supplied by Auspep Ptd. Ltd. (Parkville, Victoria). Acetonitrile (HPLC grade) was purchased from Laboratory Supply Pty. Ltd. (Coorparoo, Queensland). Deionized water was used throughout and prepared by a Milli-Q water purification system (Millipore, Waters). Screw-cap glass peptide synthesis reaction vessels (10 ml) with a sintered glass filter were obtained from Embel Scientific Glassware (Queensland). An all-Kel-F apparatus (Peptide Institute, Osaka, Japan) was used for HF cleavage. Argon, helium, and nitrogen were all ultrapure grade (CIG, Australia).
Peptide Synthesis
The sequences of the two conantokins are: conantokin-G,
Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2; and conantokin-T,
Gly-Glu-Gla-Gla-Tyr-Gln-Lys-Met-Leu-Gla-Asn-Leu-Arg-Gla-Ala-Glu-Val-Lys-Lys-Asn-Ala-NH2. The linear conantokins were assembled manually using solid
phase methodology with Boc chemistry (15). Both peptides were assembled on p-methylbenzhydrylamine-resin·HCl on a 0.2 mmol scale.
The following side chain protected amino acids were employed:
Asn(xanthyl), Lys(2-chlorobenzyloxycarbonyl), Glu(-cyclohexyl),
Arg(p-toluenesulfonyl), Ser(benzyl), Gln(xanthyl), and
Gla(
-cyclohexyl)2. A 4-fold excess of Boc amino acids
was used based on the original substitution of
p-methylbenzhydrylamine-resin·HCl.
Boc-L-Gla(
-cyclohexyl)2-OH·DCHA was coupled
with a 1.25 mol excess. Boc-Gla(
-cyclohexyl)2 was synthesized as described previously (8). All amino acids were activated
using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (0.5 M in dimethylformamide) and
N,N-diisopropylethylamine and coupled for 10 min,
except for Boc-Gla(
-cyclohexyl)2 which was coupled for
60 min. Boc protecting groups were removed using 100% trifluoroacetic
acid and the peptides were cleaved from the resin using HF.
The peptide resin (415 mg, 94.3 mmol) was treated
with HF (9 ml) in the presence of p-cresol (1 ml) at
5 °C for 2 h. Crude peptide was precipitated with cold ether,
washed with cold ether, then extracted into 20% acetic acid and
lyophilized to yield 201 mg (86%) of crude con-G. The crude peptide
was purified on a conventional reverse-phase Vydac C18
silica preparative column using a linear gradient (0-80% B) over 80 min at a flow of 8 ml/min. Solvent A was 0.1% aqueous trifluoroacetic
acid, solvent B was 90% acetonitrile/water containing 0.09%
trifluoroacetic acid. Sample absorbance was recorded at 230 nm. Highly
pure fractions were collected and lyophilized and pure con-G was
further lyophilized 3 times from deionized water. ES-MS: [M + H]+ 2264.6 (calculated 2265.2). Amino acid analysis
after hydrolysis in 6 M HCl at 105 °C for 24 h: Asx
2.43 (2), Glx 8.28 (8), Ser 0.90 (1), Gly 0.91 (1), Arg 1.00 (1), Ile
0.73 (1), Leu 1.83 (2), Lys 0.93 (1).
This peptide was prepared in a similar fashion to con-G to yield 126 mg (51%). ES-MS: [M + H]+ 2684.0 (calculated 2684.9). Amino acid analysis after hydrolysis in 6 M HCl at 105 °C for 24 h: Ala 2.01 (2), Asx 2.21 (2), Glx 7.20 (7), Gly 0.98 (1), Val 1.05 (1), Met 0.93 (1), Tyr 1.07 (1), Arg 1.03 (1), Leu 1.90 (2), Lys 3.10 (3).
Mass Spectrometry
Mass spectra were acquired on a PE-SCIEX API III triple quadrupole mass spectrometer equipped with an ionspray atmospheric pressure ionization source. Mass spectra were typically acquired in 5-15 min on an Apple Macintosh IIfx computer and processed using the software package MacSpec (Sciex, Toronto, Canada). Interpretation of the spectra was aided by MacBiospec (Sciex, Toronto, Canada).
Peptide Authentication
Venom was isolated from the dissected venom ducts of individual
C. geographus and C. tulipa
collected on the Great Barrier Reef, Australia (16). Soluble venom
components were extracted with 30% acetonitrile/water acidified with
0.1% trifluoroacetic acid, freeze-dried, and stored at 20 °C.
Venom was redissolved in 5% acetonitrile/water acidified with 0.1%
trifluoroacetic acid at
1 mg/ml prior to HPLC purification.
Comparative HPLC analyses were performed on the native peptides, the
synthetic peptides, and a mixture of native and synthetic peptides
(1:1). HPLC analyses were performed on an analytical HPLC system (ABI
140B dual syringe pump gradient HPLC system) using a Vydac
C18 5-µm column (2.1 × 250 mm). Chromatograms were
achieved using a linear gradient of 0-80% B over 160 min at a flow
rate of 1 ml/min. Solvent A was 0.1% aqueous trifluoroacetic acid,
solvent B was 90% acetonitrile/water acidified with 0.09%
trifluoroacetic acid. Synthetic samples of con-G and con-T both
co-eluted with the native samples (data not shown) which confirmed that
the synthetic and native materials were identical.
Cation Removal
Preparative HPLC was performed immediately on a calcium loaded con-G sample (CaCl2, 6.5 mM) following CD analysis. CD analysis of the repurified peptide showed no helix formation to be present. This is indicated by the lack of minima at 208 and 222 nm (data not shown). Thus HPLC chromatography under acidic conditions is sufficient to remove Ca2+ bound to con-G.
CD Studies
CD spectra were obtained using a Jasco spectrometer (J-710) with
a 0.1-cm path length cuvette at 20 °C. Both peptides were dissolved
in a 10 mM phosphate buffer (pH 7.4) at a 75 µM concentration, for all experiments. The secondary
structure was estimated by analysis of the spectrum between 190 and 250 nm. Mean residue ellipticity []MR is expressed in
degrees-square centimeter per decamole ((deg·cm2)/dmol).
Experimental estimates of helical contents were determined from the
[
]222 measurements. An estimate of helical content was determined using an equation developed by Chakrabartty et
al. (17) (
40,000 (1-2.5/n); n = number of residues), where
40,000 and 0 (deg·cm2)/dmol
are the values for 100% and 0%
-helix, respectively. This equation
is used only as a qualitative estimate of peptide helicity.
NMR Spectroscopy
All NMR spectra were recorded on a Bruker ARX 500 spectrometer
equipped with a z-gradient unit. Peptide concentrations were 2 mM. Spectra of con-G were obtained in 95%
H2O, 5% D2O with no CaCl2 (pH
5.5), 1:1 (pH 5.5) and 5:1 (pH 3.5 and 5.5) ratios of CaCl2
to peptide, and in 33% TFE. Spectra of con-T were obtained in 95%
H2O, 5% D2O with no CaCl2 (pH 5.5)
and a 5:1 (pH 5.5) ratio of CaCl2 to peptide, and in 33%
TFE. 1H NMR spectra recorded were DQF-COSY (18), NOESY (19,
20) with mixing times of 100-300 ms and TOCSY (21) with a mixing time
of 65 ms. All spectra were recorded at 280 K, except for some
additional spectra at 298 K. Spectra were run over 6024 Hz with 4 K
data points, 400-600 free induction decays, 16-64 scans, and a
recycle delay of 1 s (1.5 s for DQF-COSY). In NOESY and TOCSY
spectra, the solvent was suppressed using the WATERGATE sequence (22).
Spectra were processed using UXNMR. Free induction decays were
multiplied by a polynomial function and apodized using a 60 or 90 °
shifted sine-bell function in both dimensions prior to Fourier
transformation. Baseline correction using a fifth polynomial was
applied and chemical shift values were referenced externally to
4,4-dimethyl-4-silapentane-1-sulfonate at 0.00 ppm.
3JNH-H coupling constants were measured from
high resolution one-dimensional spectra (32 K) and compared to those
obtained from the DQF-COSY spectra which were strip transformed to
8K × 1K and extracted using the Lorentzian line-fitting routine
in the program Aurelia (Bruker GMBH).
Distance Restraints and Structure Calculations
Peak volumes in NOESY spectra were classified as strong, medium,
weak, or very weak corresponding to upper bounds on interproton distances of 2.7, 3.5, 5.0, or 6.0 Å, respectively. Lower distance bounds were set to 1.8 Å. Appropriate pseudoatom corrections (23) were
made and distances of 1.5 Å and 2.0 Å were added to the upper limits
of restraints involving methyl and phenyl protons, respectively. 3JNH-H coupling constants were used to
determine
dihedral angle restraints (24).
Structures were calculated (coordinates have been deposited with the
Brookhaven Protein Databank) using the simulated annealing protocol in
X-PLOR (25, 26) version 3.1 using the geometric forcefield,
parallhdg.pro. Starting structures were generated using random (,
) dihedral angles and energy minimized (500 steps) to produce
structures with correct local geometry. The structures were subjected
to a total of 30 ps of high temperature molecular dynamics before
cooling and energy minimization (1000 steps). Structure refinements
were performed using energy minimization (2000 steps) under the
influence of the CHARMM forcefield (27).
Data Analysis
Structures were compared using pairwise and average RMSDs for
the C, C, and N atoms (X-PLOR version 3.1) and by calculating angular order parameters for the backbone dihedral angles (28, 29).
Structure visualization was performed using INSIGHT II (Biosym
Technology Inc.).
CD spectra were used as a
qualitative gauge of peptide secondary structure. The CD spectra of
con-G and con-T both exhibit minima at 208 and 222 nm at high
concentrations of CaCl2 (9.2 and 4.5 mM,
respectively), which is the characteristic signature for -helix.
Increasing the CaCl2 concentration further did not enhance
the
-helix content in either peptide, nor did the addition of 50%
TFE to the CaCl2 (9.2 mM) solution of con-G
(Fig. 1A). For con-G, there is clearly a
CaCl2 concentration dependence. In the absence of
CaCl2 almost no helical structure was observed (7%) but on
increasing the CaCl2 concentration, the percentage of
helical structure rose steadily to a maximum of 45% (Fig.
1A). In contrast, when this experiment was performed with
con-T, no CaCl2 concentration dependence was observed.
Instead, con-T showed a high degree of helical content (50%) in buffer
solution alone (Fig. 1B). Although the addition of
CaCl2 did not affect the percentage of
-helix present in
con-T, the addition of 50% TFE to a 4.5 mM
CaCl2 solution increased the
-helical content to 63%.
The same result was obtained in 100% TFE (Fig. 1B).
CD spectra of con-G and con-T under different conditions. A, CD spectra of con-G obtained at different CaCl2 concentrations. B, CD spectra of con-T obtained at different CaCl2 concentrations. C, CD spectra of con-G obtained with different metal ions. D, CD spectra of con-T obtained with different metal ions. E, CD spectra of con-G with fixed CaCl2 concentration and different EDTA concentrations.
As the conantokin peptides apparently bind to, or affect
allosterically, the putative combined polyamine/Mg2+
binding site on the NMDA receptor (30, 31), it was of interest to
determine the effects of Mg2+ on the structure of con-G and
con-T and to explore the possibility that other positively charged ions
could stabilize the structures in the same manner as CaCl2.
The CD spectra showing the effects of cation concentration on con-G are
given in Fig. 1C. As anticipated, monovalent potassium ion
did not stabilize the structure of con-G. On the other hand,
Cu2+ stabilizes the peptide as efficiently as
Ca2+. Most striking were the effects of Mg2+
and Zn2+, which provided greater stabilization to the con-G
structure than Ca2+, with estimated -helix contents of
68 and 69%, respectively. As seen in Fig. 1D, different
cations did not significantly affect the helical content of con-T,
which was estimated to be 50%.
CD spectra showing the effects of the addition of EDTA to a fixed
concentration of peptide (con-G) and CaCl2 are given in Fig. 1E. In this experiment, the degree of -helix present
for con-G in CaCl2 is substantially decreased by the
presence of EDTA. This indicates that EDTA chelates some of the
CaCl2. However, the minimum
-helix content is greater
than that calculated for con-G in buffer alone, even when a large
excess of EDTA (20 mM) is added to the CaCl2
solution. Furthermore, it was shown that addition of 3.0 mM
EDTA to a con-G solution with no CaCl2 present had no
effect on the peptide structure.
The CD results show that the structure of con-T is predominantly helical in aqueous buffer and more so in TFE. Cations such as Ca2+, Cu2+, K+, Mg2+, and Zn2+ have no significant effect on the helix content of this peptide (Fig. 1D). In contrast, the structure of con-G, which is not highly helical in buffer, is greatly affected by the presence of Ca2+, presumably with the Gla residues being involved in the Ca2+-binding interaction. For con-G, the degree of helicity correlated to the amount of Ca2+ present. In the presence of excess EDTA, there is apparently substantial Ca2+ which remains chelated to the peptide, either inaccessible to, or in equilibrium with, EDTA. The CD studies show that con-G is selective for divalent cations, with monovalent cations having little influence on the structure of this peptide. No cation selectivity was observed for con-T. This study provides evidence that Mg2+ and Zn2+ induce greater helical content than Ca2+ and Cu2+ in con-G.
NMR Spectroscopy
Chemical Shift AssignmentDue to the large number of residues
with similar spin-types the two-dimensional spectra of con-G and, to a
lesser extent con-T, are severely overlapped under the conditions used.
Despite this, assignment was possible by a careful analysis of NOESY,
TOCSY, and DQF-COSY spectra (23). An example of the NOESY spectrum of
con-G in H2O showing the sequential assignment is given in Fig. 2. The complete chemical shift assignments of con-G
and con-T are supplied as supplementary material.
Secondary H
The deviations of H chemical shifts
from their random coil values provide information on peptide backbone
structure (23, 32). For con-G in H2O, there are some large
negative secondary H
shifts indicative of
-helix from
Asn8 to the C terminus (Fig. 3). The
magnitude of the secondary shift is greatest in region 9-13. The
addition of TFE did not alter the pattern of secondary shift, but
greatly enhanced the magnitude of secondary shift for each residue,
indicating that the
-helix is stabilized over the entire peptide.
Similarly, the addition of CaCl2 to con-G accentuated the
negative secondary shifts. At pH 3.5, the secondary shifts of residues
5-6 and 8-9 are enlarged, while at pH 5.5,
-helix is further
stabilized over the whole peptide. This is consistent with the
Ca2+-induced secondary structure stabilization.
Furthermore, since the helix content increased at a higher pH, this
suggests that the Ca2+ binds to the negatively charged Gla
side chains.
The large negative secondary H shifts of con-T (Fig.
4) indicate that this peptide is extremely helical, even
in H2O (residues 3-21) and more so in TFE (residues
2-21). The effects of Ca2+ on the H
shifts were
minimal, supporting the CD data which clearly indicated that there was
no overall structural stabilization in this case. However, an exception
to this is the secondary H
shift of Gla10, which is
shifted to a positive value, in the presence of Ca2+ at pH
5.5. It is noteworthy that this is also observed for con-G, under the
same conditions. Although this implies helix disruption, it may instead
be due to an underestimate of the random coil shift of Gla (this is not
available in the literature and the value for Glu was used in its
place).
NOEs
A summary of the observed sequential and medium-range
NOEs for con-G is given in Fig. 5. No long range NOEs
were detected under any of the conditions used. Although many
medium-range NOEs (H-H
i+3, H
-HNi+2,
and H
H
i+3) were present, consistent with
-helical
secondary structure, the peak overlap meant that it was not possible to
detect many NOEs that may be expected based on the extent of
-helix
deduced from the chemical shift data. This is most evident in the data
collected at pH 5.5 in the presence of Ca2+. In this case,
the combined effects of large line widths and peak overlap were
deleterious to spectral quality, resulting in few observable
connectivities in the NH-H
region. Increasing the temperature to 298 K sharpened the line widths, however, the accompanying decrease in
correlation time resulted in low NOE intensities. Despite this, from
decreases in the H
-NHi+1/NH-NHi+1 ratios,
and from the observation of key medium-range NOEs, it is clear that the
-helical content, present over the region encompassed by residues
5-17 in the absence of CaCl2, increased in the presence of
CaCl2 to incorporate the entire peptide. In particular, the strengths of the non-overlapped H
-H
i+3 NOEs,
i.e. from Leu5-Asn8 and
Gla10-Gla14 increase significantly in the
presence of CaCl2 at pH 5.5. At pH 3.5 and in the presence
of CaCl2 there is less difference in NOE strengths compared
with con-G at pH 5.5, providing further support that the Gla side
chains need to be charged for optimal helix stability and
Ca2+ chelation.
The two-dimensional spectra of con-T were more dispersed in the NH
region than those of con-G. Consequently, it was possible to observe
many more NOEs (Fig. 6). In H2O, the large
number of characteristic H-NHi+3,
H
-NHi+4, H
-NHi+2, and H
-H
i+3 NOEs, medium-strong NH-NHi+1 and
medium H
-NHi+1 NOEs show that con-T adopts
-helix
from residues 2 to 21. The helix enhancement in the presence of TFE is
suggested by the slightly weaker H
-NHi+1 relative to
NH-NHi+1 NOEs. However, the addition of TFE caused a
reduction in NH shift dispersion so that many connectivities are not
detectable.
3JNH-H
The 3JNH-H coupling
constant and NH slow exchange data augment the NOE information. Over
the range of solution conditions, it was possible to obtain
3JNH-H
data for all residues of both
peptides. Consistent with well defined
-helix, all measurable
3JNH-H
coupling constants are low (<6 Hz)
in con-T apart from that of Asn20. Similarly, for con-G,
all 3JNH-H
coupling constants are low except
for Ser16 and Asn17 which are only slightly
larger (<7 Hz), reflecting some degree of conformational averaging at
the C terminus. The extent and stability of helix is further
demonstrated by the large proportion (13/20) of NH protons of con-T
which were observed to be slowly exchanging in aqueous solution,
despite the relatively high pH. Similarly, several (7/16) of the NH
protons of con-G in CaCl2 also exchanged slowly with
solvent.
Three-dimensional Structure of Conantokin Peptides
The secondary structure analysis indicates that con-G adopts
transient -helix in aqueous solution which is stabilized by the
presence of Ca2+, although the CD analysis indicates that
other divalent cations will suffice or even improve helix
stabilization. In contrast, con-T forms a stable
-helix in aqueous
solution in the absence or presence of divalent counterions. The
conditions needed for these peptides to adopt what is presumably their
inherently preferred conformational state are different, however, the
qualitative study described above suggests that their three-dimensional
structures are similar. This may also be inferred from high sequence
identity in the N-terminal region and from the similarity of
their biological activity. A quantitative comparison of con-G and
con-T based on the computation of their three-dimensional structures is
described below.
A total of 161 distance restraints (58 intra residual, 60 sequential, and 43 medium range) and 14 dihedral restraints were used in the calculation of 30 structures. Of these structures 18 had no NOE violations greater than 0.2 Å and no dihedral violations greater than 3° and were chosen to represent the structure of con-G. The structures satisfy the experimental and empirical criteria as the average deviations from ideal covalent geometry and experimental restraints are low and the potential-energy and restraint energy contributions are favorable (Table I). While the backbone pairwise RMSD is high over the whole molecule, the RMSDs over each half are much lower (Table I), suggesting that the helix has some flexibility. This can also been seen from the backbone angular order parameters and RMSD values for individual residues shown in Fig. 7A. The angular order parameters are high over residues 2-15, indicating consistency in local geometry among the structures. As is usual with linear peptides, there are elevated RMSDs at the termini consistent with some helix fraying. The RMSD values are lowest in two sections of the peptide (residues 5-6 and 9-14), but elevated at residues 7-8.
|
It is evident from a series of backbone superimpositions (Fig.
8) over particular regions, that the helix of con-G is
highly defined over segments of up to 8 residues. Superimpositions of the middle segment of the peptide highlights the good definition but
leaves an exaggerated impression of frayed termini. However, superimpositions in regions around the N or C terminus show clearly that helix is relatively well defined at both termini (Fig. 8). This is
upheld by analysis of the backbone -
coordinates which shows that
all residues lie in the
-region of the Ramachandran plot. While the
individual three-dimensional structures of con-G each consist of
unbroken helix, few of the helices are linear, with most having some
form of kink or curvature. This implies that either the NMR data are
not sufficient to define the relative orientations of sections of the
helix, or that the long helix has genuine flexibility. The high degree
of curvature observed in most of our structures is not surprising, as
this is commonly observed for amphipathic helices in solution (33, 34).
Analysis of the backbone H-bonds shows that all structures contain a
mixture of i,i+3, and i,i+4 interactions, consistent with
310 and
-helix, respectively, where the 310
helix component consists of approximately 38% of the total backbone
H-bonds.
Three-dimensional Structure of Conantokin-T
A set of 17/30
structures was chosen to represent con-T, based on the criterion stated
above, using 255 distance restraints (91 intraresidual, 89 sequential,
and 75 medium range) and 19 dihedral restraints. The deviations from
ideal geometry and from experimental restraints are given in Table I,
together with backbone pairwise RMSD values. These are lower than for
con-G, reflecting the greater number of distance restraints per residue
used in the calculations. Again, the pairwise RMSD values are
substantially lower when the N terminus, middle segment, and C terminus
are treated separately. These regions are shown superimposed in Fig. 9. The backbone angular order parameters (Fig.
7B) are consistently high for residues 2-21, which all lie
in the -region of the Ramachandran plot. The backbone RMSD values
(Fig. 7B) are lowest for residues 4-9 and 16-18. The
central region 10-14 of the peptide has elevated RMSD values, most
likely due to some flexibility in this region. Similar to con-G, most
of the helices are nonlinear, with the degree of curvature varying
significantly. H-bond analysis again indicates that a mixture of
310 and
-helix is present, with the 310
helix component representing approximately 30% of the total measured
backbone H-bonds.
The CD spectra of both con-G and con-T shown in Fig. 1, A and B, indicate that these peptides are strongly helical in the presence of Ca2+. The most striking observation is that con-T is extremely helical, even in the absence of Ca2+, which may be explained by the relative positions of the Gla residues (see below). The EDTA titrations showed that some of the Ca2+ ions are bound very tightly to con-G, even when the EDTA concentration is increased to four times the concentration of CaCl2. At these relative concentrations, the peptide still exhibits residual Ca2+-induced stabilization. EDTA did not reduce the helical content below that induced by 3.0 mM CaCl2. This shows that Ca2+, in some instances, is bound more tightly to con-G than to EDTA.
Potassium ions did not stabilize the peptides (Fig. 1, C and D), due to their different ionic properties relative to Ca2+. Of the divalent cations, Mg2+ and Zn2+ provided the greatest stabilization to the structure of con-G.
Comparison of the Structures of Con-G and Con-TThe results
have shown that con-G and con-T both adopt stable helices over their
entire length, although the conditions required to achieve this are
slightly different. The three-dimensional structures of both peptides
show that although each residue is well defined locally, there are a
range of global orientations that agree with the experimental
restraints. A superimposition of con-G curved and linear structures
with corresponding structures of con-T (Fig.
10A) emphasizes a striking degree of
similarity. A surface representation of a typical curved structure for
each of con-G and con-T in identical orientations in Fig.
10B shows the hydrophilic residues, at least from residues 3 to 13, are on the exterior surface and the hydrophobic residues are on
the inner (more concave) surface, a phenomenon which is a
characteristic feature of amphipathic helices in solution. The
hydrophobic face of both peptides are alike, being composed of a
mixture of polar and hydrophobic side chains. Characteristic of
amphipathic helices, the hydrophilic face of con-T consists of
alternating regions of positive and negative charge, however, that of
con-G is unusual in that it consists almost entirely of negative
charge. Presumably, the binding of divalent cations to this region of
con-G stabilizes a conformational state that would otherwise be
unfavorable electrostatically. In contrast, con-T does not require
Ca2+ to adopt the same conformation, although it does not
necessarily follow that Gla side chains in this peptide do not bind to
divalent cations.
The three-dimensional structures of con-G and con-T are composed of a
dynamic mixture of 310 and -helix. Although theoretical studies on the relative energies of 310 versus
-helix suggest that the latter conformation is preferred in helices
of greater than 10-12 residues due to a lower degree of steric
hindrance (35, 36), this is offset in con-T by a large number of
putative electrostatic i,i+3 interactions which would favor
310 helix formation. For example, helix stabilizing
salt-bridges may be formed between Gla4-Lys7,
Lys7-Gla10,
Gla10-Arg13,
Arg13-Glu16, and
Glu16-Lys19 in the 310
conformation, but only between Gla3-Lys7 and
Gla14-Lys18 in
-helix. Furthermore, in the
-helical conformation, the Gla10-Gla14
interaction would be disruptive. The same argument may be applied to
con-G to explain why it is less helical in the absence of divalent cations. The electrostatic repulsion of the side chains of
Gla3-Gla7, Gla4-Gla7,
Gla7-Gla10, and
Gla10-Gla14 would hinder both 310
and
-helical conformations. However, the presence of
Ca2+ could promote the alignment of Gla residues so that
the above mentioned interactions become favorable.
This is the first published report on the
three-dimensional structure of con-G and con-T, however, there have
been previous CD spectroscopic studies of both peptides (4, 7). Zhou
et al. (7) suggest that con-G adopts a highly characteristic
helix with a rigid middle segment and mobile N- and C-terminal ends. They propose that the middle segment may function as a helical spacer
between the two flexible terminal domains that may bind to two separate
sites on the NMDA receptor (7). This suggestion may be regarded as
tentative, as CD spectroscopy does not provide structural information
in a sequence specific manner. The NMR data presented here provides
sequence specific structural information and shows that the helix
extends from the N to the C terminus. In addition, the helical region
is not necessarily rigid, even in the middle segment, but may have
considerable flexibility arising from -helix to
310-helix transitions. This
310
interchange was also proposed by Zhou et al. (7) on the
basis of their CD studies of con-G analogues with specific Gla
Ala,
Gla
Ser, or Gla
phospho-Ser (310 helix inducing)
substitutions.
Of significant importance is the role divalent cations play in structure-activity relationships for the conantokins. In the case of con-G, we find there is a structural dependence on divalent cation concentration, however, this is not consistent with the recent findings of Chandler et al. (4) and Zhou et al. (7). In both these earlier studies, the helix content determined by CD spectroscopy for con-G in the absence of added Ca2+ is approximately equivalent to that calculated here for the peptide in the presence of excess Ca2+. This strongly suggests that some Ca2+, or perhaps another divalent cation, was already bound to con-G prior to the addition of Ca2+. Indeed, our experiments with EDTA show that Ca2+ bound to con-G is difficult to remove. Considering this and also the range of divalent cations which have been shown here to affect the stability of the con-G structure, the validity of recent activity experiments performed in the "nominal" absence of Ca2+ are open to question (6). The role of the divalent cations seems more problematic by the finding that the structure of con-T does not appear to be affected by the addition of Ca2+. However, the lack of structural stabilization does not exclude divalent cations from binding to the Gla side chains. Furthermore, it raises questions on the biological reasons for predominance of these post-translationally modified residues, two (Gla3 and Gla4) of which have been shown to be important to the activity of these peptides (7). Since it has been demonstrated here that con-T and con-G are able to adopt the same three-dimensional structure, it is possible that the former peptide is able to adopt its biologically active conformation in the absence of divalent cations, but it requires their presence for activity.
The atomic coordinates and structure factors (codes 1ONU and 1ONT) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
Trudy Bond is gratefully acknowledged for the amino acid analysis. Marion Loughnan and Trudy Bond are gratefully acknowledged for the venom extraction.