From the Department of Molecular Pharmacology,
Physiology, and Biotechnology, Brown Medical School,
Providence, Rhode Island 02912
Received for publication, March 14, 2001, and in revised form, April 12, 2001
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ABSTRACT |
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The region encompassing residues 181-98 on the
The nicotinic acetylcholine receptor
(nAChR)1 (1) has long been a
prototype for ligand-gated ion channels. This receptor is involved in
excitatory synaptic transmission at the neuromuscular junction and also
plays an important role in the nervous system. The nAChRs are
pentameric complexes composed of homologous subunits with subunits
arranged around the central channel in a symmetrical manner. The
muscle-type nAChR contains two The snake venom-derived From its x-ray structure, Bgtx is a relatively flat, slightly concave,
disc-shaped protein with a characteristic three-finger folding motif
consisting of three loops of structure (6). Previous NMR structural
studies indicate that the solution structure of Bgtx, although
generally consistent with the x-ray structure, does reveal some
differences (7). Notably, the side chain of Trp28 in the
two structures resides on opposite sides of the major plane of the
molecule. In the solution structure, the Trp side chain is on the
concave surface, as seen with most other Previous work indicates that the main determinants for Bgtx binding to
the muscle-type nAChR lie between residues 173 and 204 of the We previously described some of the structural features revealed by an
NMR analysis of Bgtx complexed with a 12-amino acid peptide fragment
( Expression Construct--
We designed an oligonucleotide
sequence to encode for residues 181-198 (YRGWKHWVYYTCCPDTPY) of the
Cell Growth--
Cell cultures were grown in standard M9 medium
except that 15NH4Cl was used as a replacement
for normal NH4Cl. All cultures were supplemented with 100 µg/ml ampicillin (M9/Amp). A single colony from a fresh agar plate
containing ampicillin was used to inoculate 100 ml M9/Amp medium and
grown overnight at 37 °C. The overnight culture was added to 2 liters of M9/Amp medium in a VirTis benchtop fermentor. This culture
was grown at 37 °C with stirring at 600 rpm until the
A600 was 0.7-0.8.
Isopropyl- Nickel Affinity Chromatography--
After resuspension in 40 ml
of 20 mM Tris-HCl buffer (pH 7.9), cells were passed
through a French pressure cell (SLM Instruments) at 15,000 p.s.i.
Inclusion bodies were isolated by centrifugation, resuspended in
"binding buffer" (6 M guanidine-HCl, 0.5 M
NaCl, 5 mM imidazole, and 20 mM Tris-HCl (pH
7.9)), and applied to a column containing Ni2+-charged
His-Bind resin (Novagen) prepared according to the manufacturer's specifications. After washing the resin with 10 column volumes of
binding buffer and 5 column volumes of wash buffer (6 M
guanidine-HCl, 40 mM imidazole, 0.5 M NaCl, and
20 mM Tris-HCl pH 7.9), the ketosteroid isomerase fusion
protein was eluted in 5 column volumes of elution buffer (6 M guanidine-HCl, 0.3 M imidazole, 0.5 M NaCl, and 20 mM Tris-HCl (pH 7.9)).
CNBr Cleavage--
The fusion protein prepared as described
above was dialyzed against water, and the insoluble fusion protein was
isolated by centrifugation. The pellet was then resuspended in 20 ml of
80% formic acid in a round-bottom flask and mixed with 1 g of
cyanogen bromide (CNBr). After flushing the solution with helium, the
flask was sealed, and the reaction was allowed to proceed in the dark for 24 h. The reaction mixture was then diluted 1:1 with water and
applied to a C18 Sep-Pak cartridge (Waters). The peptide
was eluted with 4 ml of 35% acetonitrile in water and dried using a
SpeedVac (Savant). The dry peptide was resuspended in 50 mM sodium phosphate buffer (pH 6.0) at 37 °C for 2 days to deformylate the products.
Reverse Phase-HPLC Purification--
The peptide sample prepared
as described above was diluted 1:1 with 0.1% trifluoroacetic acid
(buffer A) and applied to a C18 reverse phase Discovery
column (Supelco). The peptides were eluted isocratically at 1 ml/min
with buffer B (25% acetonitrile with 0.1% trifluoroacetic acid). Peak
fractions were collected and then dried using a SpeedVac (Savant).
Isolated peptides were analyzed by mass spectrometry (Yale Cancer
Center Mass Spectrometry Resource and W. M. Keck Foundation
Biotechnology Resource Laboratory). The disulfide form of the peptide
with a C-terminal homoserine lactone was chosen for further structural analysis.
NMR Sample Preparation--
The 15N-labeled
disulfide homoserine lactone form of the Torpedo NMR Experiments--
All NMR spectra were recorded on a Bruker
Avance 600-MHz NMR spectrometer at a temperature of 35 °C. Chemical
shifts at this temperature were calibrated with respect to internal
3-(trimethylsilyl) tetradeutero sodium propionate (0.0 ppm). The
formation of the Bgtx·
In comparing our results with earlier, more preliminary assignments
involving an unlabeled Conformation Calculations--
The cross-peak volumes in
the two-dimensional NOESY spectra were integrated by the Gaussian
fitting protocol using SPARKY. The cross-peaks were classified into
three categories: strong, medium, and weak, with corresponding distance
ranges of 1.8-3.0, 1.8-4.0, and 1.8-5.0 Å, respectively. The
HN-H
We used Rasmol (41), MOLMOL (42), and INSIGHT II (Molecular
Simulations, Inc.) for the graphical analysis of the calculated structures. The surface charge potentials were calculated using MOLMOL.
The contact surface areas of all the final individual Bgtx· Preparation of 15N-peptide and Its
Purification--
To facilitate the assignment of the The Formation of a Stoichiometric Bgtx· NMR Assignments--
Because only the peptide is
15N-enriched, 15N three-dimensional NMR
experiments can be used to filter out Bgtx proton signals that are not
correlated to 15N. Making use of this enrichment,
three-dimensional TOCSY-HSQC, NOESY-HSQC, and HNHA experiments were
obtained to make preliminary amino acid assignments of the
Based on our three-dimensional NMR assignments, our two-dimensional NMR
data (NOESY and TOCSY), and published Bgtx assignments (7, 36), we
completed the assignment of the resonances obtained with the
Bgtx· Three-dimensional Structure Calculations and Comparison--
The
distance constraints resulting from the NOEs and the dihedral
angle restraints obtained from the HN-H
We did not find any inter-residue NOE constraints involving the last
two residues of Bgtx, Pro73 and Gly74. If these
two residues are omitted from the r.m.s.d. calculation for Bgtx, the
overall backbone r.m.s.d. is 1.98 Å for free Bgtx and 1.81 Å for
bound Bgtx in the Bgtx·
A comparison of free and bound Bgtx reveals that the proton resonances
of a number of amino acids in Bgtx undergo large chemical shift changes
upon peptide binding. These shift changes reflect significant
alterations in the chemical environment of those protons. We find that
Ala7 (C
As viewed in Fig. 6A, free Bgtx is oriented with loop I
(blue) on the left, loop III on the right, and the tip of
loop II (green) at the bottom. This view corresponds with
the so-called "concave" surface of the
To evaluate the significance of the observed change in the conformation
of Bgtx upon binding, we compared the mean pairwise backbone r.m.s.d.
within the ensemble of free structures with the mean pairwise backbone
r.m.s.d. across ensembles (i.e. each bound structure to each
free Bgtx structure). Such a comparison allows us to determine if the
two data sets, the ensembles of free and bound structures, represent
significantly different structures. The mean pairwise backbone r.m.s.d.
among the 20 free Bgtx structures is 2.94 ± 0.38 Å. Similarly,
among the 20 bound Bgtx structures, the pairwise r.m.s.d. is 2.82 ± 0.25 Å. In contrast, the mean pairwise backbone r.m.s.d. between
the 20 bound Bgtx structures and the 20 free Bgtx structures is
6.05 ± 0.37 Å. Thus, the mean pairwise r.m.s.d. across the two
ensembles differs by more than 6 S.D. from the mean pairwise r.m.s.d.
for the ensemble of free (or bound) Bgtx structures. This analysis
indicates that the ensemble of bound structures is indeed significantly
different from the ensemble of free structures.
We observed five pairs of
C Binding Interactions--
The structures indicate that Bgtx
interacts with the
The NMR studies of complexes formed between peptides and peptide
binding domains have been very instrumental in many other systems in
elucidating mechanisms of molecular recognition (45). In seeking to
understand the structural basis for the relatively high affinity
(KD ~ 65 nM) binding observed between
Bgtx and the One of our most striking observations is the binding-associated
re-orientation in several segments of Bgtx that normally are not in
contact with one another. Loop I and the C-terminal tail segment alter
their configuration with respect to the main body of the protein, and
the tip of loop II undergoes a change in its relative curvature and
shape (Fig. 7). Together, these changes suggest a considerable
coordinated reconfiguration of Bgtx upon interacting with receptor
sequences. Such a reconfiguration is consistent with the extensive
flexibility that has been noted in these and other protein toxins. It
has been suggested that such flexibility may have evolved to serve
important functional purposes (e.g. Ref. 46).
We previously reported the solution structure of the complex formed
between Bgtx and a dodecapeptide corresponding to amino acid residues
185-196 from the The Bgtx· In the Bgtx· Mutational analysis of loop I residues in the long The intermolecular NOE between Pro69 and the backbone NH of
Cys192 together with the chemical shift changes observed
within the C-terminal tail region upon formation of the
Bgtx· The proximity of the positively charged Bgtx residues
(Lys38 and Arg36) with the aromatics of the
peptide (Tyr189 and Tyr190) in the
Bgtx· An important contact role for Tyr189 is consistent with
chimeric analysis and toxin footprinting studies of nAChR Cation- In the Bgtx· In summarizing the contact information, we find that Bgtx interacts
with the 1 subunit of the muscle-type nicotinic acetylcholine receptor forms
a major determinant for the binding of
-neurotoxins. We have
prepared an 15N-enriched 18-amino acid peptide
corresponding to the sequence in this region to facilitate structural
elucidation by multidimensional NMR. Our aim was to determine the
structural basis for the high affinity, stoichiometric complex formed
between this cognate peptide and
-bungarotoxin, a long
-neurotoxin. Resonances in the complex were assigned through
heteronuclear and homonuclear NMR experiments, and the resulting
interproton distance constraints were used to generate ensemble
structures of the complex. Thr8, Pro10,
Lys38, Val39, Val40, and
Pro69 in
-bungarotoxin and Tyr189,
Tyr190, Thr191, Cys192,
Asp195, and Thr196 in the peptide participate
in major intermolecular contacts. A comparison of the free and bound
-bungarotoxin structures reveals significant conformational
rearrangements in flexible regions of
-bungarotoxin, mainly loops I,
II, and the C-terminal tail. Furthermore, several of the calculated
structures suggest that cation-
interactions may be involved in
binding. The root mean square deviation of the polypeptide backbone in
the complex is 2.07 Å. This structure provides, to date, the highest
resolution description of the contacts between a prototypic
-neurotoxin and its cognate recognition sequence.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 subunits and one each of the
1,
(
), and
subunits. The ligand binding sites are situated at
the
(
) and
subunit interfaces. The muscle-type nAChR
serves as an important model for the study of the structures and
functions of related ligand-gated ion channels (for review, see Ref.
1).
-neurotoxins fall into two categories, short
and long neurotoxins, and act as high affinity competitive antagonists
at the nAChR. Short neurotoxins (e.g. erabutoxin
a) contain 60-62 amino acid residues and 4 conserved
disulfide bridges. Long neurotoxins have 66-74 residues and 5 disulfide bonds, including four in a core region that are homologous in
position to those found in the short neurotoxins.
-Bungarotoxin
(Bgtx), obtained from the snake venom of Bungarus
multicinctus, is a long
-neurotoxin that over the years has
provided a powerful tool for the study of muscle-type nAChRs and which
has come to be viewed as somewhat of a gold standard among the
-neurotoxins. A number of the
-neurotoxins have been
heterologously expressed in recent years, allowing for investigations
using site-directed mutagenesis (2-5).
-neurotoxins containing
this highly conserved residue. In contrast, the Trp side chain is
located on the opposite face in the crystal structure (6). The
structures of several other snake venom
-neurotoxins have been
studied with NMR techniques (8-12), and all exhibit the characteristic
three-finger structure.
1
subunit (13), a region that coincides with one of three segments of the
subunit that have been implicated in agonist binding (for review,
see Ref. 1). Tyr190, along with Cys192,
Cys193, and Tyr198 are selectively cross-linked
with a variety of site-directed photoaffinity reagents (14). This
region, termed segment C, contains a conserved pair of adjacent Cys
residues, Cys192-Cys193, that form an unusual
disulfide. Studies of synthetic peptides with sequences matching those
in segment C have identified several peptides that bind Bgtx with
affinities in the micromolar to submicromolar range (15, 16). A peptide
fragment (
18-mer) with a sequence corresponding to amino acid
residues 181-198
(
-Y181RGWKHWVYYTCCPDTPY198) from the
Torpedo californica nAChR binds Bgtx with an apparent KD of ~65 nM (17). Replacing the Tyr
at position 190 with a Phe leads to a 60-fold decrease in Bgtx binding
affinity for the altered peptide, suggesting an important role for this aromatic residue in complex formation (17). Mutations of
Tyr190, when assessed in heterologous expression systems,
also result in large decreases in
-neurotoxin binding (3, 18, 19). In addition, ligand gating is also dramatically affected by mutations in Tyr190 (20, 21). Studies with recombinant receptor
fragments corresponding to the
subunit from the mongoose nAChR,
which is resistant to
-neurotoxins, suggest two subsites in the
binding domain for Bgtx; one is a proline subsite consisting of
Pro194 and Pro197, and the other is an aromatic
subsite involving positions 187 and 189 (15).
12-mer) of the Torpedo nAChR
1 subunit
(
-K185HWVYYTCCPDT196), which has an apparent
KD of ~1.4 µM (17, 22). We now
describe a more expansive NMR structural analysis of the higher
affinity complex formed with the
18-mer. The structure of this
complex may provide valuable information on the orientation of the
contact residues in the native nAChR and may help in elucidating the
essential interactions that direct the ability of the
-neurotoxins to recognize receptor sequences with remarkable affinity and specificity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 subunit from the nAChR of T. californica. Three copies
of this expression cassette were inserted downstream of a 125-amino
acid ketosteroid isomerase gene and upstream of a His-tag sequence in
plasmid pET-31b(+) (23). The final construct, ketosteroid
isomerase-Met-(
18-mer-Met)3-His-tag, contained single
Met residues separating the three cassettes from each other, from
ketosteroid isomerase, and from the C-terminal His tag. The
oligonucleotide sequence of this construct is available upon request.
The plasmid, whose insert sequence was confirmed by DNA sequence
analysis, was used to transform cells of the expression strain BL21
(DE3) (Novagen).
-D-thiogalactoside was added to a
concentration of 1 mM to initiate induction of the fusion
protein. After 3 h, cells were harvested by low speed centrifugation.
18-mer
was resuspended in 50 mM perdeuterated potassium acetate buffer (pH 4.0) with 5% D2O and 0.05% sodium azide. Bgtx
(from Sigma) was prepared in the same buffer at a concentration of 5 mM. Bgtx from this stock solution was added to the
18-mer to form a 1:1 Bgtx·
18-mer complex. The final
concentration of the Bgtx·
18-mer complex was 2.1 mM.
The "free Bgtx" NMR sample was diluted from stock Bgtx into the
same buffer to a final concentration of 2.0 mM.
18-mer complex was followed in a mole-ratio
titration using a two-dimensional 15N heteronuclear single
quantum correlation (1H-15N HSQC) (24-26)
experiment. Amino acid spin systems were identified by two-dimensional
total correlation spectroscopy (TOCSY) (24, 25, 27) and
three-dimensional TOCSY-HSQC experiments (27-31) with a mixing time of
60 ms. The assignments of the NH protons and C
H protons
of the amino acid spin systems of the peptide were further confirmed by
a three-dimensional HNHA experiment (32, 33). The three-dimensional
HNHA experiment provides the correlation between the 15NH
proton and the C
H proton of the same amino acid; these
data help confirm the identification of the NH and C
H
protons. Nuclear Overhauser effect (NOE) correlations (sequential, medium-range, and long range NOEs) were identified by two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) (24, 25) and
three-dimensional NOESY-HSQC experiments (28-31) with a mixing time of
120 ms. Spectra from these experiments were also collected at 25 °C
to facilitate the assignment of resonances. All NMR spectra were
processed and analyzed with XwinNmr (Bruker), NMRPipe (34), and
SPARKY (35).
18-mer peptide bound to Bgtx (36), we found
that most Bgtx assignments are the same or similar (chemical shifts
change less than 0.05 ppm) after calibration. However, the assignments
of Val2, His4, Ser9,
Ile11, Lys26, Cys29,
Cys33, Val40, Lys51,
Lys52, Lys70, Gln71,
Arg72, and Gly74 were significantly different;
in most cases no comparable resonances were observed in our
two-dimensional NOESY spectra. On the other hand, new resonance peaks
appear elsewhere in the spectra. All these new resonances involving
these residues were re-assigned based on sequential NOE connectivity.
We think it most likely that the observed chemical shift differences
between the two samples are caused by a difference in the ionic
strength of the two samples even though both were prepared at pH 4.0 and spectra were acquired at 35 °C. Our present sample is dissolved
in 50 mM potassium acetate, whereas the earlier sample was
simply adjusted to pH 4.0 with the addition of HCl. A change in the
ionic environment could have significant effects on electrostatic
interactions between side chains of charged residues, leading to
changes in the chemical environment of a subset of spins. Similarly, it
was necessary to re-assign most of the
18-mer peptide resonances. Of
the eight previously assigned peptide residues, only Asp195
and Thr196 are unchanged between the two samples. However,
the C
H proton and C
H proton of
Thr196 were erroneously assigned previously (36). The new
swapped assignments incorporate the results from the three-dimensional HNHA experiment.
3J coupling
constants of the
18-mer peptide were obtained from the
three-dimensional HNHA experiment (37). The 3J
coupling constants were converted to dihedral angle restraints using
previously described methods (38). For 3J < 6 Hz, the dihedral angle restraint was assigned to
60° ± 30°;
for 3J > 8 Hz, the dihedral angle
restraint was
120° ± 40°. All structures were calculated with
distance geometry and simulated annealing protocols using the dg_sa.inp
script of the NMR structure calculation program, CNSsolve (39). The
following is the potential energy function used in these calculations:
Ftotal = Fbon + Fang + Fimp + Fvdw + Fnoe + Fcdih, where Fbon relates
to bond length, Fang and
Fimp to bond angles, Fvdw
relates to the van der Waals repulsion term,
Fnoe relates to NOE distance constraints, and
Fcdih relates to dihedral angles. Pseudoatoms
were used for protons that could not be stereospecifically assigned.
The pseudoatom correction feature of CNSsolve was used to adjust the
NOE distance constraint range automatically. In each batch of
calculations, a different random seed number was used to initiate the
calculation of a set of 50 structures. From the pool of calculated
structures, only those structures lacking any NOE violation (>0.5 Å)
were selected as "acceptable" for further analysis. As a result of
the weighting of the Fnoe term in CNSsolve, none
of the other energy terms were as critical as
Fnoe in determining an acceptable structure. In total, 120 acceptable structures of the Bgtx·
18-mer complex were obtained from 6 batches of independent calculations (i.e.
300 total structures), and 122 acceptable structures of free Bgtx were
obtained from 8 batches of independent calculations (i.e. 400 total structures). The 20 lowest-energy structures out of the
acceptable structures for free Bgtx and for the Bgtx·
18-mer complex were selected to form an ensemble of representative final structures. The mean structure corresponding to each ensemble was
calculated by a program written by Dr. Christian Rölz (40). The
two mean structures (free Bgtx and Bgtx·
18-mer complex) were further partially energy-minimized using DISCOVER (Molecular
Simulations, Inc.) to create representative structures complete with
side chains. All structures depicted in Fig. 7 have been deposited into
the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics. The four files corresponding to the mean and ensemble
structures for the Bgtx·
18-mer complex and for free Bgtx have been
assigned the identifiers 1IDG, 1IDH, 1IDI, 1IDL.
18-mer
complex structures were calculated by contacts of structural units
(CSU) using CSU software (43). The energetically significant cation-
interaction analysis of the Bgtx·
18-mer complex structures was
performed using the CaPTURE program (44).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
18-mer
peptide resonances while complexed with Bgtx, the peptide corresponding
to Torpedo nAChR
1 subunit residues 181-198 was
expressed heterologously in Escherichia coli as part of an
insoluble fusion protein under conditions where
15NH4Cl was used as sole source of nitrogen.
After isolation of the fusion protein, CNBr cleavage at engineered Met
sites was used to release the desired peptide, which contained the 18 residues of
1 subunit with an additional C-terminal residue derived
from the engineered Met. As expected for CNBr cleavage of Met sites, the peptides isolated are a mix of the C-terminal homoserine form of
the peptide and its corresponding dehydrated homoserine lactone form.
HPLC analysis revealed three major peptide peaks which were further
characterized (Fig. 1). Preliminary
solid-phase binding studies indicated that all three peptide fractions
bind Bgtx to an extent comparable with that obtained with a similar
synthetic
18-mer peptide lacking the C-terminal homoserine (data not
shown). All three peptide fractions were resistant to thiol alkylation with N-ethylmaleimide except after prior incubation of the
peptide with dithiothreitol. These results suggest that the adjacent
cysteines, Cys192 and Cys193, are in the
disulfide state in the isolated peptides. Mass spectrometric analysis
revealed that P2 corresponds to the C-terminal homoserine lactone form of the
18-mer, whereas P1 is the C-terminal
homoserine form. The P2 peptide was chosen for the
production of a Bgtx·
18-mer complex.
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Fig. 1.
C18-reverse phase HPLC
purification of the 18-mer. The peptide
peak labeled P1 corresponds to the disulfide homoserine
form of the
18-mer; P2 is the disulfide homoserine
lactone form of the
18-mer that was collected and used in this NMR
study. P4 is an unidentified form of the
18-mer, which
slowly converts to P2. See "Experimental Procedures"
for a detailed description of chromatographic conditions.
18-mer
Complex--
The pure
18-mer (P2) was resuspended in 50 mM perdeuterated potassium acetate buffer (pH 4.0) and
analyzed with a two-dimensional 1H-15N HSQC
experiment that is designed to acquire signal only from protons bound
to 15N (Fig. 2A).
An equimolar amount of Bgtx was then added, and the sample was again
analyzed by HSQC (Fig. 2B). A comparison of these spectra
(Fig. 2) clearly demonstrates the formation of a stoichiometric complex
between the
18-mer and Bgtx. The free peptide (Fig. 2A) appears to be largely unstructured; all the NH resonances are poorly
dispersed in chemical shift and vary in intensity. In contrast, after
binding to Bgtx (Fig. 2B), nearly all the NH resonances undergo large chemical shift changes, and there is little evidence of
any free peptide remaining based on the disappearance of resonances seen in the free peptide. These observations suggest that the
18-mer
adopts a defined structure upon binding to Bgtx. Furthermore, in
mole-ratio titration studies with less than stoichiometric concentrations of Bgtx, NH resonances corresponding to both the bound
and the free peptide are present, and the chemical shift of the NH
resonances for the bound peptide are fixed and do not vary with Bgtx
concentrations (data not shown). These results indicate that the
Bgtx·
18-mer complex is in slow exchange.
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Fig. 2.
Comparison of the HSQC spectra of free
18-mer and of the
Bgtx·
18-mer complex. A, HSQC
spectrum of free
18-mer at a concentration of 1.0 mM.
B, HSQC spectrum of the stoichiometric Bgtx·
18-mer
complex at a concentration of 2.1 mM. The NH resonance
assignments were determined as described in the text. W184
S. C. is the side chain NH of Trp184; W187
S. C. is the side chain NH of Trp187.
18-mer in
its bound form. Fig. 3 illustrates a
representative strip analysis used to identify the resonances of
Lys185. Three-dimensional TOCSY-HSQC analysis is used to
identify the resonances correlated by through-bound scalar connectivity
to the 15NH (i.e. C
H proton,
C
H proton, etc.). Using these three-dimensional NMR
experiments, we assigned the observable resonances for all of the amino
acid residues in the
18-mer except for the N-terminal
Tyr181, which has an exchangeable NH, the C-terminal
homoserine lactone, whose mobility may cause its signals to be too weak
to be identified, and the two prolines, which lack amide protons (Fig.
2B). These assignments of the peptide resonances greatly
facilitated the assignment of the Bgtx resonances in the homonuclear
two-dimensional NMR data obtained with the complex.
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Fig. 3.
Multi-dimensional NMR strip analysis
of the 15N-enriched 18-mer
illustrating the assignment of Lys185. Strip A is from
a three-dimensional TOCSY-HSQC experiment that identifies the amino
acid spin system of Lys185. Strip B is from a
three-dimensional NOESY-HSQC experiment that depicts the short and
medium range NOEs involving the NH of Lys185, and strip C
is from a three-dimensional HNHA experiment showing the correlation
between the NH proton and the C
H proton of
Lys185. NH, amide proton;
d
H, the sequential C
H
proton from Trp184;
H, the C
H
proton; d
H, the sequential C
H
proton from Trp184;
Hs, the C
H
protons.
18-mer complex. Fig. 4
summarizes the C
Hi proton to
NHi+1 proton NOEs (sequential NOE), the
C
Hi proton to
NHi+1 proton NOEs, and the NHi proton to NHi+1
proton NOEs in the Bgtx·
18-mer complex used to complete the
connectivity of the polypeptide backbone. In addition, 11 unambiguous
intermolecular NOEs on the NH region of the peptide were assigned
(Table I). Additional intermolecular NOEs
in the C
H proton and C
H proton regions
were observed but have not as yet been unambiguously assigned. We
performed a similar analysis on free Bgtx to generate the structure
important for comparison of the free and bound forms of Bgtx.
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Fig. 4.
Structurally relevant NOEs from the
two-dimensional NOESY spectra of the
Bgtx· 18-mer complex. The thickness of
the bars indicates the NOE intensity (strong,
d < 3.0 Å; medium, d < 4.0 Å; weak,
d < 5.0 Å).
Observed intermolecular NOEs
3J couplings (Table
II) were then incorporated into CNSsolve
for structure determination. These calculations utilized both distance geometry and simulated annealing protocols. Twenty structures of free
Bgtx and of the Bgtx·
18-mer complex with the lowest potential energy and no NOE violation larger than 0.5 Å are superimposed and
shown as an ensemble in Fig. 5. In Fig.
6, the mean structure of each ensemble,
partially energy-minimized, is shown to illustrate some of the key
structural features. The overall backbone atomic root mean square
deviation (r.m.s.d.) between the individual structures and the mean
structure of free Bgtx is 2.03 Å, whereas that of the Bgtx·
18-mer
complex is 2.07 Å. The pairwise r.m.s.d. between the two mean
structures is 6.05 Å.
Structural statistics for free Bgtx and for the Bgtx · 18-mer
complex
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Fig. 5.
Structural comparison between
free-Bgtx and the Bgtx· 18-mer complex.
A, twenty superimposed structures calculated based on
distance constraints in free Bgtx. The red lines mark those
Bgtx regions that undergo large chemical shifts upon
18-mer binding.
B, twenty superimposed structures calculated based on
distance constraints for the Bgtx·
18-mer complex. The blue
lines correspond to Bgtx, and the green lines signify
the
18-mer. The black lines and the labels N
and C mark the N and C termini of Bgtx. The labels
N' and C' mark the N and C termini of the
18-mer. Only backbone traces are shown. The figures were prepared
using the program MOLMOL (42).
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Fig. 6.
Stereo views of the mean calculated
structures for free Bgtx and for the
Bgtx· 18-mer complex. A, free
Bgtx. B, the Bgtx·
18-mer complex. These are mean
structures calculated from the appropriate ensemble of the 20 lowest
energy structures (see Fig. 5). The structures have been partially
energy minimized to remove artifacts from averaging.
18-mer complex. A comparison of the
r.m.s.d. determinations for various sequence segments within Bgtx
indicates considerable regional variation in r.m.s.d. as summarized in
Table II. The stem region of loop II is well defined (r.m.s.d. no
greater than 0.90 Å in Bgtx·
18-mer and 1.19 Å in free Bgtx)
compared with loop II when considered in its entirety (r.m.s.d. of 1.56 Å in Bgtx·
18-mer complex and 1.51 Å in free Bgtx). The tip of
loop II can also be reasonably well superimposed (r.m.s.d. of 1.29 Å in Bgtx·
18-mer complex or 1.14 Å in free Bgtx).
H), Ser9
(C
H, C
H), Ile11 (NH,
C
H, C
H, C
H),
Trp28 (NH), Cys29 (NH, C
H),
Asp30 (NH), Phe32 (NH), Cys33
(C
H), Arg36 (NH), Gly37 (NH,
C
H), Lys38 (NH, C
H,
C
H), Val39 (C
H),
Val40 (NH, C
H), Lys52
(C
H), Val57 (C
H),
Lys64 (C
H), Asn66
(C
H), Lys70 (NH, C
H),
Gln71 (C
H), and Arg72
(C
H, C
H, C
H) all are
characterized by chemical shift changes greater than 0.2 ppm upon
binding. These amino acid residues are highlighted in red in the
backbone structures shown in Fig. 5A. The changes in
Ala7, Ser9, and Ile11 suggest that
the outer tip of loop I participates in binding the
18-mer. This is
substantiated by the intermolecular NOEs observed between the
C
H and C
H protons of Pro10
and the NH protons of Asp195 and Thr196 in the
peptide (Table I). Similarly, the chemical shift changes of
Arg36-Val40 are correlated with intermolecular
NOEs involving Lys38, Val39, and
Val40 in Bgtx loop II. In the C-terminal tail region, the
chemical shift changes in Lys70-Arg72 accompany
intermolecular NOEs between the C
H protons of
Pro69 and the NH of Cys192 in the peptide
(Table I). In contrast to these examples of shift changes correlated
with intermolecular contacts, several other shift changes in Bgtx
residues appear to be due to conformational changes involving possible
reorientations about the central triple-stranded
-sheet common to
all
-neurotoxins. We believe that the chemical shift changes in the
Trp28-Asp30, Phe32,
Cys33 region and in Val57 are caused by general
movement about the
-strand involving loop II and the proximal
portion of loop III. Such secondary effects could also explain the
chemical shift change of the side-chain C
H proton in
Lys52 of loop III. Finally the chemical shift changes
involving the side chains of Lys64 and Asn66 at
the beginning of the C-terminal tail may reflect a change in chemical
environment due to the apparent relocation of the distal C-terminal
region to make a contact with the
18-mer peptide bridging the gap
between Bgtx loop I and loop II (Fig. 5).
-neurotoxins (6, 12). In
the free form of Bgtx, the tip of loop II forms the lower rim of this
concave surface, and the loop I and loop II of free Bgtx are well
separated with no inter-loop NOE constraints. The distance between the
N of Ser34 and the N of Pro10 in free Bgtx is
~31 Å. Upon peptide binding, both loop I and loop II interact with
peptide residues (see below), as revealed by intermolecular NOEs
(Table I). As a consequence of binding, loop I and II move closer to
each other; the average distance between the N of Ser34 and
the N of Pro10 narrows to ~24 Å. In addition, the tip of
loop II switches to a convex conformation upon binding (Fig. 6 and Fig.
7, A-D). The C-terminal tail
region of free Bgtx is relatively unconstrained in free Bgtx but
participates in peptide interaction in the bound state (see
below). The rest of the structure appears to change little upon
complex formation (Fig. 6B). To provide a full comparison of
binding-induced structural changes in Bgtx, we have superimposed the
mean backbone structures (Fig. 7, B and D) and
the full ensemble of structures (Fig. 7, A and C)
as stereo images. The red traces correspond to the backbone
of free Bgtx, whereas the blue traces refer to the backbone
of bound Bgtx. The
18-mer backbone is colored green. In
the front view (Fig. 7, A and B), loop I and loop
II move toward each other and toward the peptide, whereas loop III is
largely unaltered. In the left-profile view, the
18-mer has been
removed for a better view of the changes in the Bgtx backbone (Fig. 7,
C and D). The C-terminal tail is shifted to the
right (toward the peptide) in the bound state. The tip of loop II moves left, highlighting the change in general orientation in this region from a concave to a convex surface.
View larger version (46K):
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Fig. 7.
Stereo views comparing the polypeptide
backbone traces of bound and free Bgtx. A and
C, a view of the ensemble of structures for free Bgtx and
for the Bgtx· 18-mer complex. B and D, a view
of the two mean structures calculated from the ensembles. In
A and B, the views correspond to the concave
surface of Bgtx. In C and D, Bgtx is rotated
~90° to the right as compared with the view in A and
B (the
18-mer is not shown). The red traces
correspond to free Bgtx, the blue traces correspond to Bgtx
in the Bgtx·
18-mer complex, and the green traces
correspond to the
18-mer. The figures were prepared using the
program MOLMOL (42).
H-C
H long-range NOEs in the
two-dimensional NMR study of the
18-mer complex. These included the following pairs of residues: Cys23-Cys44,
Tyr24-Cys59,
Arg25-Leu42,
Lys26-Val57, and
Met27-Val40. These NOEs were previously
reported as evidence for an anti-parallel triple-stranded
-sheet in
the solution structure of Bgtx (7), and similar
-sheet defining,
inter-strand NOEs have been observed in the solution structures of
other
-neurotoxins (12).
18-mer at three sites. These are the tip of loop
I, the C-terminal tail region (intermolecular NOEs are obtained with
Thr8, Pro10, and Pro69), and loop
II residues facing loop I including Val39,
Val40, and Lys38. Although Loop III of some
-neurotoxins (3) has been reported to be involved in binding to the
nAChR, we did not observe any direct intermolecular NOEs between the
18-mer and loop III of Bgtx. The chemical shifts of most residues in
loop III change little upon binding of the
18-mer, consistent with a
lack of involvement of Bgtx loop III in
18-mer binding.
18-mer residues responsible for the intermolecular NOEs with the
Bgtx sites are Tyr189, Tyr190,
Thr191, Cys192, Asp195, and
Thr196. The contact zone is ~18 Å in length (from N of
Tyr189 to N of Thr196), and the total van der
Waals contact surface areas range from 500 to 690 Å2, with
a mean of 580 Å2. It is noteworthy that there is a cluster
of four positive charged Bgtx residues (Arg36,
Lys38, Lys70, Arg72) near the
contact zone. In close apposition in the complex are found peptide
residues Trp187, Tyr189, Tyr190
(Fig. 8). This arrangement suggests the
possibility of cation-
interactions (44) contributing energetically
to the formation of the Bgtx·
18-mer complex. A cation-
interaction analysis carried out on the 120 Bgtx·
18-mer-calculated
structures revealed that a total of 24 individual structures, including
two from the 20 best structures, have energetically significant
cation-
interactions as determined by CaPTURE (44). In this
analysis, cation-
interactions are selected as energetically
significant if the electrostatic energy is <
2.0 kcal/mol or,
alternatively, if the electrostatic energy is <
1.0 kcal/mol and the
van der Waals interaction for the pair is <
1.0 kcal/mol (44). As
shown in Table III, the following candidate cation-
pairings were observed:
Lys38/Tyr190,
Lys38/Tyr189,
Lys38/Trp187,
Arg36/Tyr189,
Arg36/Trp187, and
Lys70/Tyr190. The
Lys38/Tyr190 pairing was observed in 12 of
these 24 structures, and one of these structures is shown in Fig.
9.
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Fig. 8.
Stereo view of the surface charge profile of
the Bgtx· 18-mer complex. Surface charge
potentials were calculated as described under "Experimental
Procedures." Blue regions show positive charge, and
red regions show negative charge. See Fig. 5B for
orientation. The figure was prepared using the program MOLMOL
(42).
Observed cation- interaction pairs
18-mer complex for cation-
interactions. Two of the
24 structures identified contained two cation-
interaction pairs. In
both cases, the two pairs were Lys38/Tyr190 and
Lys38/Trp187.
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Fig. 9.
Orientation of a suggested
Tyr190-Lys38 cation-
interaction. The two side chains are taken from one of the
20 ensemble Bgtx·
18-mer structures depicted in Fig. 5.
a, the distance between the NZ of Lys38 and the
CE2 of Tyr190 is 5.49 Å; b, the distance
between the NZ of Lys38 and the CE1 of Tyr190
is 5.85 Å; c, the distance between the CG of
Tyr190 and the NZ of Lys38 is 5.53 Å.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
18-mer, we identified a number of NOE distance
constraints that reveal important information about the interaction
between the
18-mer and Bgtx and that also define conformational
changes in Bgtx upon complex formation. These constraints have been
used to generate the structural features presented in this study. The disc-like shape of Bgtx and of the complex limits the total number of
long-distance NOEs available (NOEs between residues distant in primary
sequence), and this in turn limits the final resolution of the
structures obtained (Table II). This limitation is applicable to all
members of the
-neurotoxin family (11, 12). Finally, we compare the
structural features of the binding between Bgtx and the cognate
18-mer to the biochemical- and mutagenesis-based observations made
with the intact nAChR.
subunit of the Torpedo nAChR (22). The
apparent affinity of Bgtx for the
12-mer is about 15-20-fold lower
than for the
18-mer. In both structures, Tyr189 and
Tyr190 lie in a similar position, close to
Val39 and Val40 of Bgtx. In addition, in both
structures the peptides are relatively elongated. There are significant
differences, however, in the orientation of the two peptides relative
to Bgtx. In the Bgtx·
12-mer complex, the polypeptide backbone of
the five identified amino acid residues is between loop I and loop II,
in a position roughly parallel to loop II. In the Bgtx·
18-mer
complex, the peptide is in a more perpendicular orientation with
respect to loop II and the tip of loop I (Fig. 6).
12-mer complex may represent an intermediate stage in
binding with the aromatics of the peptide, Tyr189 and
Tyr190, forming a nucleation site for further interactions.
Although the Bgtx·
18-mer complex is more energetically favored,
the total contact surface areas are very similar, ~ 600 Å2, for the two complexes. A library-derived peptide
selected for its ability to bind Bgtx with high affinity also contains
two adjacent Tyr residues, and these residues contribute the largest contact area in the complex (47). Although this 13-residue
library-derived peptide adopts a more globular or sphere-like
conformation, it is also found localized to the cleft formed between
loop I and loop II with close apposition of the C-terminal region
(47).
18-mer complex, the intermolecular NOEs between
Pro10 and both Asp195 and Thr196
and between Thr8 and Cys192 (Table I)
demonstrate the involvement of Bgtx loop I in the formation of the
peptide complex. This finding is consistent with mutagenesis studies of
erabutoxin a, a short
-neurotoxin. Loop I mutations, S8T
and Q10A, in erabutoxin a result in a very large reduction
in binding affinity for the Torpedo nAChR (2). Recently, a
double-mutant cycle analysis involving the related short
-neurotoxin, Naja mossambica mossambica I
(NmmI), and the mouse nAChR has revealed an interaction
between Ser8 in NmmI and Tyr198 on
the
subunit at the
site (3). In contrast, no evidence of an
interaction was observed between Ser8 and
Val188 in the NmmI study. In addition,
Val188 can be energetically coupled to Arg33
and Arg36 in NmmI (19). Based on sequence
alignment, the corresponding Bgtx residues would be Arg36
and Lys38, respectively. These observations are entirely
consistent with the structure of the Bgtx·
18-mer complex; the tip
of loop I is in closer proximity to the peptide residues C-terminal to
Thr196 (Tyr198 is located at the extreme left
in Fig. 6B), whereas Val188 is removed from loop
I and in close proximity to the loop II residue, Val39
(Table I).
-neurotoxin,
-cobratoxin from Naja kaouthia venom, failed to detect a significant role for this region in binding to the Torpedo
nAChR (2). Because the sequence of the loop I region differs greatly between Bgtx and this
-cobratoxin and because loop I is two residues longer in Bgtx (12 versus 10 between the corresponding Cys
residues delimiting loop I), it is possible that these two toxins
differ in this region in their mode of interaction with the nAChR. In addition, recent comparisons of short and long
-neurotoxins suggest significant differences between these two families of toxins in their
detailed mode of interaction with the nAChR (2, 18).
18-mer complex clearly indicate that the C-terminal tail
region of Bgtx also plays a role in peptide binding. This observation
is consistent with biochemical and mutagenesis studies examining the
role of the C-terminal tail region in binding to native nAChRs where
binding affinity was decreased by 7-15-fold when C-terminal residues
were removed (4, 48).
18-mer complex suggests an important functional contact.
Mutagenesis studies in Bgtx and related toxins also point to important
roles for Arg36 and Lys38. Ala-substitution of
Arg36 in Bgtx leads to a 90-fold decrease in Bgtx binding
affinity as measured with heterologously expressed mouse nAChR (4), whereas charge reversal studies in
-cobratoxin demonstrate that R33E
(position corresponds to Arg36 in Bgtx) causes a 767-fold
decrease in binding affinity for Torpedo nAChR (2).
Ala-substitution at Arg36, the position corresponding to
Lys38 in Bgtx, reduces binding affinity by 7.4-fold
(2).
subunits. The
3 subunit, which shows no sensitivity to Bgtx block,
acquires a significant sub-micromolar affinity for Bgtx with a single
point mutation, K189Y, involving the introduction of an aromatic
residue at position 189 (49). In a footprinting protection study using Cys-substituted mutations in the heterologously expressed mouse
1
subunit, the introduced thiol of
F189C is protected by Bgtx from
reaction with a hydrophilic biotinylmaleimide (18). Similarly, the
functional importance of Tyr190 in
-neurotoxin binding
is supported by mutagenesis studies where large decreases in Bgtx and
NmmI toxin binding affinity are observed (3, 18, 19). A
double-mutant cycle analysis with NmmI toxin revealed
pairwise contacts between Tyr190 and R33E and R36E, with a
greater coupling energy to R36E (3, 19). In the present study, we
document an intermolecular NOE constraint between Lys38
(position corresponds to Arg36 in NmmI toxin)
and Thr191, one residue removed from Tyr190 in
the Bgtx·
18-mer complex.
interactions are interactions between a cationic group, such
as the side chain of Arg and Lys, with the electronegativity of an
aromatic
cloud (44). Cation-
interaction pairs within the
Bgtx·
18-mer complex were identified using CaPTURE (44). Of the 120 acceptable structures, 24 showed evidence of cation-
interactions
including two of the 20 best structures (Table III). One of these
latter two structures was chosen to illustrate one such candidate
cation-
interaction (Fig. 9). In this example, the NZ nitrogen of
Bgtx Lys38 is oriented within 6 Å of the aromatic ring of
Tyr190. We speculate that cation-
interactions may be
involved in Bgtx binding to the nAChR just as cation-
interactions
involving
Trp149 may be important in the binding of
acetylcholine to the nAChR (50). Additional high resolution structures
with better resolution of the side-chain positions would be needed to
test this proposal further.
18-mer complex, Cys192 and
Cys193 of the peptide are located in between loop I and
loop II and adjacent to the C-terminal tail of Bgtx. This position
correlates well with recent biochemical cross-linking data concerning
the spatial orientation of the reduced disulfide bond between
Cys192 and Cys193 at the
interface of
the Torpedo nAChR (51). Several Cys-substituted mutants of
Naja nigricollis
-neurotoxin, a short-chain neurotoxin, were cross-linked to the Torpedo
subunit with various
efficiencies depending on the spacer length of the dimaleimide
derivative used and the site of toxin mutagenesis. It was concluded
that Cys192 and Cys193 were located under the
tip of the first loop, ~11.5 Å from the
-carbon at toxin position
10, and close to the second loop, ~ 15.5 Å from the
-carbon at
toxin position 33 (51). This orientation is entirely consistent with
the structure shown in Fig. 6B.
18-mer primarily through three contact regions. Arg36-Val40 in loop II appears to be a core
binding site that is common to both the Bgtx·
18-mer complex and
the earlier Bgtx·
12-mer complex. We also find that the tip of loop
I is involved in peptide binding. Lys70 and
Arg72 of the C-terminal tail provide the third binding
region that serves to function as a physical continuum between the two
other binding sites. As described above, there is a remarkable
correlation between the conclusions drawn from various mutagenesis and
biochemical cross-linking studies and the structures presented here for
the Bgtx·
18-mer complex. Although it is clear that additional
receptor contacts are required to achieve the Bgtx binding affinity
observed with the native receptor, the evidence to date nevertheless
suggests that the
18-mer complex with Bgtx may serve as a useful and
accessible model for studying the structural and energetic basis of the
high affinity toxin-receptor interaction.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Dale F. Mierke for critical reading of and comments on the manuscript. We acknowledge the assistance of Andrea Piserchio with the NMR experiments. We also thank Dr. Vladimir J. Basus for helpful discussions.
![]() |
FOOTNOTES |
---|
* This research was supported by National Institutes of Health Research Grants GM32629 and NS34348 (to E. H.). NMR instrumentation was funded by National Institutes of Health Grant RR08240 and National Science Foundation Grant DBI-9723282.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1IDG, 1IDH, 1IDI, and 1IDL ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ This work was done in partial fulfillment of the requirements for a Ph.D. degree from Brown University.
¶ Present address: Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Research East 319, 41 Ave. Louis Pasteur, Boston, Massachusetts 02215.
To whom correspondence should be addressed: Dept. of Molecular
Pharmacology, Physiology, and Biotechnology, Brown Medical School, Box
G-B391, Providence, RI 02912. Tel.: 401-863-1034; Fax: 401-863-1595;
E-mail: Edward_Hawrot@brown.edu.
Published, JBC Papers in Press, April 18, 2001, DOI 10.1074/jbc.M102300200
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ABBREVIATIONS |
---|
The abbreviations used are:
nAChR, nicotinic
acetylcholine receptor;
Bgtx, -bungarotoxin;
HPLC, high performance
liquid chromatography;
CNBr, cyanogen bromide;
HSQC, heteronuclear
single quantum correlation;
TOCSY, total correlation
spectroscopy;
NOE, nuclear Overhauser effect;
NOESY, nuclear Overhauser
enhancement spectroscopy;
r.m.s.d., root mean square deviation;
NmmI, Naja mossambica mossambica I.
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