From the
Using site-directed mutagenesis, we previously identified some
residues that probably belong to the site by which Erabutoxin a (Ea), a
sea snake toxin, recognizes the nicotinic acetylcholine receptor
(AcChoR) (Pillet, L., Trémeau, O., Ducancel, F. Drevet, P.,
Zinn-Justin, S., Pinkasfeld, S., Boulain, J.-C., and Ménez, A.
(1993) J. Biol. Chem. 268, 909-916). We have now studied
the effect of mutating 26 new positions on the affinity of Ea for
AcChoR. The mutations are F4A, N5V, H6A, Q7L, S9G, Q10A, P11N, Q12A,
T13V, T14A, K15A, T16A,
Toxic proteins from animals are known to perturb physiological
processes by binding to key elements, most often a receptor, an ion
channel, or an enzyme. Understanding, at the molecular level, of the
mode of action of a toxin requires, at least in a first step, an
identification of the site by which it recognizes its target. In this
respect, the functional site of snake curaremimetic toxins has been the
subject of extensive studies. This site is associated with the capacity
of curaremimetic toxins to bind to the nicotinic acetylcholine receptor
(AcChoR)
Snake venom
curaremimetic toxins are classified as short chain toxins (60-62
residues and four disulfides) and long chain toxins (66-74
residues and five disulfides). Despite their differences in length
these toxins bind to AcChoRs in a mutually exclusive manner (Endo and
Tamiya, 1991) and have a similar folding (Agard and Stroud, 1982;
Laplante et al., 1990; Love and Stroud, 1986; Low et
al., 1976, Low, 1979; Tsernoglou and Petsko, 1976, 1977;
Walkinshaw et al., 1980; Zinn-Justin et al., 1992).
On the basis of various chemical derivatizations, some residues
implicated in the AcChoR-binding site of several curaremimetic toxins
were tentatively identified (Endo and Tamiya, 1991). More recently, we
submitted erabutoxin a (Ea) to a mutational analysis, an approach which
proved well suited to map the functional site of a protein (Cunningham
and Wells, 1993; Kam-Morgan et al., 1993; Prasad et
al., 1993). Ea is a short chain toxin from venom of the sea snake
Laticauda semifasciata (Sato and Tamiya, 1971). It possesses
62 amino acids and binds with great specificity and high affinity
( K
Two lines of evidence suggest that the
functional site of Ea may include additional amino acids. First, it was
noted (Pillet et al., 1993) that the previously identified
functional residues cover a surface that is smaller than that currently
observed in protein-protein contact areas (Janin and Chothia, 1990).
Second, previous experiments made with toxin variants (Harvey et
al., 1984) and receptor fragments (Basus et al., 1993;
Ruan et al., 1990; Fulachier et al., 1994) suggested
that several residues of the first loop may contribute to the capacity
of curaremimetic toxins to recognize AcChoR. Such a possibility
appeared intriguing since the first loop residues can be variable
within the family of curaremimetic toxins.
By mutational analysis,
we now explored the role of 26 new toxin residues located at proximity
of those previously recognized as being functionally important (Pillet
et al., 1993). Thus, we (i) completed the analysis of loop I
by modifying Phe-4, Asn-5, His-6, Gln-7, Ser-9, Gln-10, Pro-11, Gln-12,
Thr-13, Thr-14, Lys-15, and Thr-16; (ii) mutated Ser-18 and Glu-21 in
the large turn between loops I and II; (iii) modified 5 residues in
loop II, i.e. Tyr-25, Gln-28, Ser-30, Thr-35, and Ile-36; (iv)
completed the analysis of loop III by changing Pro-44, Thr-45, Val-46,
Lys-47, Pro-48, Ile-50, and Ser-53. In addition, we reinvestigated the
previously tested Ser-8, Asp-31, Arg-33, and Glu-38. For each mutant,
we determined the secondary structure and equilibrium binding affinity
toward AcChoR from T. marmorata by, respectively,
monitoring its far ultraviolet circular dichroic spectrum and measuring
its ability to compete for the receptor with a tritium-labeled toxin.
The data presented in this paper enabled us to propose a delineation of
the functional site of Ea. The identified surface is compatible with
that expected for a protein-protein contact area. Unexpectedly, it
includes residues that are not conserved in all curaremimetic toxins.
The probes were synthesized
using an Applied Biosystems 381A synthesizer and purified on denaturing
polyacrylamide gel electrophoresis. Site-directed mutagenesis was
performed using the Muta-Gene M13 in vitro mutagenesis kit
from Bio-Rad. Mutated codons are underlined, except in the case of the
deletion of Ser-18 indicated as Ea
In
the case of cytoplasmic expression, the pET expression system was used
as follows. The ZZ-mutant toxin hybrid protein, without the signal
sequence, was cloned in the pET3a vector, and E. coli BL21(DE3) was used as a host. Culture was made in TSB medium
supplemented with 5 g/liter of glucose and 200 mg/liter of ampicillin.
Induction was triggered with
isopropyl-1-thio-
Dichroic spectra
were recorded at 22 °C using a CD III or CD VI Jobin-Yvon
dicrograph.
Specific binding of toxin mutants to acetylcholine
receptor-rich membranes was determined from competition experiments
(Faure et al., 1983) using a
Choice of the Mutations
Curaremimetic toxins constitute a large family of proteins that bind
to AcChoR. They share several common amino acids (Endo and Tamiya,
1991). Ten of them were recently mutated in Ea from L.
semifasciata, a typical short chain toxin (Pillet et
al., 1993). Mutations at Ser-8, Lys-27, Trp-29, Asp-31, Arg-33,
and to a lesser extent at Phe-32, Gly-34, and Glu-38 resulted in a
decrease in binding affinity of Ea for AcChoR, whereas mutations of
Gly-49 and Leu-52 had no effect on Ea binding affinity. We now mutated
several additional residues which, as indicated in Fig. 1, are often in
proximity to those previously investigated and spread on the three
toxin loops, including both faces of the toxin (Corfield et
al., 1989). Mutations were chosen so as to produce the maximal
change in the original chemical function and the minimal structural
perturbations. The newly introduced residues were therefore often
chosen for their propensity to adopt the same secondary structure as
that of the wild-type residues (Chou and Fasman, 1978; Sibanda et
al., 1989; Wilmot and Thornthon, 1988, 1990). Preparation and Characterization of the Mutants
The method, that consists of producing a weakly active fusion toxin
in bacteria, is labor intensive but is compatible with the rules of our
National Control Committee for Biohazards. Previously, we fused the
toxin to a large protein A fragment, with the resultant fusion protein
accumulated in the periplasm of the bacteria (Ducancel et al.,
1989; Pillet et al., 1993). We now replaced protein A by its
smaller two IgG-binding domains (ZZ) (Löwenadler et al.,
1987; Nilson et al., 1987), so that the synthesized ZZ toxin
hybrids were directly secreted into the growth medium of E. coli HB101, making them easy to purify by affinity chromatography on an
IgG-Sepharose column. Treatment with CNBr (Boyot et al. 1990)
followed by HPLC led to highly purified recombinant toxins with
production yields routinely ranging between 0.025 and 0.5 mg
toxin/liter of culture. This procedure was quite appropriate, since the
resulting recombinant Ea is indistinguishable from Ea purified from
venom, regarding both the biological properties (Boyot et al.,
1990; Hervé et al., 1992; Pillet et al., 1993)
and three-dimensional structure (Arnoux et al., 1994).
We
prepared 30 new mutants of Ea. In experiments not reported in this
paper, we determined that all these mutants (i) were encoded by genes
having the expected nucleotide sequences; (ii) had the expected amino
acid compositions and amino acid sequences at least up to the site of
mutation. In addition, all chemical analyses (reverse phase-HPLC and
SDS-polyacrylamide gel electrophoresis) indicated that the mutants had
the expected molecular weight. In most cases, we met no particular
difficulty with production of the mutants. However, when Phe-4 was
changed to Ala, we detected no ZZ mutant in the medium. We therefore
produced this mutant using the cytoplasmic expression system pET-3a
described under ``Materials and Methods.'' The hybrid was
retained on an IgG column and oxidation of the disulfides was
conveniently performed by applying a mixture of oxidized and reduced
glutathione to the column. The hybrid was cleaved by CNBr, and 2 mg of
purified mutant were obtained per liter of culture. That the EaF4A
mutant could not be produced using a secretion system which was
otherwise appropriate for most mutants might indicate that the Phe-4
plays a key role in the dynamic of in vivo folding of the
toxin and/or its translocation process.
The far ultraviolet circular
dichroic spectra of all mutants were recorded. These spectra will not
be presented in this paper; however, they are available upon request.
All spectra were virtually superimposable with that of the wild-type
toxin, a result that indicates that the secondary structure of each
mutant is similar to that of the wild-type Ea and that underlines the
high permissivity of the toxin structure toward mutagenesis.
Binding
affinities of all mutants for AcChoR from the electric fish T.
marmorata were determined on the basis of competition
experiments, using a protocol previously described in detail (Pillet
et al., 1993). As an illustration of our experimental data,
Fig. 2 shows the competition binding curves obtained with some mutants
modified at a single residue of the first loop of Ea. It was previously
reported that such binding data nicely correlate with the toxic and
probably therefore with the curaremimetic function of the toxin
(Ishikawa et al., 1977). Mutational Analysis
Second, mutations at the tip of the first loop, i.e. in the stretch His-6-Gln-7-Ser-8-Ser-9-Gln-10, resulted in
affinity decreases (). We previously showed that mutation
of Ser-8 to Gly induces a 176-fold affinity decrease (Pillet et
al., 1993). We confirmed the critical role of this residue which
occupies the i+1 position in the 7-10
In
conclusion, the loop I possesses functionally important residues,
especially Gln-7, Ser-8, and Gln-10, which all belong to the tip the
loop.
That Ile-36 might play some role in the function of
curaremimetic toxins has been previously anticipated (Ménez
et al., 1982, 1984). We mutated Ile-36 into Arg, a side chain
frequently found at this position in other toxins, and strikingly we
observed a substantial increase in toxin affinity. Although competition
experiments do not allow an accurate evaluation of such increments, we
estimate it to be close to 7-fold. This is the first time that an
affinity increase is seen upon individual mutation of an Ea residue.
Recently, Mori and Tu (1991) demonstrated that Arg-36 of Lapemis neurotoxin becomes inaccessible to an arginine-specific reagent
upon complexation of the toxin with AcChoR. This observation together
with our own findings suggest that position 36 is functionally
important, at least for some curaremimetic toxins. That the I36R
mutation provokes a substantial affinity enhancement indicates that the
interface between the wild-type toxin and AcChoR does not correspond to
an optimized binding.
Previously, we showed that mutation of Asp-31
to His causes an affinity decrease of Ea for AcChoR by a factor of 46
(Pillet et al., 1993). In contrast, Rosenthal et al. (1994) found that mutation of Asp-31 to Ala does not affect the
affinity of
Pillet et al. (1993)
showed that mutations of Arg-33 to Lys or Glu caused respectively 25-
and 318-fold affinity decreases, indicating the functional importance
of this residue and in particular of its positive charge. This is
confirmed by the new mutation of Arg-33 to the neutral Gln which causes
a nearly 200-fold affinity decrease.
Finally, we mutated a number of
residues which, in contrast to those that have been so far
investigated, have their side chains on the toxin convex face
(Fig. 1). Thus, we mutated Gln-28, Ser-30, and Thr-35 to Ala and
found no alteration of Ea affinity. This result agrees with the
previous observation that Eb, a natural variant differing from Ea by a
single mutation at position 26 (Asn in Ea is replaced by His in Eb
(Sato and Tamiya, 1971)) has virtually the same affinity as Ea for
AcChoR (Ishikawa et al., 1977). Together, all these results
indicate that residues on the convex side of loop II are not
functionally important.
Second, we identified a
number of residues forming a homogeneous surface and whose mutations
induced substantial affinity changes. These are Gln-7, Ser-8, Gln-10,
Lys-27, Trp-29, Asp-31, Arg-33, Ile-36, Glu-38, and Lys-47, among which
Ser-8, Gln-10, Lys-27, and Arg-33 appear to constitute a subset of more
crucial residues for binding affinity. Nicely surrounding the important
functional side chains are residues whose mutations caused moderate
affinity decreases. These are His-6, Ser-9, Tyr-25, Phe-32, and Gly-34
which belong to the first and second loops.
Based on all available
data, Figs. 3 and 4 show the functional site of Ea. It is spread on the
three toxin loops, mostly on its concave face, where it forms a
homogeneous surface of approximately 680 Å
The most remarkable feature emerging from our
result is that the functional site unexpectedly overlaps the first
loop, in contrast to previous proposals (Low, 1979; Ménez et
al., 1982, 1984). Among the residues that were not predicted to be
functionally important are Gln-7 and Gln-10 which are not present in
long chain toxins and which were not even considered as being conserved
within the family of short chain toxins (Endo and Tamiya (1991). These
residues are sometimes replaced in short chain toxins by Pro or Met and
Thr or Glu, respectively. Ile-36 is another functional variable residue
which is nearly totally absent in long chain toxins where it is
frequently replaced by an arginine and sometimes by a valine.
Therefore, the site by which the short chain toxin Ea binds to AcChoR
from T. marmorata includes variant residues.
It is
well documented that both the short and long chain toxins bind with
high affinity to AcChoR isolated from electric organ of Torpedo fish (Endo and Tamiya, 1991). This binding can be first
rationalized by our finding that approximately 60% of the residues that
have been identified as functionally important for Ea are also present
in nearly all curaremimetic toxins. These residues may provide all
these toxins with a basic moderate affinity for AcChoR from
Torpedo. In Ea, the additional functional residues mostly
belong to loop I. Strikingly, they are not present in long chain toxins
whose loop I greatly differs from that of short chain toxins in terms
of size, conformation, and amino acid composition (Endo and Tamiya,
1991). To exert their high affinity for Torpedo AcChoR, the
long chain toxins are therefore anticipated to possess other functional
residues, presumably also variable in nature. This suggestion is
supported by a recent NMR study depicting the three-dimensional
structure of the complex formed between the long chain toxin
At present, 36 individual positions of Ea have been submitted
to site-directed mutagenesis, two others (positions 1 and 51) have been
mono-modified by chemical means, and one (position 26) has been
substituted in a natural mutant. If one excludes the four disulfides
whose structural role is highly probable (Rydén et al.,
1973; Ménez et al., 1982, 1984), nearly 72% of the Ea
residues underwent mutational analysis. Considering that (i) the
functionally identified residues are located within a continuous
surface surrounded by a large number of excluded residues; (ii) the
number of functional residues and the surface (680 Å) covered by
them is close to that currently observed for a protein determinant, we
believe that most of the ``curaremimetic'' site of Ea is now
identified. However. it remains to study the individual contributions
of the identified residues in the formation of the toxin
Mutations made in
the first loop and in the stretch that joins loops I and II. For symbol
notations see legend of Table II.
The values
were determined from competition data similar to those shown in Fig. 2
according to Ishikawa et al. (1977). The second column shows
the ratio of the affinity constant of each mutant to that of the
wild-type Ea. The residues for which mutations induced important
affinity changes are emphasized by bold letters. The notation (a)
indicates that the mutation occurs naturally in the variant Eb which
differs from Ea by this single substitution at position 26. The
notation (b) indicates that the mutation occurs naturally in the
variant called Ec which differs from Ea by two mutations, one at
position 26 where Asn is replaced by His and the other at position 51
where Lys is replaced by Asn. Note that the chemical derivative of Ea
selectively monoacetylated at position 51 has a similar affinity as
compared to Ec,
We thank Drs. Sophie Zinn-Justin, Sylvaine Gasparini,
Denis Servent, and Bernard Gilquin for their kind help and their
constant interest. We are also indebted to Evelyne Lajeunesse for her
technical assistance. Biohazards associated with the experiments
described in this publication have been examined previously by the
French National Control Committee.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
S18, E21A, Y25F, Q28A, S30A, T35A, I36R,
P44V, T45A, V46A, K47A, P48Q, I50Q, and S53A. Binding affinity
decreases upon mutation at Gln-7, Gln-10 and to a lesser extent at
His-6, Ser-9 and Tyr-25 whereas it increases upon mutation at Ile-36.
Other mutations have no effect on Ea affinity. In addition, new
mutations of the previously explored Ser-8, Asp-31, Arg-33, and Glu-38
better explain the functional role of these residues in Ea. The
previous and present mutational analysis suggest that the
``functional'' site of Ea covers a homogeneous surface of at
least 680 A
, encompassing the three toxin loops, and
includes both conserved and variant residues. The variable residues
might contribute to the selectivity of Ea for some AcChoRs, including
those from fish, the prey of sea snakes.
(
)
and hence to alter nerve-muscle
transmission (Changeux, 1990). Thus, curaremimetic toxins provoke
paralysis of skeletal muscles, including diaphragm, and induce death as
a result of respiratory failure (Chang, 1979).
= 7. 10
M) to AcChoR from Torpedo marmorata (Ishikawa
et al., 1977; Pillet et al., 1993). Its
three-dimensional structure was elucidated by x-ray crystallography
(Corfield et al., 1989). The cDNA encoding Ea was cloned
(Tamiya et al., 1985) and expressed in Escherichia coli as a fusion protein (Ducancel et al., 1989). Cleavage by
CNBr led to a recombinant toxin having biological properties (Boyot
et al., 1990) and a three-dimensional structure (Arnoux et
al., 1994) indistinguishable from those of the wild-type toxin.
Therefore, 10 amino acids that are commonly found in most curaremimetic
toxins were submitted to site-directed mutagenesis in Ea (Hervé
et al., 1992; Pillet et al., 1993). This preliminary
mutational analysis together with chemical modification
experiments
(
)
indicated the functional role of
Ser-8, Lys-27, Trp-29, Asp-31, Arg-33, Lys-47 (probed by chemical
modification), and to a lesser extent of Phe-32, Gly-34, and Glu-38.
These residues form an homogeneous surface in which all residues but
one (Ser-8) have their side chain oriented on the same concave face of
Ea
-sheet. These residues were proposed to belong to the
functional site of Ea.
Probes for Site-directed Mutagenesis
We used the
following nucleotide probes for mutagenesis: F4A
(5`-ATGAGGATATGTGCTAACCATCAGTCA-3`), N5V
(5`-AGGATATGTTTTGTCCATCAGTCATCG-3`), H6A
(5`-ATATGTTTTAACGCTCAGTCATCGCAA-3`), Q7L
(5`-TGTTTTAACCATCTGTCATCGCAACCG-3`), S8T
(5`-TTTAACCATCAGACCTCGCAACCGCAA-3`), S9G
(5`-AACCATCAGTCAGGTCAACCGCAAACC-3`), Q10A
(5`-CATCAGTCATCGGCTCCGCAAACCACT-3`), P11N
(5`-CAGTCATCGCAAAACCAAACCACTAAA-3`), Q12A
(5`-TCATCGCAACCGGCTACCACTAAAACT-3`), T13V
(5`-TCGCAACCGCAAGTGACTAAAACTTGT-3`), T14A
(5`-CAACCGCAAACCGCTAAAACTTGTTCA-3`), K15A
(5`-CCGCAAACCACTGCTACTTGTTCACCT-3`), T16A
(5`-CAAACCACTAAAGCTTGTTCACCTGGG-3`), EaS18
(5`-ACTAAAACTTGTCCTGGGGAGAGC-3`), E21A
(5`-TGTTCACCTGGGGCTAGCTCTTGCTAT-3`), Y25F
(5`-GAGAGCTCTTGCTTCAACAAGCAATGG-3`), Q28A
(5`-TGCTATAACAAGGCATGGAGCGATTTC-3`), I36R
(5`-TTCCGTGGAACTCGTATTGAAAGGGGA-3`), E38L
(5`-GGAACTATAATTCTGAGGGGATGTGGT-3`), P44V
(5`-GGATGTGGTTGCGTCACAGTGAAGCCC-3`) I50Q
(5`-GTGAAGCCCGGTCAGAAACTCAGTTGT-3`).
S18. After digestion with
EcoRI and BamHI, the 0.4-kilobase fragments encoding
mutated Ea cDNAs were purified and cloned into either the
expression/secretion vector pEZZ 18 (Protein A gene Fusion Vector
purchased from Pharmacia) or the cytoplasmic expression vector pET3a
(Studier and Moffatt, 1986).
Expression and Purification of Fused Mutants
The
bacterial host used for expression of ZZ-Ea hybrid mutants was
E. coli HB 101 (Boyer and Roulland-Dussoix, 1969).
Bacteria were grown in a 5-liter fermentor (LSL Biolafitte) with an
initial working volume of 4 liters of TSB (Tryptic Soy Broth) medium
(Difco) supplemented with 5 g/liters of glucose and 200 mg/liters of
ampicillin. The temperature was kept at 37 °C, the pH was
maintained at 7.2 with 5
M NaOH, and oxygen concentration was
adjusted to 80% saturation by adding an increasing proportion of pure
oxygen to the air supply. When the initial glucose concentration fell
to 0.5 g/liter, feeding was initiated by adding glucose (40%) as carbon
source. The feeding rate was adjusted to give a growth rate of 0.3
h. Culture was stopped at the early stationary
phase. Cells were discarded by centrifugation, the culture supernatant
was filtered through a 0.2-µm filter, and concentrated by
ultrafiltration on a membrane with a cut-off mass of 10 kDa (FILTRON).
The solution was then applied on top of a 10-ml IgG-Sepharose column as
described previously (Ducancel et al., 1989). The hybrid was
lyophilized and dissolved in HCl 0.1
N in the presence of a
500-fold excess of CNBr for 24 h. The resulting recombinant toxin was
separated from ZZ moiety and other side products by chromatography on a
reverse phase HPLC column (Vydac, C
, 5 µm 10
250 mm). The column was equilibrated in 0.1% trifluoroacetic acid, and
elution was performed using a trifluoroacetic
acid/CH
CN/H
O gradient system. The recombinant
toxin was further purified by chromatography on a Mono S (Pharmacia)
HPLC column equilibrated in ammonium acetate 0.01
M, pH 4.5,
and eluted by a gradient of 0.01
M to 1.5
M ammonium
acetate at the same pH. Purity of the hybrid mutant was checked by
SDS-polyacrylamide gel electrophoresis and isoelectric focusing.
-
D-galactopyranoside (0.5 m
M final concentration) when the OD
reached 0.5.
After 3 h cells were harvested by centrifugation and resuspended in
lysis buffer (30 m
M Tris, 5 m
M EDTA, 20% sucrose, 0.1
mg/ml lysozyme, 0.1 mg/ml DNase I, pH 8). Cells walls were disrupted by
three cycles of freezing and thawing, the supernatant was clarified by
centrifugation for 30 mn at 10,000 rpm, and applied to an IgG-Sepharose
column. The column was washed with 0.1
M phosphate buffer (pH
8) containing 0.1% Tween 20. Washing was continued until the A
reached base line. Then two volumes of renaturation buffer (0.1
M phosphate buffer, 5 m
M EDTA, 2:4 m
M GSH/GSSG) were passed through the column which was then incubated
24 h at room temperature. The gel was subsequently washed with 5 m
M ammonium acetate (pH 5), and the hybrid protein was eluted with
0.5
M acetate (pH 3.4). The eluted fraction was then submitted
to cleavage with CNBr, and the resulting recombinant toxin was
chromatographed on two successive HPLC columns, as indicated above.
Preparation and Characterization of the
Mutants
Each mutant was submitted to amino acid sequencing,
amino acid analysis, and isoelectric focusing. Protein concentrations
were determined in solution from molar absorbancies at 278 nm evaluated
as described previously (Pillet et al., 1993). This value was
equal to 9000 for all mutants and 7500 for Ea Y25F.
H-labeled toxin as a
radioactive tracer (Ménez et al., 1971). Equilibrium
dissociation constants were determined from competition binding
experiments according to Ishikawa et al. (1977). This was done
by assuming that the two toxin-binding sites present per AcChoR
molecule (Changeux, 1990) are equivalent and in particular that their
affinities for the toxin and each mutant are identical. The precision
of the experiments is compatible with such a hypothesis, and indeed all
competition binding data fit nicely with theoretical curves in which a
homogeneous class of binding sites was assumed to be present per
molecule of AcChoR (see ``Results'').
Definition of a Functional Residue
Residues implicated
in contact surfaces of protein-protein complexes can be accurately
identified by structural approaches, especially x-ray analyses (Janin
and Chothia, 1990). On the other hand, residues that may be important
for protein-protein interactions can be identified by functional
assays, such as mutational studies. At present, the relationship
between determinants defined by structural and functional analyses is
not entirely clear. However, determinants characterizing
protein-antibody (Prasad et al., 1993) and hormone-receptor
(Cunningham and Wells, 1993) complexes have been identified using
structural and functional approaches. These studies demonstrate that
determinants identified by both types of methods do largely overlap.
The structurally defined determinants are apparently somewhat larger,
but this difference might be associated with the observation that, in a
small number of particular cases, more than a single mutation is needed
to identify the actual functionality of a residue (Kam-Morgan et
al., 1993). The comparative analysis described by Prasad et
al. (1993) reveals that functional residues for which mutations
cause more than 10-fold affinity changes also belong to the
structurally defined determinant, whereas residues for which mutations
cause lower affinity changes are at the border of the structural
determinants. Finally, several reports suggested that only a small
subset of side chains, i.e. three to four, dominate the
binding affinity of protein-protein interactions (Jin et al.,
1992; Cunningham and Wells, 1993; Nuss et al., 1993; Prasad
et al., 1993). Therefore, in the following, we will consider
as functional, a residue whose mutation causes substantial affinity
change of Ea for AcChoR. In view of the above observations, we infer
that this residue has a great probability to belong to the interacting
area between Ea and AcChoR; however, one should keep in mind that its
actual contribution to the receptor binding remains to be determined.
Loop I
Loop I of Ea has 13 residues located
between Cys-3 and Cys-17, forming a two-stranded -sheet joined by
a
-turn (Corfield et al., 1989). The effects of mutations
of these residues on binding affinities for AcChoR can be classified in
two categories (Table I). First, changing Phe-4, Asn-5, Pro-11, Gln-12,
Thr-13, Thr-14, Lys-15, and Thr-16 virtually produced no effect on the
affinity. Therefore, these residues are likely to be excluded from the
toxin-receptor interaction. Previously, it was shown that
neutralization by acylation of the positive charge of Lys-15 of Eb, an
isoform of Ea (Hori and Tamiya, 1976) or of Ea
has no
effect on toxin activity. The present data confirm this finding and
further show that replacement of Lys-15 by the smaller alanine can be
also achieved without altering the toxin affinity. Therefore, most
residues at the base of loop I may not be implicated in the functional
site of Ea.
-turn, since
changing Ser-8 to Thr causes an even greater affinity decrease of
nearly three orders of magnitude
(780) . This is the largest
effect observed so far for any single mutation in Ea. The dichroic
spectrum of this mutant was superimposable with that of Ea (not shown),
excluding the possibility that the large affinity change that is
observed is associated with major modification of the toxin secondary
structure. Clearly, a Ser to Thr change cannot be considered as
functionally equivalent, despite the presence in both cases of a
hydroxyl group. Gln-7 and Gln-10, which respectively occupy positions i
and i+3 in the
-turn, are also critical residues as inferred
from the substantial affinity decreases caused by their respective
mutations. Strikingly, neither of these 2 residues had ever been
suggested to play a role in the function of a curaremimetic toxin. The
neighboring Ser-9 is less important since its mutation into Gly causes
only a 9-fold affinity decrease. That mutation of the adjacent Ser-8
into Gly causes a much larger affinity decrease (Pillet et
al., 1993), emphasizes the fine specificity of the mutational
analysis and suggests that of the 2 adjacent serine residues Ser-8 and
Ser-9 at the tip of the first loop only Ser-8 is highly important for
the binding function of Ea. Another residue of loop I whose mutation
moderately affects Ea affinity is His-6. This residue appears
inaccessible to solvent since it has a low p K around 3, a low
exchange rate with D
O when in solution (Inagaki et
al., 1978), and a weak chemical reactivity toward iodine (Sato and
Tamiya, 1970). Despite its structural involvement, His-6 can be changed
into Ala without causing more than a 6-fold affinity decrease.
The Stretch between Loop I and Loop II
The stretch
between Cys-17 and Cys-24 forms a large turn. In Ea, it comprises 6
residues whereas in other toxins it sometimes includes one or two
deletions (Endo and Tamiya, 1991). We therefore deleted Ser-18, a
residue which is located at position i of the -turn 18-21 in
Ea (Corfield et al., 1989). A recent structural and
immunological analysis suggested that this deletion is likely to
provoke a shift of the type II
-turn from residues 18-21 to
residues 17-20 (Zinn-Justin et al., 1994).
Notwithstanding this substantial structural change, the deletion has no
consequence on Ea binding affinity (), indicating that
Ser-18 and its vicinal region are not implicated in the functional site
of Ea. Accordingly, mutation of Glu-21, a residue located at position
i+3 of the
-turn, also has no effect on toxin affinity
(). We suggest, therefore, that the whole stretch
17-24 is not implicated in the functional site of Ea.
Loop II
Lys-27, Trp-29, Asp-31, Arg-33 and to a
much lesser extent Phe-32, Gly-34, and Glu-38 were previously shown to
be functionally important residues (Pillet et al., 1993). Most
of them have their side chains on the toxin concave face. We now probed
the two remaining side chains of this face of loop II, i.e. Tyr-25 and Ile-36. Tyr-25 is highly conserved among toxins that
adopt a three-finger structure (Endo and Tamiya, 1991; Dufton and
Hider, 1991). Its high p Ksuggests that
its hydroxyl is hydrogen bonded, probably to Glu-38 as indicated from
x-ray data of Ea (Corfield et al., 1989) or the isotoxin Eb
(Bourne et al., 1985). Since this bond was previously
anticipated to be important for maintaining the biologically active
conformation of some toxins (Endo and Tamiya, 1991), we replaced Tyr-25
by Phe. Not only was the mutant found to have a circular dichroic
spectrum similar to that of the native toxin (not shown), but also, as
shown in Table II, the mutation has little effect on the toxin binding
affinity. The hydroxyl group of Tyr-25 and its hydrogen bond with
Glu-38 do not constitute critical requirements for Ea to fold into a
functional conformation. Why then do so many toxins possess a tyrosine
at position 25? We are currently addressing this question with new
mutants.
-bungarotoxin for AcChoR. In agreement with this
result, we found that substitution of Asp-31 to Asn does not affect Ea
affinity. At least two explanations can account for the observation
that Ea affinity is uniquely affected by introduction of His at
position 31. First, Asp-31 may play no functional role but the presence
of the imidazole ring could induce some local conformational change.
Since it is not detected by CD analysis, we infer that the putative
change would be small and that Asp-31 would be at proximity of the
functional site. Second, Asp-31 may be functionally important, but only
mutation to His is capable of highlighting it. Interestingly, such a
situation has been previously encountered by Kam-Morgan et al. (1993). These authors observed that mutation in lysozyme (HEL) of
Asp-101
to Gly had virtually no effect on the affinity of
HEL for a HEL-specific antibody, whereas mutations of Asp-101
to bulkier residues such as Arg or Phe, provoked affinity
decreases by factors of 36 and 39, respectively. Notwithstanding this
selective mutational sensitivity, the side chain and backbone atoms of
Asp-101
formed clear contacts with the antibody, as
judged from the crystal structure of the HEL-antibody complex
(Kam-Morgan et al., 1993). Although no definite explanation
accounts for their observations, these authors demonstrated that a
single mutation does not necessarily inform about the actual role of an
amino acid. Similarly, mutation of the highly conserved Glu-38 of Ea
into Gln and Lys was previously shown to cause no and moderate effect
on Ea affinity, respectively (Pillet et al., 1993), whereas
introduction of a Leu residue, whose side chain cannot establish
hydrogen bonds, produces a 25-fold affinity decrease (Table II). As in
the case of Asp-31 it is clear that more than one mutation is required
at position 38 to observe a substantial decrease in affinity. In view
of the high degree of conservation of Asp-31 and Glu-38, especially in
short chain toxins (Endo and Tamiya, 1991), we wish to suggest that the
affinity decreases that are selectively observed upon mutation to His
and Leu, somehow reflect the functional character of both residues,
although the actual nature of these functionalities remains unknown. We
can only conclude in both cases that the negative charge is unlikely to
be a critical functional parameter.
Figure 1:
Three-dimensional structure of the
polypeptide chain of Ea from Laticaudata semifasciata (Corfield et al., 1989; Arnoux et al., 1994).
The four disulfides are indicated by gray lines. Residues that
have been mutated in our previous study (Pillet et al., 1993)
or chemically modified (Lys-47 and Lys-51)are indicated by
black circles. New positions that have been submitted to
mutagenesis are indicated by gray circles. The side chains of
residues oriented toward the reader are indicated by an arrow pointing to the upper part of the figure. * indicates that Asn-26
is naturally changed to His in the isotoxin Eb (Sato and Tamiya, 1971;
Saludjian et al., 1992).
Loop III
The third loop of Ea includes 10 residues
located between the half-cystines 43 and 54. Previous chemical
modifications of individual lysine residues revealed that the Lys-47
derivatized Ea has lower affinity for AcChoR as compared to unmodified
Ea. This result agrees with both similar experiments made with other
curaremimetic toxins (Ishikawa et al., 1977; Faure et
al., 1983; Endo and Tamiya, 1991) and the observation that
mutation of Lys-47 to Ala markedly decreases the binding affinity of Ea
(). Chemical modification of Lys-51 does not affect the
binding affinity of Ea.Neither the natural mutation of
Lys-51 to Asn, as it occurs in the natural variant Ec, affects the
toxin affinity. This result further confirms that the residue 51, whose
side chain is on the convex face, is not functionally important. Pillet
et al. (1993) have observed that mutations of Gly-49 and
Leu-52 have no effect on Ea binding function. We now observe that
mutations of Pro-44, Thr-45, Val-46, Pro-48, Ile-50, and Ile-53 have no
effect on the binding affinity of Ea for AcChoR ().
Therefore, the present data suggest that of all the residues that
constitute the loop III of Ea only the concave face-orientated Lys-47
is functionally important. On the Functional Site of Ea
General Description
Two complementary pieces of
evidence allowed us to delineate the ``functional site'' of
Ea. First, we identified a large set of probably excluded residues, as
inferred from the mutants displaying a similar affinity as compared to
the wild toxin (Tables I and II). These residues are Phe-4, Asn-5,
Pro-11, Gln-12, Thr-13, Thr-14, Lys-15, Thr-16, Ser-18, Glu-21, Gln-28,
Ser-30, Thr-35, Pro-44, Thr-45, Val-46, Pro-48, Gly-49, Ile-50, Leu-52,
and Ser-53. This is also the case of the N-terminal group and Lys-51
which were chemically modifiedand of Asn-26 which is
naturally mutated in His in the nearly equipotent Eb (Ishikawa et
al., 1977). Therefore, of the 62 residues of Ea, at least 24 seem
not to belong to the toxin functional site. Interestingly enough, a
large number of them, i.e. the N-terminal group Phe-4, Gln-12,
Thr-14, Thr-16, Asn-26, Gln-28, Ser-30, Thr-35, Val-46, Lys-51, and
Ser-53 are located on the convex side of the toxin and widely spread on
loops I, II, or III. This observation strongly suggests that the convex
face of the
-sheet formed by the three loops of Ea is not
implicated in the functional site. Moreover, a substantial proportion
of the concave face also seems to be excluded from the site. It
concerns the residues that are localized on the
-sheet strands of
loop I, i.e. Asn-5, Pro-11, Thr-13, Lys-15, between loops I
and II, i.e. Ser-18 and Glu-21 and in loop III, i.e. Thr-45, Gly-49, Ile-50, and Leu-52.
, a value
which is compatible with that expected for a protein-protein contact
area (Janin and Chothia, 1990). As emphasized in Fig. 3, the site
comprises three polar clusters, Lys-27-Glu-38 and Arg-33-Asp-31 in loop
II, and Gln-7-Gln-10 in loop I, which all interact with the central
hydrophobic cluster Trp-29-Ile-36 in loop II. Ser-8 establishes the
only main chain-main chain contact between the loops I and II in Ea,
with its hydroxyl group forming an hydrogen bond with the NH group of
Ile-37 (Corfield et al., 1989; Bourne et al., 1985;
Low and Corfield, 1986) and Lys-47 looks surprisingly isolated on loop
III.
Figure 3:
Representation of the residues whose
mutations causes a change in the affinity of Ea for AcChoR. In
yellow are those side chains whose mutations cause a
3-9-fold affinity decrease. The side chains colored orange are residues for which at least one mutation causes an affinity
decrease by a factor equal to or greater than 10. In blue is
indicated the residue, Ile-36, whose mutation causes an increase in
affinity. To further classify the differential effects of the
mutations, we indicated by bold lines the side chains whose
mutations caused more than 100-fold affinity decrease. These are Ser-8,
Gln-10, Lys-27, and Arg-33. Note that Asp-31 and Glu-38 have been
indicated as functional residues on the basis of the observation that
at least one, although not all tested mutations causes more than a
10-fold affinity decrease.
The functional site of Ea may not be completely delineated,
however. As indicated by our mutational analysis of Asp-31 and Glu-38
and by other studies (Kam-Morgan et al., 1993), more than a
single mutation may be required to establish the functional importance
of a residue. Since most residues that are considered to be excluded
from the functional determinant have undergone a single mutation, they
need to be submitted to further mutagenesis. In particular, it is
intriguing that only Lys-47 plays a functional role in loop III. This
is all the more surprising as it was recently suggested that a fragment
of AcChoR specifically recognizes loop III (Fulachier et al.,
1994), and it is unlikely that such a recognition could be specifically
exerted with a single positively charged residue. Neither does our
analysis inform about the interactions that the backbone atoms might
establish with the receptor. Finally, the actual individual
contributions of the functional residues to the establishment of
EaAcChoR complex remain to be determined. In this respect, it
would be of interest not only to estimate the on and off rate constants
of the mutant-receptor complexes but also to determine the effects of
the mutations on the thermodynamic parameters, i.e.
H and
S, involved in Ea
AcChoR interactions.
The Functional Site of Curaremimetic Toxins Has Both
Invariant and Variant Residues
Curaremimetic toxins are
classified as short chain toxins which, like Ea, contain 60-62
residues and four disulfides and as long chain toxins with 66-74
residues and five disulfides (Endo and Tamiya, 1991). Despite their
length differences, curaremimetic toxins share a similar overall
folding, as revealed by both NMR and x-ray crystallography (Agard and
Stroud, 1982; Laplante et al., 1990; Love and Stroud, 1986;
Low et al., 1976; Low, 1979; Tsernoglou and Petsko, 1976,
1977; Walkinshaw et al., 1980; Zinn-Justin et al.,
1992). Using the common presence of eight half-cystines, the amino acid
sequences of curaremimetic toxins can be aligned, revealing the
existence of a number of residues that are invariant or type invariant
in short chain toxins, in long chain toxins, or in both categories of
toxins, as proposed by Endo and Tamiya (1991). Among the residues
recognized to be highly important for the function of Ea, 4 are highly
conserved in both toxin categories. These are Lys-27, Trp-29, Arg-33,
and Lys-47. These invariant residues might constitute a ``common
functional core'' through which curaremimetic toxins establish
conservative contacts with AcChoRs. In addition, it is possible that
the invariant Asp-31 and Glu-38, whose roles remain however to be
clarified, may also contribute to the conserved functionality of
curaremimetic toxins.
-bungarotoxin and the fragment 185-196 from the
-subunit of Torpedo californica (Basus et al.,
1993). This study shows that several interacting residues of
-bungarotoxin are mutated or even do not exist in Ea. This is the
case, for example, of Thr-5, Thr-6, Ala-7, and Ile-11 which belong to
the loop I of
-bungarotoxin and which all differ in Ea. This is
also the case for His-68 which is uniquely located at the C-terminal
tail of
-bungarotoxin. Clearly, therefore, variant residues
especially localized in loop I substantially contribute to the high
binding affinity of curaremimetic toxins. This finding implies that
there is not a unique ``functional site'' for all toxins but
rather a sort of ``variation on a theme'' around a common
functional core. Interestingly enough, implication of variable residues
to AcChoR binding offers an explanation as to why short chain toxins
are usually more toxic in rodents and birds whereas the long chain
toxins tend to be more effective against receptors from humans and from
some reptiles and amphibians (Lee and Chen, 1976; Chang, 1979; Burden
et al., 1975; Ishikawa et al. 1985). Finally, we
noted that the 3 important residues Gln-7/Ser-8/Gln-10 of Ea, a sea
snake toxin, are also present in more than 80% of the other short chain
toxins from sea snakes (Endo and Tamiya, 1991). This observation
suggests that these residues are evolutionarily conserved to provide
the short chain toxins with a high binding affinity toward AcChoR from
fish, the well-known prey of sea snakes (Heatwole, 1987).
AcChoR
complex and to identify the receptor residues that recognize the toxin
functional site. Nevertheless, the present knowledge can already be
exploited in various practical directions, including the preparation of
controlled toxoids and the design of new curare compounds for clinical
use.
Table:
Indication of the apparent Kvalues of the wild-type and mutants of Ea
Table:
Mutations made in loops I and II
further confirming the nonimportance of
Lys-51 in Ea function. Asterisks indicate data from Pillet et al. (1993).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.