(Received for publication, January 6, 1996; and in revised form, March 5, 1996)
From the
An approach to obtain new active proteins is the incorporation
of all or a part of a well defined active site onto a natural structure
acting as a structural scaffold. According to this strategy we
tentatively engineered a new curaremimetic molecule by transferring the
functional central loop of a snake toxin, sequence 26-37,
sandwiched between two hairpins, onto the structurally similar
-hairpin of the scorpion toxin charybdotoxin, stabilized by a
short helix. The resulting chimeric molecule, only 31 amino acids long,
was produced by solid phase synthesis, refolded, and purified to
homogeneity. As shown by structural analysis performed by CD and NMR
spectroscopy, the chimera maintained the expected
/
fold
characteristic of scorpion toxins and presented a remarkable structural
stability. The chimera competitively displaces the snake curaremimetic
toxin
from the acetylcholine receptor at 10
M concentrations. Antibodies, elicited in rabbits
against the chimera, recognize the parent snake toxin and prevent its
binding to the acetylcholine receptor, thus neutralizing its toxic
function. All these data demonstrate that the strategy of active site
transfer to the charybdotoxin scaffold has general applications in the
engineering of novel ligands for membrane receptors and in vaccine
design.
The design of proteins with novel functions represents an
exciting potential of protein engineering. Simple protein architectures
or scaffolds(1, 2, 3, 4, 5, 6) have been
obtained by de novo protein design, but only a few have been
harnessed with a function, such as in the case of the heme-binding and
redox-active proteins (7, 8) and of the DNA-binding
proteins(9, 10) . Genuine successes in the generation
of functionally useful novel proteins, however, have been obtained by a
more conservative approach which exploits the structure of some natural
proteins as appropriate structural scaffolds and manipulates surface
loops and insert new functional sequences in permissive regions. As an
illustration of this strategy, a human-mouse chimera was obtained by
the transfer of the complementarity-determining regions of a mouse
antibody into the corresponding one of a human myeloma protein, with
concomitant reproduction of antibody specificity into the human protein (11) . The resulting ``humanized'' antibodies are not
recognized as foreign by the immune system and then possess great
therapeutic potential. Other examples include the insertion of elastase
inhibition activity in interleukin 1 by transfer of the protease
inhibitor loop into a structurally compatible loop of the cytokine
scaffold(12) , the recruitment of RNase activity in angiogenin (13) or angiogenic activity in RNase A (14) by
exchanging surface loops, the recruitment of carboxypeptidase activity
into RTEM-
lactamase by introduction of a 28-amino acid segment of
carboxypeptidase(15) , the substitutions of few key residues in
human prolactin to generate binding to the receptor of the homologous
growth hormone(16) , the stabilization of the Bacillus
subtilis neutral protease obtained by the insertion of the
10-residue calcium-binding loop of thermolysin into the corresponding
region of the bacterial protease(17) , and the stabilization of
subtilisin BPN` by incorporation of a Ca
-binding loop
from the homologous thermophilic thermitase(18) .
Small disulfide-rich structures are interesting candidates as structural scaffolds for protein engineering studies, since in many cases the disulfide bridges provide most of the stabilizing energy, leaving a large part of the sequence available for substitutions or insertions. Kunitz trypsin inhibitor was transformed into a powerful neutrophil elastase inhibitor by replacing the active loop with a new sequence deduced on the basis of phage display and selection methodology(19) ; a highly efficient inhibitor of human leukocyte elastase was engineered on the scaffold of human pancreatic trypsin inhibitor (20) and a bisheaded inhibitor of trypsin and carboxypeptidase A on the scaffold of a squash inhibitor (21) on the basis of a rational structure-based design.
Recently, we proposed the small disulfide-stabilized structure of
the scorpion charybdotoxin as a basic scaffold, able to present guest
sequences in a defined and well ordered conformation as a means to
engineer novel proteins(22) . The scorpion toxin structure (Fig. 1), containing only 37 amino acids, consists of a short
-helix and a triple stranded
-sheet, with three disulfide
bridges forming most of the interior core. This structural motif is
common to all scorpion toxins, irrespective of whether they contain
60-65 amino acids, as in long scorpion toxins, active on
Na
channels or contain 30-40 amino acids, as in
short scorpion toxins, active on K
channels and
Cl
channels(23, 24) . The same
structural motif is also present in insect defensins(24) ,
expressing antimicrobial activity through membrane permeabilization and
in plant
-thionins(25) . The only amino acid residues
common to all these proteins are the six cysteines forming the three
disulfide bridges(23, 24) . This simple, compact, and
well organized structural motif seems to have been naturally selected
for its high sequence permissiveness (as shown by its compatibility
with hundreds of different sequences from scorpion toxins and insect
defensins) and functional versatility (as shown by its compatibility
with Na
, K
, Cl
channel blockage activity in scorpion toxins and antibacterial
activity in defensins). A major difference of this structural scaffold
from the previously reported ones is that a large part of its molecular
surface might be substituted by a novel sequence and not just the
exposed loops.
Figure 1:
Three-dimensional structure of the
scorpion charybdotoxin (left; PDB code, 2CRD) and the snake
toxin (right; PDB code, 1NEA). The structure models were
generated by the Molscript program(76) . Numbers indicate the chain termini and the location of the snake
curaremimetic loop and of the host scorpion
-hairpin.
On the basis of these considerations, we thought that
it was possible to artificially engineer many more functions on this
fold than those that are naturally selected. Thus, by transferring the
carbonic anhydrase metal binding site into the -sheet of the
scorpion scaffold, we engineered a new metal binding activity with
affinity and selectivity for transition metals(22) . We here
report the engineering of a novel binding activity toward a membrane
receptor, the acetylcholine receptor (AchoR), (
)by
re-designing the
-sheet molecular surface of the charybdotoxin
scaffold and taking advantage of the knowledge of the structural
similarity between the charybdotoxin
-sheet region and that of
natural ligands of this receptor contained in snake venoms.
Snake
venom contains toxins that bind to the AchoR at the postsynapic
membranes of skeletal muscle, thus provoking flaccid paralysis. This
activity is similar to that shown by the alcaloid curare and is of
potential clinical interest in anesthesia. Curaremimetic neurotoxins
constitute a family of proteins with a high sequence similarity that
present a similar overall folding, consisting of three adjacent loops
forming a -pleated sheet, which emerge from a globular core
containing four conserved disulfides(26, 27) . On the
basis of chemical modification and mutational analysis studies, several
amino acid residues implicated in the AchoR binding of one of these
toxins, erabutoxin a, were identified(28, 29) . These
residues define a continuous surface, forming the curaremimetic site of
snake neurotoxins, centered around the central loop (residues
26-37) of the concave face of the
-sheet platform,
containing six of the ten most ``active'' residues. Toxin
, from the venom of Naja nigricollis, has high sequence
similarity and the same residues in the concave side of the central
loop as erabutoxin a, besides the substitution His
Phe. Its three-dimensional structure (Fig. 1) has been recently
solved on the basis of NMR data (27) and has been shown to be
highly similar to the crystal structure of erabutoxin a(30) .
The mutational studies of curaremimetic toxins suggest that being
able to fix the sequence of the central loop of toxin in its
native conformation should result in a molecule possessing some
affinity for AchoR. On the basis of a backbone structural similarity
between the loop sequence 26-37 of toxin
and the
-hairpin 25-36 of the charybdotoxin structure, we
transferred the snake sequence into the scorpion framework. The newly
designed chimeric protein was produced by solid phase synthesis. Its
structure was analyzed by CD and NMR spectroscopy. Its function was
investigated by competitive binding to AchoR and by analyzing the
specificity of the antibodies produced by immunization of this chimeric
construction in rabbits.
For amino acid analysis, peptide samples (50 µg) were hydrolyzed in ultrapure 6 N HCl for 16 h at 120 °C in sealed evacuated tubes. The hydrolysates were then analyzed on an Applied Biosystems model 130A automatic analyzer equipped with an on-line model 420A derivatizer for the conversion of the free amino acids into their phenylthiocarbamoyl derivatives.
Mass analysis was performed in a Nermag R10-10 mass spectrometer, coupled to an Analitica of Branford electrospray source. The quadrupole was scanning over the range m/z 300-2000. A HP-ChemStation software (Hewlett-Packard) was used to drive the spectrometer and to acquire the data.
For peptide mapping,
the folded oxidized purified product (70 µg), dissolved in 100
µl of 0.1 M TrisHCl buffer, pH 7.6, containing 1
mM iodoacetamide (to prevent disulfide scrambling) was
digested for 15 h at 30 °C with a mixture of trypsin (3 µg) and
chymotrypsin (3 µg). Digest was analyzed by reverse phase HPLC on a
Vydac C18 column (25
0.46 cm) using a 30-min linear gradient of
1-25% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of
1 ml/min. The eluate was monitored at 214 nm. Peaks were collected,
lyophilized and analyzed by electrospray mass spectrometry.
Circular dichroism spectra were
obtained with a Jobin-Yvon CD6 dichrograph, driven by an IBM-PC
operating with a CD6 data acquisition and manipulation program. Spectra
were run at 20 °C in 5 mM sodium phosphate, pH 7.0, and
from 180 to 255 nm, with a 0.1-cm quartz cell and a protein
concentration 1.5-2.0 10
M;
from 250 to 320 nm with a 1.0-cm quartz cell and a 1.0-1.5
10
M protein concentration.
For
the NMR experiments, 9 mg of protein were dissolved in 0.4 ml of
solvent so that the final concentration was 7 mM. The solvents
used were either a mixture of 95% (v/v) HO and 5% (v/v)
D
O at pH 3.5 or 100% D
O at pD 3.5. Chemical
shifts were measured relative to
3-trimethylsilyl-(2,2,3,3-2H4)propionate. Proton two-dimensional
NMR spectra were recorded at 600 MHz with a Bruker AMX600 spectrometer.
Spin system identification was achieved using COSY(39) ,
DQF-COSY(40) , TOCSY(41) , and NOESY (42) spectra recorded at two temperatures (20 and 30 °C).
273 distance restraints were obtained from the volume integration of
NOESY cross-peaks. 23 angle restraints were defined from the analysis
of the COSY and DQF-COSY spectra. 18 three-dimensional solution
structures consistent with the distance and angle restraints were
calculated using a hybrid DIANA/X-PLOR
protocol(43, 44) . A final minimization of the
structures was carried out in a force field derived from CHARMM22 (file
parallh22x.pro and topallh22x.pro in X-PLOR 3.1).
All the structures were displayed, analyzed, and compared on a Silicon Graphics 4D/25 station using the SYBYL package (Tripos Associates, Inc.).
Antibodies directed against the chimeric construction were obtained by immunizing, subcutaneously and at multiple sites, two rabbits (blanc du Bouscat, Wiss) with 200 µg of chimera in complete Freund's adjuvants. Rabbits were reimmunized three times in incomplete Freund's adjuvants with the same quantity of peptide at 21-day interval. Animals were bled 15 days after each boosting, and their sera were tested for specific antibody production by enzyme-linked immunosorbent assay (ELISA).
Elicited
antibodies were titrated against either the chimera and toxin ,
coated on microtiter plates (1.0 and 0.1 µg/well, respectively).
After overnight incubation at 4 °C, wells were then saturated with
PBSA (saturating buffer), 100 µl of rabbit antiserum, diluted 1/50
in saturating buffer, were added to the well (serially diluted 1/3) and
incubated for 2 h at room temperature. After washing, 100 µl of
goat anti-rabbit IgG conjugated to HRP (Sigma), diluted 1/6000, was
added to each well for 1 h at room temperature. Bound antibodies were
detected as above using ABTS as substrate.
Pooled anti-chimera sera
were purified by affinity chromatography through a toxin
-Sepharose 4B column (3.5
1 cm), prepared by coupling the
toxin
(4.0 mg) to CNBr-activated Sepharose 4B (1.0 g; Pharmacia
Biotech Inc.).
Relative affinities of the purified antibodies for
the chimera and native toxin were tested by competitive
inhibition ELISA. These tests were performed by coating the purified
anti-chimera antibodies into the wells (0.5 µg/well) and, after
saturation and washing, by titrating the soluble antigens (50 µl)
into the wells. Starting concentrations were 10
M for chimera and toxin
and 10
M for the denatured chimera. A constant dilution
(corresponding to 50% binding in direct binding assays) of HRP-toxin
in 50 µl was then added to the wells, and the plate was
incubated for 2 h at room temperature. After extensive washing the
bound HRP-toxin
was detected as described above.
Antibody
neutralization of toxin binding to AchoR was performed by using
anti-chimera antibodies. Microtiter plates were coated with solubilized
AchoR as described above. Serial dilutions of 50 µl of either the
affinity-purified anti-chimera antibodies or the monoclonal antibodies
M
(48) or M
(49) were added to the wells. The starting concentration
for all three antibodies was 5
10
M. A constant dilution of HRP-toxin
in 50 µl
was then added to the wells, and the plate was incubated for 3 h at
room temperature. Bound HRP-toxin
was detected as described
above. The specificity of the binding of anti-chimera antibodies to
toxin
was tested by incubating the antibodies with
10
M chimera 2 h before adding the
HRP-toxin
conjugate.
Figure 2:
Sequence of the scorpion charybdotoxin (top), the curaremimetic loop 26-37 of the snake toxin
(middle), and the designed snake-scorpion curaremimetic
chimera (bottom). The structurally important cysteine residues
of the scorpion scaffold are underlined, and the functionally
important residues of the curaremimetic loop are in boldface. Z is 5-oxoproline (pyroglutamic
acid).
Figure 3:
Three
chromatography steps of purification of the synthetic chimera. Reverse
phase HPLC of crude (left), refolded (middle), and
purified chimera (right). A Vydac C18 column (0.46 15
cm) was used, eluted with a 30-min 5-30% acetonitrile gradient in
0.1% trifluoroacetic acid at 1 ml/min flow
rate.
Figure 4: Conformation and stability of the chimera. Far-UV (A) and near-UV (B) circular dichroism spectra of the chimera (solid line) and of charybdotoxin (dashed line). C, far-UV circular dichroism spectra of the chimera (solid line) and charybdotoxin (dashed line) at 90 °C.
The chimeric protein is
soluble in water at millimolar concentration, thus allowing H NMR characterization. Assignment of proton resonances was
achieved according to the standard method developed by
Wüthrich and co-workers(52) . The backbone
proton chemical shifts are close to those of charybdotoxin: differences
higher than 0.25 ppm concern only a few residues, located at the N
terminus (Cys
, Thr
, Thr
) or in the
mutated part of the sequence (Gly
, Trp
,
Asp
, Arg
, Gly, (
)Ile
,
Ser
).
The secondary structure of the
chimeric protein was established by analyzing the backbone proton NOEs
and the J
coupling constants.
The pattern of strong sequential and long range dNN connectivities for
residues 12-21 indicates the presence of a helical conformation,
confirming the CD analysis. Furthermore, the presence of strong
sequential d
N NOEs, the typical pattern of long range d
,
dNN, and d
N connectivities, and the large values of the coupling
constants indicate that residues 25-28 and 33-36 are
involved in a two-strand antiparallel
-sheet. Thus, the secondary
structure elements of charybdotoxin (23) are found at the same
positions in the new protein. The similarity of the chemical shifts and
the conservation of the secondary structure elements in the new protein
suggests that the tertiary structures of the two proteins are very
similar. Full description of NMR data, three-dimensional structure
resolution, and analysis will be reported elsewhere.
Charybdotoxin is characterized by a remarkably high
conformational stability: CD spectroscopy reveals no significant
conformational changes when the protein is heated up to 90 °C (Fig. 4C) or is dissolved in 5 M guanidine
HCl. When the chimera is treated under the same strong denaturing
conditions, the far-UV CD spectrum (Fig. 4C) indicates
the protein is still substantially folded. Thus, in spite of the fact
that all the surface of the -sheet has been re-designed and the
N-terminal segment of charybdotoxin deleted, the chimera exhibits an
exceptional conformational stability, comparable with that of the
original scorpion scaffold.
Figure 5:
Acetylcholine receptor binding activity of
the chimera. Inhibition of binding of HRP-labeled toxin to AchoR
in the presence of varying amounts of native toxin
(
), the
chimera (
), and the denatured chimera (
). Incubation time
was 24 h at 20 °C. The receptor was coated in microtiter plates and
revelation performed colorimetrically as described under
``Experimental Procedures.''
Charybdotoxin is a powerful
blocker of K channels, activated by both intracellular
Ca
increase and membrane
depolarization(54, 55, 56) . The chimera,
which maintains a folding similar to charybdotoxin, has been tested on
rat skeletal muscle Ca
-activated K
channels inserted in a lipid bilayer(57) : no evidence of
blockage was recorded up to a chimera concentration of 1
µM. (
)Thus, the chimera, which presents a
completely redesigned
-sheet sequence (and a N-terminal
truncation), does not retain any affinity for the K
channel, while it has acquired a new affinity for a different
receptor, the AchoR.
When tested in direct binding assays, the anti-chimera-elicited
antibodies were able to recognize coated toxin (not shown). In
order to select a more specific antibody population recognizing
uniquely the transferred
-hairpin, we purified the antiserum by
affinity chromatography on a column containing immobilized toxin
.
Furthermore, to avoid partial unfolding of the chimera during the
coating procedure and to minimize artifacts due to the ELISA plates, we
used a solution assay in which the chimera and the toxin
were in
solution free to compete with labeled toxin
for binding to the
coated antibodies(45) . The results of this binding inhibition
experiment (Fig. 6A) reveal that the chimera and the
parent toxin are equally well recognized by those antibodies,
indicating a full antigenic equivalence between the chimera and the
toxin
for the selected antibody population. Absence of reaction
with denatured chimera and native charybdotoxin (Fig. 6A) demonstrates that the antibody subpopulation
is indeed recognizing, in a conformation restricted manner, a surface
region that is shared by both toxin
and the chimera: this region
is the part of the toxin
curaremimetic site that has been
transferred from the snake toxin to the scorpion scaffold.
Figure 6:
Anti-chimera antibodies analysis. A, competition inhibition of binding of HRP-labeled toxin
to affinity chromatography purified anti-chimera antibodies in
the presence of varying amounts of native toxin
(
), the
chimera (
), the denatured chimera (
), and charybdotoxin
(
). B, inhibition of binding of native toxin
to
the acetylcholine receptor by the affinity chromatography-purified
anti-chimera antibodies (
), monoclonal M
2-3 (
),
monoclonal M14-1-1 (
) and anti-chimera antibodies in the presence
of 10
M chimera
(
).
All of our results show that, by redesigning a part of the
molecular surface of the natural scaffold of charybdotoxin, we were
able to engineer a chimeric protein exhibiting a new function.
Naturally, the structural scaffold used in our design has evolved to
block the K channel (at nanomolar concentration). By
systematic mutagenesis studies, the
-sheet region of
charybdotoxin, comprising the residues 25-36, has been mapped as
the molecular surface intimately interacting with the K
channel mouth (58) , thus physically obstructing the
K
ion flux through the ionic pore. Other scorpion
toxins also seem to use this region to interact with other K
channel subtypes, with different affinities. In any case,
although the active molecular surfaces of the various scorpion toxins
are different as defined by their amino acid sequences, their overall
structure is similar, as shown by the published three-dimensional
structure of charybdotoxin(23, 24) ,
iberiotoxin(59) , PO5(60) , kaliotoxin(61) ,
margatoxin(62) , chlorotoxin(63) ,
agitoxin(64) , and noxiustoxin(65) . We adopted
charybdotoxin as a structural scaffold and replaced the amino acid side
chains forming the exposed functional
-sheet surface with those of
a loop from a curaremimetic neurotoxin presenting a structurally
similar
-hairpin. This way we succeeded in generating a novel and
artificial ligand, with micromolar affinity for AchoR, which is
completely unrelated to the K
channel.
The present
results confirm previous examples reported in the literature (see (66) and (67) for two recent reviews), showing that
novel activities can be generated on protein scaffolds by replacing
exposed functional loops. However, two major differences characterize
our engineering work. First, most of the literature examples include a
loop transplantation between structurally homologous host proteins. In
our case no overall structural homology exists between charybdotoxin
and toxin . Local structural similarity, however, exists between
the two exchanged loops: they form two regular
-hairpins (Fig. 1), superimposable with little differences in the spatial
disposition of their backbone atoms. Second, although Hynes et al.(68) and Wolfson et al.(12) showed that
loops can be transferred from one structural context to another as
structural cassette without perturbating their own structural and
functional properties, a notable difference exists between these two
examples and our work: the transferred loop 26-37 in our chimeric
construction is fixed to the scaffold by three covalent disulfide bonds
and not just held by the N and C termini. These bonds, involving
cysteine residues within the transferred loop, are supposed to limit
considerably the conformational space of the transferred loop. Even if
we cannot disprove that solely the four residues of the turn, between
Cys
and Cys
, are responsible of the observed
functional properties of the chimeric construction, we believe the
entire transferred sequence is responsible of that. The fact that no
receptor binding or antibody recognition is exhibited by the unfolded
linear chimera ( Fig. 5and Fig. 6) stresses, by itself,
the importance of the disulfide bonds in determining the structural and
functional properties of our chimeric construction. The two above
differences are the direct consequences of two major properties of the
scorpion scaffold, advantageous for protein engineering: (i) functional
versatility and (ii) sequence permissiveness. These two characteristics
are directly related and the second may explain the first: in fact,
permissiveness in sequence mutation may allow this fold to adapt its
structure to the different functions of blocking the different ionic
channel types (in toxins) and also of forming holes in membranes (in
defensins).
Insertion of exogenous sequences in protein structures
most often results in a decrease of overall conformational stability of
the host scaffold(68, 14) . This is one of the major
difficulties, for example, in the engineering of the immunoglobulin
scaffold and in rat antibody humanization. The /
scorpion
scaffold does not suffer from this weakness: even after multiple
substitutions (8 residues upon 31), it maintains its exceptional
conformational stability. The previously designed metal binding
protein(22) , containing the same scorpion fold, also maintains
such a stability. This stability makes a powerful structural solution
of the scorpion scaffold, which naturally evolved to be compatible with
a great variety of sequences and functions. We believe that the
presence of an interior core formed essentially by three disulfides (69) may explain not only the sequence permissiveness but also
the structural stability of the scorpion scaffold.
We have
determined the three-dimensional structure of the chimera on the basis
of H NMR data: detailed description of this structure will
be reported elsewhere.
The analysis of this structure
confirmed the presence of the
/
fold in the chimera, as
suggested by CD data, and of a conformation in the transferred loop
similar to that of the central loop of toxin
, as suggested by the
strong competition between the chimera and the toxin
, seen in the
immunological characterization. However, structural differences exist
at the level of some amino acid side chain orientations. It is well
documented that single point mutations of biologically active surface
may be enough to cause the loss of several order of magnitude in
biological potency, e.g. in erabutoxin a the substitution
Arg
Glu result by itself in a 318-fold lower
affinity molecule(28) , with consequent loss of 3.8
Kcal
mol
in receptor binding energy.
Differences in the orientation of some critical side chains thus may be
responsible for the low potency of the chimera as AchoR ligand.
However, to account for the large difference in biological activity of
the chimera (10
-fold difference in affinity, which accounts
for a loss of 7.0 Kcal
mol
) as compared with
toxin
, other factors must play additional important roles. Recent
mutagenesis data (29) have defined the functional site of
erabutoxin a as an approximately 700-Å
surface,
including residues from loops I, II, and III (Fig. 7). Our
chimeric construction contains only the active residues of loop II,
corresponding to the central part of the curaremimetic site (colored in red in Fig. 7). The absence in the chimera of the
functional residues of loops I and III (in orange in Fig. 7), by itself, may explain its lower affinity. For example,
from the data of Trémeau et al.(29) , we calculated that the contribution to binding free
energy of Ser
and Gln
of loop I and Lys
of loop III accounts for 3.1, 3.2, and 2.0
Kcal
mol
, respectively. Thus, the deletion of
these residues is predicted to account for a total energy loss of 8.3
Kcal
mol
: this figure has to be compared with
the 7.0 Kcal
mol
lower binding energy exhibited
by the chimera. These calculations assume a simple additivity in free
energy of binding(70) . We did not experimentally prove the
additivity of mutational effects in the case of curaremimetic toxins;
however, this simple calculation may serve to indicate that, even if
the structural resemblance of the transferred site to the original one
might be improved in future designs, the biological activity of the
actual chimera may be close to that expected by the central loop of
toxin
alone. This consideration suggests that a substantial
improvement of the binding activity of the chimera may be obtained if,
in addition to the modifications that might be suggested by its
detailed structural analysis, we also combine a nonrational
combinatorial approach. The substitution Ile
Arg in
erabutoxin a has already been shown to increase the binding affinity by
a factor of 7(29) : similar substitutions may be also selected,
if single positions of the curaremimetic site of the chimera will be
subjected to random substitution by combinatorial chemistry and the
best binder to the AchoR selected on the basis of screening tests. A
recent work on the minimization of the atrial natriuretic peptide
hormone (71) has shown that indeed affinity loss due to
deletion of active residues may be rectified by optimizing the
remaining residues by means of phage display and selection methodology.
Figure 7:
Space-filling structure of the chimera (left) and the snake toxin (right). In red are the residues corresponding to the transferred site in the loop
II of toxin
(left) and in the chimera (right).
In orange are the residues of loops I and III of the toxin
active site (28, 29) that contribute more than 1
Kcal
mol
to the energy of acetylcholine
receptor binding.
A close structural resemblance between the transferred site in the chimera and that present in the parent snake toxin is clearly apparent from the immunological characterization of the antibodies, elicited by immunization with the chimeric construction. This resemblance is the basis of the efficient recognition of the snake toxin and the prevention of its binding to AchoR by these antibodies. Clearly, the chimera may represent a less toxic, but equally effective, immunogen than snake toxins themselves in the production of toxin neutralizing antibodies. Faithful reproduction or molecular mimicry of a protein antigenic site is of fundamental importance in the design of novel constructs able to elicit high affinity antibodies for use as vaccines. Due to the high chain flexibility, simple peptides are not able to mimic the conformational aspects of protein epitopes. Insertions of antigenic epitopes in carrier proteins (72, 73) or in de novo designed scaffolds (74, 75) have been already used in an attempt to mimic discontinuous determinants for the purpose of activating B-cells specific for native proteins. The results presented here demonstrate that immunization with a highly constrained peptide immunogen is sufficient to induce high titer antibodies, with high affinity and specificity for a toxic protein antigen. Our immunogen, which is able to reproduce (or mimic) the secondary and tertiary conformation of a toxin protein epitope, has been constructed on the basis of an epitope transfer to a permissive, versatile, and stable host scaffold. Utilization of this approach and similar ones are significant in the rational design of synthetic vaccines.
In our
previous work(22) , we proposed the /
scorpion fold
as a natural structural scaffold for protein engineering. By
transferring the carbonic anhydrase metal binding site in it, we
engineered a novel metal-binding protein, exibiting affinities and
selectivity for different metal ions. The present results confirm that
this fold is a useful structural scaffold and host plate form for the
construction of highly stable miniproteins, presenting transplanted
sites in a fixed and well defined conformations, thus extending its
scopes and potentialities to the generation of biologically active
proteins involved in relevant protein-protein recognition processes.
Furthermore, the presence in this fold of structural motifs
(-helix,
-sheet, loops) generally found in globular proteins,
the tolerance for sequence mutations, and its retained stability after
multiple substitutions appear as unique qualities of this fold and
extremely valuable for protein engineering. In addition, in the
creation of novel proteins, since apparently any region can be
engineered with new sequences, a combinatorial and functional selection
approach can be suitably used to improve the design. Furthermore, given
the reduced size of the scaffold, the chemical methods can increase the
structure diversity of novel proteins by using unnatural amino acids,
peptide mimetics, or labeled compounds.