 |
INTRODUCTION |
The nicotinic acetylcholine receptor (nAChR)1 (1) is a
ligand-gated ion channel that mediates excitatory transmission at the neuromuscular junction and at synapses in the central and peripheral nervous systems. It is the
most intensely studied member of the ligand-gated ion channel
superfamily and serves as a model for understanding the structure and
function of related ion conducting channels including glycine,
-aminobutyric acid type A, -aminobutyric acid type C, and
type 3 serotonin receptors. nAChRs are pentameric complexes that
assemble in the membrane with 5-fold symmetry. Each subunit contains an
N-terminal extracellular domain about 200 amino acids long followed by
four membrane-spanning segments (M1-M4) with an intracellular loop of
variable length between M3 and M4. The second transmembrane region from
each subunit contributes to the formation of the wall lining the
channel pore. In muscle and Torpedo electric organ, the
subunit composition is (
1)2
and
(1)2
in embryonic and adult tissue,
respectively (for review, see Ref. 1). Neuronal nAChR subunits
(2-10 and 2-4) apparently can assemble in various
combinations giving rise to multiple receptor subtypes. In heterologous
expression systems, particular subunit combinations form functional
hetero-pentamers (e.g.
(4)2(2)3) whereas the 7-9 subunits
form functional homo-pentamers (2, 3).
Snake venom-derived -neurotoxins bind the muscle-type and, in some
cases, homo-pentameric neuronal nAChRs with high affinity (4, 5) and
are grouped into two families (4). Short chain neurotoxins have 60-62
amino acids. Long chain toxins have 66-74 amino acids and a fifth
disulfide at the tip of the second loop. Solution NMR and x-ray
crystallographic studies (e.g. Refs. 6-8) show that all
-neurotoxins share a tertiary structure known as the three-finger
fold, a four-disulfide globular core from which emerge three loops or
fingers and a C-terminal tail. An NMR dynamics study of a short
-neurotoxin reveals "disorder" at the tips of fingers I and II
reflecting localized mobility on the picosecond to nanosecond time
scales (9). The significance of this mobility is highlighted by
evidence that mutation of residues at these locations in other related
toxins produces large effects on binding (11).
-Bungarotoxin (Bgtx), a long neurotoxin from the venom of
Bungarus multicinctus, has been an important tool in many
biochemical and functional studies of nAChRs including the
homo-pentameric nAChRs. The three-dimensional structure of Bgtx,
determined by x-ray crystallography (12), differed from solution and
x-ray structures of related short and long toxins particularly in the length of the highly conserved central -sheet in finger II (6-8). In contrast, an NMR-based investigation of the secondary structure in
Bgtx indicated that the structure of Bgtx in solution most likely
resembles that of other -neurotoxins (13). Although this study paved
the way to a better understanding of the solution structure of Bgtx, an
adequately high resolution structure of Bgtx has not been available
until now. The high resolution three-dimensional structure of unbound
Bgtx presented here greatly facilitates the investigation of
conformational changes that Bgtx undergoes as it binds nAChR-derived
and engineered sequences, the subject of recent structural studies
(14-20).
Photolabeling and mutagenesis studies indicate that the ligand-binding
site on the nAChR is formed at subunit interfaces in the N-terminal
extracellular region of the receptor (1). The major structural
determinants of ligand binding are found on the subunit with
additional contributions made by residues of adjoining subunits.
Strongly conserved residues from three discontinuous regions of the subunit (designated loops A-C) contribute to the binding pocket. In
the crystal structure of the acetylcholine-binding protein (AChBP), a
snail homologue of the extracellular domain of a homo-pentameric nAChR,
loops A-C contribute to a relatively hydrophobic cavity at the subunit
interface (21). Earlier studies (22) have shown that the major
determinants of Bgtx binding lie between positions 173 and 204 on the
1 subunit, coincident with loop C. This region includes the highly
conserved aromatic residues Tyr190 and Tyr198
and, in addition, Cys192 and Cys193 (in 1
numbering) which form an uncommon disulfide. Synthetic peptide binding
studies have suggested that the Bgtx-binding site on the 7 subunit
is in the homologous region (7 178-196) (23). The importance of
this region in toxin binding was further highlighted by the observation
that six residues from this region of 7 could confer Bgtx
sensitivity when placed into the corresponding positions of the
Bgtx-insensitive 3 subunit (24). Additionally, mutation of loop C
residues, Tyr187 and Tyr194 of 7, to Phe
reduced Bgtx blockade of ACh-evoked currents in heterologously
expressed receptors (25). This is consistent with a 60-fold reduction
in Bgtx binding observed in synthetic peptides where Tyr190
of 1 (which corresponds to Tyr187 of 7) was mutated
to Phe, showing the important role of Tyr190 in binding
Bgtx (26).
Previously, we reported NMR solution structures for two 1
subunit-derived peptides, an 12-mer (185-196) and an 18-mer (181-198), in complex with Bgtx (14-16). Here we extend our
analysis of the Bgtx-binding site to the corresponding 19-amino acid
segment on the neuronal nAChR 7 subunit (7 178-196) with an
original solution NMR structure of the 7 19-mer in complex with
Bgtx. Our primary goals are to determine an energetically favorable conformation for a region of the neuronal nAChR important in agonist and antagonist binding and to understand the structural basis for the
strong interaction between Bgtx and the 7 nAChR. In addition, we
present a new high resolution analysis of the secondary structure-rich core of Bgtx, significantly extending the initial NMR structural studies of this toxin (13).
| |
EXPERIMENTAL PROCEDURES |
Expression and Purification of the 7 19-mer--
We
prepared a synthetic gene encoding residues 178-196
(IPGKRTESFYECCKEPYPD) of the chick neuronal nAChR 7 subunit (27) using mutually priming oligonucleotides. The oligonucleotides were
designed according to the specifications of the pET-31 Peptide Expression System (Novagen) with 3' overhangs encoding methionine (28).
The sequences of the two oligonucleotides are
5'-ATTCCGGGCAAACGTACCGAAAGCTTCTATGAATGCTGCAAAGAACCGTATCCGGATATG-3' and
5'-ATCCGGATACGGTTCTTTGCAGCATTCATAGAAGCTTTCGGTACGTTTGCCCGGAATCAT-3'. Two
copies of this expression cassette were ligated in tandem into
pET-31b(+), and the construct was authenticated by DNA sequencing in
the forward and reverse directions. The 7 19-mer was expressed as a
ketosteroid isomerase fusion protein with a C-terminal polyhistidine tag in Escherichia coli BL21(DE3)pLysS cells (Novagen).
Isotopically labeled 7 19-mer was produced in E. coli
using M9 minimal medium with
(15NH4)2SO4 and
13C6-glucose (Cambridge Isotope
Laboratories) as the sole sources of nitrogen and carbon,
respectively (29). The medium was supplemented with vitamins
according to Ref. 30. Detailed methods for expression and purification
of the 7 19-mer have been reported previously (16) for a similar
nAChR recombinant peptide. Briefly, ketosteroid isomerase-7
19-mer fusion protein was isolated from resolubilized inclusion bodies
by nickel affinity chromatography. The fusion protein was then treated
with CNBr and the reaction products separated by RP-HPLC. 7 19-mer
was isocratically eluted from a semi-preparative C18
RP-HPLC column (Vydac) at 5 ml/min in 20% acetonitrile, 0.1% trifluoroacetic acid. Mass spectrometric analysis of HPLC fractions identified purified 7 19-mer (HHMI Biopolymer/Keck Foundation Biotechnology Resource Laboratory, Yale University School of
Medicine). The redox status of the adjacent cysteine residues,
Cys189-Cys190, was determined by RP-HPLC
analysis of N-ethylmaleimide-treated 7 19-mer in its
condition after purification and following pretreatment with
dithiothreitol. Typical yields of isotopically labeled 7 19-mer were
2-4 mg/liter. Purified peptide was lyophilized and stored at
20 °C.
Binding Experiments--
The KD value for the
7 19-mer-Bgtx interaction was determined by measurement of
inhibition of the initial rate of Bgtx binding to nAChR-enriched
Torpedo membranes following preincubation of Bgtx with 7
19-mer (26). 125I-Labeled Bgtx (2.5 nM) was
incubated over a wide range of concentrations of 7 19-mer in 0.2%
bovine serum albumin, 30 mM sodium phosphate, pH 7.4, for
18 h at room temperature. 100 µl of the peptide/toxin mixture
was added to microtiter plate wells coated with 2 µg of Torpedo membranes that were pre-blocked with 200 µl of 2%
bovine serum albumin for 1 h. Following a 5-min incubation, the
binding reaction was aspirated, and wells were washed 4 times with 200 µl of 0.2% bovine serum albumin, 30 mM sodium phosphate,
pH 7.4. Torpedo membrane-bound 125I-Bgtx was
measured in a counter. All measurements were done in triplicate.
The effect of pH was also assessed by preparing samples in 30 mM sodium phosphate, pH 5.5, to replicate the buffer conditions of the NMR sample (see below).
NMR Sample Preparation--
Hydrated Bgtx (Sigma) was used to
resuspend the 7 19-mer in order to facilitate formation of a 1:1
peptide-toxin complex. Uniformly 15N-labeled and uniformly
15N,13C-double-labeled 7 19-mer·Bgtx
samples were prepared at a concentration of 1.5-2.0 mM.
These samples contained 30 mM sodium phosphate and 50 µM sodium azide in 95% H2O, 5%
D2O at pH 5.5. Sodium 3-trimethylsilylpropionate (Cambridge
Isotope Laboratories) was added at 50 µM as an internal calibration standard. For deuterium exchange experiments, the complex
was lyophilized and reconstituted in 99.9% D2O at pH 5.5 (isotope effect unaccounted). Preparation of the free Bgtx samples was
described previously (13).
NMR Experiments--
NMR experiments were carried out at
1H frequencies of 400 and 600 MHz on Bruker Avance
spectrometers at Brown University and at 500 MHz on a GE spectrometer
equipped either with a GN console with a Nicolet computer or an Omega
console with a Sun 3/160 computer at the University of California, San Francisco.
For free Bgtx, the following spectra in H2O were acquired
at 15, 25, and 35 °C, and pH 5.79: DQF-COSY (31), TOCSY (70 ms MLEV-17 spin-lock sequence) (32), and NOESY spectra (160-ms mixing
time) (33) using the water suppression scheme described in Basus (34).
No significant shifts in free Bgtx proton resonances were observed
between samples at pH 5.5 and 5.79, suggesting that the chemical
environment is comparable at both pH conditions. In addition, NOESY
(160-ms mixing time) and E-COSY spectra (35) at 25 and 35 °C in
D2O were acquired.
Distance restraints were calculated from experimental NOESY intensities
using the program MARDIGRAS version 2.0 (36, 37) which uses the
complete relaxation matrix to produce an upper and lower distance bound
for each experimental intensity. To make use of this procedure that
determines accurate distances from the integrated intensities of the
NOESY cross-peaks, a value for the rotational correlation time,
c, must be defined. For free Bgtx the correlation time
was determined by measurement of the 13C
T1 and T2
relaxation times at natural abundance, using a sample dissolved in
99.96% D2O. These experiments were carried out using a
double-DEPT technique with proton detection for maximum sensitivity. For T1, we used the double-DEPT sequence with
inversion recovery (38), and for T2, we used the
double-DEPT sequence with a Carr-Purcell-Meiboom-Gill modification
using a series of 180° pulses with a repetition rate of 1 ms to
replace the single 180° refocusing pulse in the sequence of Nirmala
and Wagner (39). The sum of the resonances at different portions of the
spectra was used to determine the relaxation times. The data were
analyzed by fitting to a single exponential decay function. Peak
volumes from D2O and H2O NOESY spectra were
obtained by fitting the peak or peaks to be integrated to a Gaussian
line shape in Sparky. In the case of peaks with low signal to noise ratio, the points within a manually selected rectangular or elliptical area surrounding the cross-peak were summed using Sparky (40). In NOESY
spectra the 3JHN
coupling
constants were determined by line fitting as described above, using
Sparky. These values were used as the minimum values, because the
apparent coupling constant in NOESY spectra will be smaller than the
actual coupling constant. In DQF-COSY spectra, coupling constants were
determined by line fitting the antiphase multiplets, followed by
measurement of the separation between the simulated anti-phase
multiplets (35). These values were used as maximum values, because the
apparent coupling in DQF-COSY spectra is larger than the actual
coupling constant due to the summation of anti-phase cross-peaks.
Exchange rates were measured at 25 °C for Bgtx lyophilized from
H2O and re-dissolved in D2O. Immediately
following this procedure, the sample was placed in the spectrometer,
and one-dimensional and TOCSY spectra (40 ms mixing time) were
alternately acquired. The first one-dimensional spectrum was acquired
14 min after dissolving the lyophilized powder in D2O, and
the first TOCSY spectrum was started 1 min later. Several spectra were
obtained up to 36 h after starting the exchange, and further
spectra were obtained 1 and 2 weeks later with the sample maintained at
25 °C. The cross-peaks in the resulting spectra were integrated, and
cross-peaks between - and -protons were integrated for use as
intensity references to eliminate variations of the spectrometer
conditions after the sample was removed and re-inserted in the magnet
for the last time points.
For the 7 19-mer·Bgtx complex, 1H-15N
three-dimensional NOESY-HSQC (120-ms mixing time) (41),
1H-15N three-dimensional TOCSY-HSQC (60-ms
MLEV-17 spin-lock sequence) (41), and three-dimensional HNHA (42)
experiments were collected at 35 °C using a uniformly
15N-labeled 7 19-mer·Bgtx sample in order to assign
resonances. To clarify ambiguities in the assignments, an HNCA
experiment (43) was performed using uniformly
15N/13C double-labeled 7 19-mer.
1H homonuclear NOESY (120-ms mixing time) (33) and TOCSY
(60-ms MLEV-17 spin-lock sequence) (32) experiments were performed with
15N decoupling to assign bound Bgtx resonances. NOESY and
TOCSY experiments were performed at 15, 25, and 35 °C and with
different mixing times to resolve ambiguities and facilitate the
assignment process. Water was suppressed using the WATERGATE method,
incorporating a 3-9-19 refocusing pulse sequence with pulsed field
gradients (44). Deuterium exchange was performed to identify slowly
exchanging amide protons involved in secondary structure. Following a
two-dimensional 1H-15N HSQC (45), five
(sequential) homonuclear TOCSY spectra were collected over 16 h
following reconstitution of the 7 19-mer·Bgtx complex in
D2O. The stoichiometry of the 7 19-mer·Bgtx complex was determined in a mole ratio titration of the 7 19-mer using two-dimensional 1H-15N HSQC experiments. NMR
data were processed by XWIN-NMR (Bruker) or NMRPipe (46), and resonance
assignments were made in Sparky (40). 1H chemical shifts
were referenced to sodium 3-trimethylsilylpropionate (0.0 ppm). The
1H, 15N, and 13C assignments will
be deposited in the BioMagResBank chemical shift data base.
Structure Calculations--
For free Bgtx, distance geometry
calculations were performed on the Cray Y-MP at the San Diego
Supercomputer Center using the distance geometry program VEMBED (47), a
vectorized version of EMBED (48). Restrained molecular dynamics
calculations were carried out with the four-dimensional modifications
(49) to the GROMOS-87 programs (50) using a SparcIPX work station. The 37D4 force field was used, and all the calculations were performed in vacuo with all charged groups neutralized and with the
mass of the hydrogens set to 12 atomic mass units. The distance
restraint constant, Kdis, was set to 10,000 kJ
mol
1 nm2, with an initial temperature of
800 K. Three ps of dynamics was run followed by 5 ps during which the
three-dimensional projection force constant K3d
was increased from 0 to 5,000 kJ mol1 nm2.
At the same time, the temperature was slowly lowered in an annealing procedure over the next 11 ps with the final temperature set to 0.1 K. The time constant tc for coupling to the thermal bath was
set to 0.005 ps.
For the 7 19-mer·Bgtx complex, distance constraints were derived
from a two-dimensional homonuclear NOESY experiment (120-ms mixing
time; 35 °C). NOEs were manually classified as strong, medium, or
weak according to intensity. The distance ranges corresponding to the
categories are as follows: 1.8-3.0, 1.8-4.0, and 1.8-5.0 Å,
respectively. When stereospecific assignment of methylene and methyl
protons was not possible, a pseudoatom correction was employed. Slowly
exchanging amide protons involved in a network of NOEs characteristic
of -sheets were identified as hydrogen bond donors and assigned the
following constraints for residues (i,j):
HNi-Oj 1.6-2.5 Å and
Ni-Oj 2.5-3.3 Å. Dihedral angle constraints were
based on 3JHN coupling constants
calculated from HNHA data (42). For
3JHN > 8 Hz, 
was restrained
to 120 ± 40°; for 3JHN < 6 Hz, was restrained to 60 ± 30°. The peptide bond between adjacent cysteines involved in a disulfide deviates from the
typical trans configuration (51). Accordingly, the 
dihedral angle between Cys189 and Cys190 was
unrestrained to enable calculation of an energetically favorable conformation. Structures were calculated starting from a random conformation model using the distance geometry/simulated annealing program in CNSsolve (52). Distance restraints were used with a
square-well potential. The force constant on the NOE restraints was 50 kcal mol1 and on the dihedral restraints 200 kcal
mol1 radians2. The van der Waals
energy was represented by a repel function whose force constant varied
during the cooling stage from 0.003 to 4 kcal mol1
Å4. From multiple rounds of calculations, each initiated
with a random seed number, acceptable structures were defined as those with no NOE violations exceeding a 0.5-Å cutoff. The 10 lowest energy
structures from a pool of over 100 accepted structures were selected
for calculation of an energy-minimized average structure using the
accept.inp program of CNSsolve. The average structure was
further energy-minimized using 100 steps of steepest descents in
DISCOVER (Molecular Simulations, Inc.) to remove averaging artifacts.
The structures were visualized in MOLMOL (53). Intermolecular contact
surface areas of the 7 19-mer·Bgtx complex were calculated by
Contact of Structural Units software (54), and hydrogen bond analysis
was performed by the DSSP program (55). All structure coordinates for
Bgtx and the 7 19-mer·Bgtx complex were deposited in the Research
Collaboratory for Structural Bioinformatics Protein Data Bank. The
ensemble structures of Bgtx have been assigned the identifier 1KFH and
the ensemble structures and minimized average structure of the 7
19-mer·Bgtx complex 1KC4 and 1KL8, respectively.
| |
RESULTS |
Determination of the Structure of Bgtx--
Integrated volumes
from cross-peaks in NOESY spectra of Bgtx were used in the program
MARDIGRAS (36, 37) to calculate accurate distance constraints taking
into account spin-diffusion effects. These calculations were carried
out using a correlation time of 3.3 ns and were repeated at 3.7 and 2.9 ns to include the range of uncertainty in the correlation time
determination. Based on the dipole-dipole relaxation mechanism due to
the directly attached proton, the correlation time was determined from
13C T1 and T2
relaxation times. Indirect measurement of the 13C
T1 relaxation time yielded an average value of
0.6 ± 0.06 s for the -carbons; the 13C
T2 relaxation time was 73 ± 7 ms. Extended
starting structures were used in the distance calculations initially,
and subsequent calculations began with structures obtained in the
previous round of calculations. The largest distances thus calculated
were used as the upper bound and the lowest as the lower bound for each distance restraint.
Initial structure calculations revealed a triple-stranded -sheet
whose hydrogen bonds were confirmed by H/D exchange experiments (Table
I and see below). The nine most
unambiguous hydrogen bonds were used as additional restraints in the
final structure calculations, which included 588 NOE restraints.
Stereospecific assignments were obtained for 10 diastereotopic pairs of
-protons. From the HN-H coupling
constants, 43 angle constraints were obtained, and from E-COSY
spectra -proton coupling constants were obtained to yield
constraints for 17 
1 angles. The relevant structural
statistics are shown in Table II. From 20 distance geometry structures calculated, 13 were selected that had the
correct global fold, as indicated by the lower energies and smaller
number of distance violations. To overcome local barriers in the
restrained molecular dynamics calculations, the four-dimensional version of GROMOS (49) was used. The final structures have a large
r.m.s.d. (2.11 Å), when all backbone atoms are included, and several
regions show large local r.m.s.d. If we exclude these regions,
primarily the C-terminal segment and the end of finger II, the
remaining region consisting of residues 1-16, 22-28, 39-48, and
54-68 can be matched together to yield, for the backbone atoms, an
r.m.s.d. to the average of 0.58 Å (Fig.
1). Two regions with regular secondary
structure are well defined, the -sheet in finger I (residues 1-16)
and the triple-stranded -sheet formed by residues 22-28, 39-45,
and 56-60. The poorly defined regions have few NOEs, and in
particular, finger II has sequential NOEs that are smaller than
expected, indicating the possibility of additional local motion in this
region. Those regions poorly defined in free Bgtx are better defined in
the 7 19-mer·Bgtx complex as indicated by the lower overall
r.m.s.d. (Table II and see below).
View larger version (41K):
[in this window]
[in a new window]
|
Fig. 1.
Stereo views of the well defined
regions of free Bgtx. Top, residues 1-16 forming
finger I; middle, the triple-stranded -sheet;
bottom, the region of Val57 to
Pro67. The structures were matched to the rigid portions of
Bgtx, i.e. to the backbone atoms of residues 1-16, 22-28,
39-48, and 54-68, collectively. The main chain is dark
blue with the side chains red, except for the aromatic
side chains Tyr and Trp in green, Ile, Leu, and Val in
light blue, and the cysteines in orange. Backbone
carbonyl oxygen atoms are colored magenta.
|
|
H/D Exchange Rates for Free Bgtx--
H/D exchange in a sample of
lyophilized Bgtx dissolved in D2O at 25 °C and pH 4.0 was followed using TOCSY and one-dimensional spectra. A total of 30 amide protons were observed to be in slow exchange (Table I). The
experimentally determined set of slow exchanging amide protons that
denote potential hydrogen bond donors was compared with the set of
predicted hydrogen bonds calculated by DSSP from the Bgtx coordinates
(Table I). DSSP predicts hydrogen bonding based on the distance between
hydrogen-bonding partners and their orientation. In general, there is
good agreement between the hydrogen bonds detected in the structures
and the amide protons slowly exchanging with deuterium in
D2O. The only slowly exchanging amide proton with a long
exchange time that did not form a hydrogen bond in the structures is
Thr47-HN. Only two hydrogen bonds detected in
all structures with low hydrogen bond energy, which were not slowly
exchanging, occur between Glu56 and Met27 and
between Asp30 and Gly37. Mobility at the tip of
finger II may explain this observation as both are found at the end of
the triple-stranded -sheet in finger II.
Expression and Purification of Metabolically Labeled 7
19-mer--
Homonuclear NMR studies of Bgtx complexed with unlabeled
synthetic peptides derived from the nAChR 1 subunit resulted in incomplete assignment of peptide proton resonances because of signal
overlap (14, 15). This precluded a structure determination of the
entire peptide sequence and a complete description of the peptide/toxin
interface. To overcome this problem, we adopted a recombinant approach
to produce the 7 19-mer uniformly labeled with the half-spin
isotopes 15N and 13C for heteronuclear NMR
studies. The 7 19-mer was expressed in E. coli as a
fusion protein (see "Experimental Procedures"). The fusion protein
precipitated in inclusion bodies that were isolated and resolubilized
for nickel affinity purification using the C-terminal polyhistidine tag
of the fusion protein. Purified fusion protein was treated with CNBr to
liberate the 7 19-mer from its fusion partner and from the
polyhistidine tag, taking advantage of engineered methionine residues
flanking the 7 19-mer sequence. The 7 19-mer was purified by
RP-HPLC. As expected of CNBr-digested proteins, two related and
interconvertible peptides were obtained differing only at their C
terminus (56). As confirmed by mass spectrometric analysis, one
contains a homoserine and the other has the dehydrated homoserine
lactone. In solid-phase competition binding studies, there was no
difference between the recombinant 7 19-mer and a synthetic 7
19-mer peptide lacking a C-terminal homoserine. Cys189 and
Cys190 readily form a disulfide in the purified peptides as
evidenced by resistance to N-ethylmaleimide alkylation and
susceptibility to N-ethylmaleimide alkylation following
treatment with dithiothreitol. Both 7 19-mer HPLC peak fractions
were combined to prepare NMR samples as homoserine and homoserine
lactone are in pH-dependent equilibrium (56).
Affinity of the 7 19-mer·Bgtx Complex--
The affinity of
the 7 19-mer for Bgtx was determined in a solution-based assay that
is rooted in the finding that synthetic peptides derived from the nAChR
compete with intact Torpedo nAChR for binding Bgtx (24).
Following equilibration of 125I-labeled Bgtx with varying
concentrations of the 7 19-mer, the percentage of Bgtx that remained
unbound was determined by the amount of binding to the nAChR under
initial rate conditions. The concentration of 7 19-mer that
diminished the initial rate of binding by 50% (KD)
was 30 µM (data not shown). The KD
value was identical at pH 7.4 and pH 5.5.
Stoichiometric Interaction between the 7 19-mer and
Bgtx--
CD studies of 7 19-mer alone in solution showed that the
peptide structure is random coil (data not shown). Hence, we did not
undertake NMR studies of free 7 19-mer and proceeded directly to
determine its structure in complex with Bgtx. The stoichiometry of the
7 19-mer-Bgtx interaction was determined by titrating U-15N-labeled 7 19-mer with Bgtx and monitoring chemical
shift changes in 1H-15N HSQC spectra. These NMR
experiments track changes in the chemical environment of peptide amide
protons as Bgtx is incrementally introduced. In the absence of Bgtx,
the peptide amide proton resonances lie within a narrow chemical shift
range, indicative of a lack of secondary or tertiary structure (Fig.
2A). Upon addition of Bgtx,
virtually all the amide proton resonances undergo a change in chemical
shift (Fig. 2, B and C). New peaks, corresponding to bound peptide, appear in previously unpopulated environments. The
intensities of the "bound" peaks progressively increase as the
added toxin approaches equimolar ratio leading to the conclusion that
7 19-mer and Bgtx form a 1:1 complex (Fig. 2, C and
D).
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
Stoichiometric interaction between the
7 19-mer and Bgtx. Changes in the amide proton
environment of the 7 19-mer upon titration with Bgtx as monitored by
1H-15N HSQC experiments. Ratio of Bgtx to 7
19-mer: A, 0:1; B, 0.5:1; and C, 1:1.
The filled arrow highlights a "free" 7 19-mer amide
proton resonance and the open arrow a "bound" amide
proton resonance. D, plot of percent maximum free
(filled arrow) and bound (open arrow) peak
intensities over the mole ratio titration for the two peaks highlighted
in A-C.
|
|
Discrete sets of peaks corresponding to free and bound 7 19-mer
indicate that the peptide-toxin complex is in slow exchange (57). This
is further supported by the observation that the bound peak line
widths and chemical shifts do not change upon incremental addition of
Bgtx.
Assignment of 7 19-mer and Bgtx Resonances--
We used
U-15N-labeled 7 19-mer to distinguish unambiguously
peptide resonances from those of Bgtx in the NMR spectrum fingerprint region. Three-dimensional TOCSY-HSQC and NOESY-HSQC spectra were collected to assign 15N-correlated resonances using the
sequential resonance assignment strategy of Wüthrich (58).
Additionally, correlation of 7 19-mer intraresidue amide and
-protons was confirmed in an HNHA experiment. Of the 20 amino
acids of the peptide, only 16 were assignable by the
15N-edited experiments because the N-terminal NH is rapidly
exchangeable and three residues are prolines with no amide proton. The
sequential NOE assignments are summarized in Fig.
3.
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Sequential proton-proton NOE connectivities
of the 7 19-mer·Bgtx complex. The
intensities of dN,
d N, and dNN sequential
NOEs (d , d,
and dN for Xaa-Pro dipeptides and
dN, dN, and
dN for Pro-Xaa dipeptides) are indicated by
bar thickness: strong, d < 3.0 Å; medium,
d < 4.0 Å; and weak, d < 5.0 Å.
|
|
Assignment of 7 19-mer resonances by heteronuclear NMR methods
greatly simplified the assignment of Bgtx as several peptide resonances
overlapped with those of Bgtx in homonuclear spectra. Bound Bgtx
resonances were assigned by the sequential assignment strategy of
Wüthrich (58) using two-dimensional homonuclear TOCSY and NOESY
data (Fig. 3).
Structure Calculations--
For the 7 19-mer·Bgtx complex,
671 NOE-derived distance constraints were used in distance geometry and
simulated annealing calculations, including 548 intra-toxin, 95 intra-peptide, and 28 intermolecular constraints. Five HNHA-derived angle restraints for the 7 19-mer were introduced, as well as 20 hydrogen bond constraints within Bgtx based on assignment of hydrogen
bond donors determined by deuterium exchange. The 10 lowest energy
structures that fit the experimentally derived constraints with no NOE
violations exceeding a 0.5-Å cut-off are shown in Fig.
4. Relevant structural statistics are
shown in Table II. The backbone r.m.s.d. value calculated between the
10 structures and the mean structure is 1.04 Å in the well defined
regions of the complex as follows: Ile1-Cys16,
Leu22-Trp28,
Val39-Thr48, and
Tyr54-His68 in Bgtx and
Ser185-Tyr194 in the 7 19-mer. In Bgtx,
these regions include slow exchanging amide protons and numerous long
range NOEs; in the 7 19-mer, we find intermolecular and long range
intrapeptide NOEs (see below). The corresponding heavy atom r.m.s.d. is
1.55 Å. The backbone r.m.s.d. for 7
19-mer(Ser185-Tyr194) and the entire toxin is
1.38 Å, for 7 19-mer(Ser185-Tyr194) alone
is 1.10 Å, and for the entire 7 19-mer and Bgtx is 1.53 Å.
View larger version (32K):
[in this window]
[in a new window]
|
Fig. 4.
Stereo view of the 7
19-mer·Bgtx complex. Ten NMR-derived backbone traces of Bgtx
(blue) and the 7 19-mer (red) are
superimposed. N-terminal residues 178-184 of the 7 19-mer are
unconstrained and were removed in this figure. For orientation, the N
termini of Bgtx and the 7 19-mer, as presented, are colored
black. The C-terminal tail of Bgtx (residues 69-74) is
colored green for clarity. The figure was prepared using the
program MOLMOL (53).
|
|
Structure of 7 19-mer-bound Bgtx--
The overall
-neurotoxin three-finger fold is preserved in 7 19-mer-bound
Bgtx, as shown in Fig. 4. This view of the complex presents the concave
surface of Bgtx with finger I on the left and finger III on the right.
The central three-stranded -sheet that is conserved among
-neurotoxins is evidenced by a network of long range
H-H and H-HN
NOEs, as well as 10 slowly exchanging amide protons. Similarly, a
two-stranded antiparallel -sheet in finger I is observed in many
calculated structures. Notably, 7 19-mer binding induces chemical
shift perturbations greater than 0.2 ppm in Bgtx resonances for at
least 15 residues consistent with their involvement in intermolecular
contacts or conformational rearrangements (Table III). Fig.
5A illustrates the
conformational changes that occur in Bgtx upon 7 19-mer binding. In
comparison to unbound Bgtx, the tip of finger I in bound Bgtx is
extended and parallel to the central -sheet, owing to intermolecular
contacts in this region (Table IV). A
hydrogen bond between Ser9-O and
Ile11-HN, absent in the uncomplexed
form, stabilizes the tip of finger I in 8 of the 10 ensemble
structures. The flexible tip of finger II undergoes two significant and
stabilizing conformational changes upon binding. The fifth disulfide
loop (Cys29-Cys33) adopts a convex
conformation, whereas Gly34-Lys38 move closer
to the 7 19-mer, placing Arg36 and Lys38 in
position to make intermolecular contacts. These qualitative observations are quantitated in a comparison of the calculated per
residue r.m.s.d. for free and 7 19-mer-bound Bgtx using the respective average structures (Fig. 5B).
View this table:
[in this window]
[in a new window]
|
Table III
Chemical shift perturbations in Bgtx induced by 7 19-mer binding
The resonances of the protons listed are shifted by  0.2 ppm upon
complex formation. Proton designations follow IUPAC-recommended
nomenclature.
|
|
View larger version (35K):
[in this window]
[in a new window]
|
Fig. 5.
Structural comparison of free and
7 19-mer-bound Bgtx. A,
superposition of the ensemble of structures of free (red)
and bound (blue) Bgtx shown in a stereo view. This
presentation of Bgtx with finger I on the left and finger
III on the right corresponds to the concave surface of
-neurotoxins. For clarity, the C-terminal tail is not shown.
B, a plot of r.m.s.d. as a function of residue position in
Bgtx was derived from the average structure for Bgtx, free (open
circles), and bound to the 7 19-mer (filled
squares). Structures were matched globally to the backbone atoms
of residues 1-74.
|
|
Three-dimensional Structure of the 7 19-mer--
The 7
19-mer exhibits no regular secondary structure upon binding Bgtx
according to the NMR data as no long range intrapeptide backbone NOE
networks nor slowly exchanging peptide amide protons characteristic of
-helical or -sheet structure were observed. Rather, the 7
19-mer is primarily constrained by contacts with Bgtx over a 9-residue
stretch from Phe186 to Tyr194 (see below) and
long range intrapeptide NOEs involving backbone proton interactions
between Ser185 and Tyr194 and between
Phe186 and Tyr194. We also observe long range
intrapeptide ring proton NOEs between Tyr187 and
Tyr194. In addition, medium range NOEs involving
Tyr187/Cys189 and
Glu188/Cys190 constrain the 7 19-mer
describing a hairpin-like turn about the vicinal disulfide. Only
sequential NOEs are observed for residues flanking the
Ser185-Tyr194 region, leaving the two ends of
the 7 19-mer unconstrained.
We find that the sequential NOEs connecting Cys189 and
Cys190 are characteristic of a trans peptide
bond conformation. We observe strong dN and
dNN NOEs typical of a trans
conformation, and there is no evidence for
d and dN NOEs
that would be expected for a cis conformation (58).
The 7 19-mer·Bgtx Interface--
An extensive network of
intermolecular NOEs ranging from Phe186 to
Tyr194 on the 7 19-mer positions the 7 19-mer between
fingers I and II of Bgtx on the concave face of the toxin and forms a
contact area of ~650 Å2 (Fig. 4 and Table IV). The
aromatic ring of Phe186 alone is involved in contacts with
Ala7, Ser9, and Ile11 of the first
finger in Bgtx (Table IV). Interestingly, we observe a 0.6 ppm upfield
shift of the Ile11 methyl protons (Table III). Our
structures indicate that these protons are close to the ring
protons of the Phe186 aromatic ring, suggestive of a ring
current-induced shift. A similar shift in the
Ile11(H1)3 signal involving the
corresponding residue Tyr190 in 1 was observed in the
12-mer·Bgtx structure (14) and is thought to suggest a hydrophobic
contact. More expansive contacts are made between the 7 19-mer and
the side of finger II proximal to finger I involving
Phe186-Glu188 of the 7 19-mer and
Lys38-Val40 in Bgtx. Phe186 and
Tyr187 make hydrophobic contacts with Val39 and
Val40. This hydrophobic patch is continuous with additional
intratoxin hydrophobic contacts between Val39 and
Trp28 and between Val40 and His68.
Glu188 makes contacts with Lys38, possibly
forming an electrostatic pair and, in half of the ensemble structures,
donates its amide proton in a backbone hydrogen bond with
Arg36. Furthermore, we observe NOEs involving the protons of Arg36 at the tip of finger II in Bgtx and the
protons of both Phe186 and Tyr194 in 7
19-mer, suggestive of additional hydrophobic contacts. Cation-
interactions have been suggested to stabilize ligand interactions with
the nAChR (59). NOEs that are characteristic of cation- interactions
(60) are not observed between Bgtx and the 7 19-mer. The positive
charge of Arg36 points away from the 7 19-mer,
suggesting that it may make contact with residues elsewhere in the
intact 7 receptor (e.g. Trp149). No
intermolecular NOEs are observed involving Cys189 and
Cys190 of the 7 19-mer, consistent with biochemical
evidence that Bgtx binding does not depend on these residues (61).
Finally, the N terminus of Bgtx is distant from the intermolecular
contact zone, consistent with the observation of identical apparent
affinities of N-terminal polyhistidine-tagged recombinant Bgtx and
venom-derived Bgtx for both Torpedo and mouse muscle nAChRs
(62).
| |
DISCUSSION |
Structure of Free Bgtx--
The three-dimensional structure of
Bgtx presented here builds on an earlier NMR structural study aimed
primarily at determining elements of secondary structure (13). The
present structure provides a high resolution view of the disulfide core
and -sheet regions that are the structure-defining features of
three-finger proteins (6-8). This structure, with a backbone r.m.s.d.
from the mean of 0.58 Å in the structured regions (residues 1-16,
22-28, 39-48, 54-68), improves on a recent NMR solution structure of Bgtx that has a backbone r.m.s.d. of 1.76 Å for the corresponding regions (16; Research Collaboratory for Structural Bioinformatics Protein Data Base code 1IDL). Additionally, the new structure now makes
apparent the structural disorder at the tip of finger II that is
thought to be important in recognition of the nAChR, as illustrated in
the 7 19-mer·Bgtx complex (see below).
The Bgtx NMR structure is most similar to the x-ray structure of the
long chain toxin, -cobratoxin (Cbtx) (8), with an average backbone
atom r.m.s.d. between Cbtx and the 13 Bgtx structures of 1.33 Å, for
the segment of the well defined regions of Bgtx that are best matched
to the corresponding regions in Cbtx (residues 1-6, 11-16, 22-28,
39-48, and 54-68 in Bgtx; residues 1-6, 9-14, 19-25, 36-45, and
51-65 in Cbtx). A comparison of the sequences reveals that finger I in
Bgtx is two residues longer than in Cbtx. These additional residues
appear as an extra bulge in finger I without changing the overall
conformation of the finger. The sequences of Bgtx and Cbtx at the tip
of finger II (residues 29-38 in Bgtx; 26-35 in Cbtx) are nearly
identical with only two residues different out of ten. Although the
overall structure of Bgtx suggests this region is disordered (Fig.
5A), a match of Bgtx residues 29-38 indicates that it has
elements of autonomous structure. Furthermore, the local structure
between residues 29 and 38 agrees well with the x-ray structure of Cbtx
(Fig. 6) with an average backbone r.m.s.d. of 1.74 Å. The local structure of the corresponding loop of
the long neurotoxin LSIII from Laticauda semifasciata (9) also shows a similar structure, with an average backbone r.m.s.d. of
1.6 Å between Cbtx and LSIII for residues 26-35 in both toxins. Like
the Bgtx and LSIII solution structures, the NMR solution structure of
Cbtx (7) also exhibits a large r.m.s.d. for finger II with respect to
the overall structure, with a local structure for residues 26-35
similar to its crystal structure. This suggests that the tip of finger
II may move as a rigid body relative to the triple stranded -sheet
to which it is connected.
View larger version (35K):
[in this window]
[in a new window]
|
Fig. 6.
Structure of finger II. Stereo view
showing residues 29-38 corresponding to finger II for 10 of the 13 structures of Bgtx (gray) matched to the corresponding
region comprising residues 26-35 of the x-ray structure of Cbtx (8)
(black). The labels indicate the location of the -carbons
of some of the residues in this loop for the Bgtx structures. With the
exception of the cysteines of Bgtx, only the backbone atoms are
shown.
|
|
The 7 19-mer-Bgtx Interaction--
The structure of the 7
19-mer·Bgtx complex reveals several important intermolecular contacts
that form the structural basis for the high affinity interaction
between nAChRs and -neurotoxins. Previously, indirect structural
information as determined in mutagenesis studies has helped identify
-neurotoxin binding determinants on the neuronal nAChR (24, 63). The
results presented here provide the first direct structural evidence for
the contact zone involving an -neurotoxin and a neuronal nAChR
sequence. Additionally, the structure obtained may represent an
energetically favorable conformation for a region of the nAChR
important in agonist and antagonist binding and provide an
understanding of differences between -neurotoxin recognition of
muscle-type and neuronal nAChRs.
The 7 19-mer on its own lacks stable secondary structure as
demonstrated by its CD profile (data not shown) and narrow dispersion of its HN resonances (Fig. 2A). Bgtx binding
induces a transition whereby the 7 19-mer takes on a hairpin-like
structure (Fig. 4) that is temporally stabilized, meaning that on the
NMR time scale, the 7 19-mer does not rapidly exchange between the
free and bound states (Fig. 2B). Such slow exchange
kinetics, although often associated with complexes of higher affinity
than the 30 µM KD of the 7 19-mer
(57), has also been observed in the interaction between
cyclin-dependent kinase 2 and a fragment of its inhibitor p21Waf1/Cip1/Sdi1, which is characterized by an affinity in
the 105 M range (64). The p21 fragment
undergoes a binding-induced transition from its native unfolded state
to a folded bound state. Disorder-to-order folding transitions have
been observed recently in other proteins that are involved in
protein-protein or protein-nucleic acid interactions such as
fibronectin-binding protein (65) and the transcriptional activation
domain of the herpesvirus protein VP16 (66). The binding domains of
these proteins are unfolded in the native state and form -helical or
-hairpin structures upon interacting with their respective target
molecules. In thermodynamic terms, disorder-to-order transitions are
accompanied by a large, negative change in conformational entropy. It
is likely that the large entropic cost involved in stabilizing the 7
19-mer upon complex formation comes at the expense of association
energy. A decrease in conformational entropy has been associated with increased specificity and weaker affinity in protein-protein and protein-DNA interactions (64, 67). Indeed, the affinity of Bgtx for the
7 19-mer is relatively weak in solution. Interestingly, when the
7 19-mer is tethered to a solid surface, we observed a 250 nM IC50 value in competition studies between
125I-labeled Bgtx and unlabeled Bgtx (data not shown). The
increased affinity observed in solid-phase studies may be explained by
conformational constraint of the 7 19-mer that lowers the entropic
cost of complex formation of an otherwise random coil peptide.
Consistent with this reasoning, the 18-mer (Fig. 8), which binds
Bgtx with a KD of 65 nM (24) and shows
no discrepancy between solution-based and solid-phase assays (data not
shown), exhibits significant -sheet secondary structure in CD
studies.2
Comparison of nAChR Peptide·Bgtx Complexes--
The 7 19-mer
binds Bgtx between fingers I and II like all nAChR-derived peptides
studied by solution NMR. This is illustrated in a comparison of the
18-mer·Bgtx (Research Collaboratory for Structural Bioinformatics
Protein Data Bank code 1IDH) and 7 19-mer·Bgtx complexes (Fig.
7). The contact zone on the 7 19-mer,
involving chiefly Phe186-Glu188 and
Tyr194, is consistent with a two-site model of the
Bgtx-nAChR interaction within the major determinant of Bgtx binding
(68). We find that Ala7, Ser9, and
Ile11 at the tip of finger I in Bgtx make extensive
contacts with Phe186 of the 7 19-mer, a strongly
conserved residue among Bgtx-sensitive -subunits (Fig.
8 and Table IV). Similar intermolecular
NOEs are observed between finger I residues of Bgtx and the homologous aromatic residue of the 12-mer, a phage display library-derived 13-mer (named LLPep) and two high affinity engineered 13-mers based on
the library lead (named HAPep and HAP; Fig. 8) (14, 17, 18, 20).
Interestingly, mutation of finger I residues in the related long toxin
Cbtx does not significantly affect its affinity for the 7 nAChR
(63), possibly because finger I of Cbtx is two residues shorter than
that of Bgtx. The importance of the length of the first finger for
making contacts with the muscle-type nAChR is also reflected in short
-neurotoxins that are distinguished from long toxins by the
increased length of their first finger. Mutations at the tip of the
first finger of the short toxins erabutoxin a and Naja mossambica
mossambica I (NmmI) cause a decrease in affinity for the
Torpedo nAChR (11, 69).
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Structural comparison of the
18-mer·Bgtx and 7
19-mer·Bgtx complexes. Backbone traces of the 18-mer·Bgtx
complex on the left and the 7 19-mer·Bgtx complex on
the right display Bgtx in blue and the peptides
in red. Shown here is the most representative member of each
of the 18-mer·Bgtx (PDB code 1IDH) and 7 19-mer·Bgtx
ensembles (PDB code 1KC4), as calculated by NMRCLUST (82). Only
residues involved in intermolecular contacts with Bgtx or in long range
intrapeptide contacts are presented:
Tyr189-Thr196 in the 18-mer (1
numbering, Tyr186-Thr193 by 7 numbering)
and Ser185-Tyr194 in the 7 19-mer (7
numbering). The N terminus of each peptide is colored green
and the adjacent cysteine residues in yellow. The figure was
prepared using the program MOLMOL (53).
|
|
View larger version (32K):
[in this window]
[in a new window]
|
Fig. 8.
Sequence comparison of Bgtx-binding
proteins. Amino acids are numbered according to the chick 7
sequence (27). Those residues shaded in light gray are
conserved across species and between 1 and 7 nAChR subunits. They
also have been localized to the binding site by chemical modification
or photoaffinity labeling (1). A, alignment of nAChR and
AChBP sequences. Glu188 (dark gray shading), a
residue that interacts with Lys38 on the convex side of
Bgtx, is conserved among 7 subunits of various species.
B, alignment of Bgtx-binding peptides studied by NMR and
x-ray crystallography. 12-mer (14), 18-mer (15, 16), and p25
(19) sequences are derived from the Torpedo 1 subunit.
LLPep is a phage display library-derived peptide isolated
for its ability to bind Bgtx (17), and HAPep and HAP are high affinity
engineered peptides based on the library lead (18, 20).
|
|
On finger II of Bgtx, at Val39 and Val40, we
find that multiple contacts are made with Phe186 and
Tyr187 in the 7 19-mer. Our structural data confirm the
importance of similar hydrophobic interactions observed between
aromatic residues at 1 homologous receptor positions 189 and 190 and
Val39 and Val40 of Bgtx in complexes with the
12-mer, 18-mer, LLPep, and HAPep (see Refs. 14 and 16-18; Fig.
8). Taken together, these results indicate that Val39 and
Val40 in Bgtx are important for binding multiple nAChR
subtypes through interactions with these adjacent aromatic residues,
highly conserved among Bgtx-sensitive -subunits. As hydrophobic and,
in particular, aromatic residues are directly involved in many
protein-protein interactions (70), these residues may provide binding
stability and specificity and may serve as a nucleating site for
subsequent nAChR subtype-specific interactions
-Bungarotoxin and Its Complex with
the Principal 
§,
, and
TOP
ABSTRACT
INTRODUCTION
