(Received for publication, May 7, 1996, and in revised form, November 8, 1996)
From the Département d'Ingénierie et d'Etudes des Protéines, CEA, Saclay, 91191 Gif-sur-Yvette Cedex, France and the § Department of Physiology and Pharmacology, University of Strathclyde, Glasgow G1 1XW, United Kingdom
BgK is a K+ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residue protein that adopts a novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed the functional importance of five residues, which include a critical lysine and an aromatic residue separated by 6.6 ± 1.0 Å. The same diad is found in the three known homologous toxins from sea anemones. More strikingly, a similar functional diad is present in all K+ channel-blocking toxins from scorpions, although these toxins adopt a distinct scaffold. Moreover, the functional diads of potassium channel-blocking toxins from sea anemone and scorpions superimpose in the three-dimensional structures. Therefore, toxins that have unrelated structures but similar functions possess conserved key functional residues, organized in an identical topology, suggesting a convergent functional evolution for these small proteins.
Functional properties of proteins are frequently associated with a small number of important residues. For example, enzyme activities depend on a few residues that are essential for catalysis. Also, protein-protein recognition processes have been predicted (1) and recently demonstrated (2) to be energetically driven by a small proportion of the residues forming the contacting areas in protein-protein complexes, as identified by x-ray studies (3, 4). Among the proteins whose major functions require protein-protein interactions are animal toxins, which bind to various molecular targets, such as receptors or ion channels, using a small number of binding residues (5-8). As has been shown for enzymes (9), toxins with different architectures are capable of exerting similar functions (10). However, in contrast to enzymes, the molecular basis associated with the conservation of the function in structurally unrelated toxins remains unknown. In this paper, we show that two families of animal toxins with different folding patterns but a comparable capacity to bind to potassium channels include similar functional diads, composed of a critical lysine and an aromatic amino acid separated from each other by 6.6 ± 1.0 Å.
The amino acid sequence of
BgK1 was proposed a few years ago (11).
However, chemical synthesis attempts, based on these data, systematically failed. The proposed amino acid sequence was therefore questioned, re-examined, and ultimately
corrected.2 The revised amino acid sequence
of BgK from Bunodosoma granulifera is:
VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCELC. BgK and each
alanine-substituted analog were synthesized by solid phase synthesis
using an Applied Biosystems model 431A peptide synthesizer, starting
from 0.1 mmol of Rink-resin
(4-(2,4
-dimethoxyphenylhydroxymethylphenoxy resin; 0.48 mmol/g). A
10-fold excess (1 mmol) of Fmoc
(N-(9-fluorenyl)methoxycarbonyl)-protected amino acid was
used and coupled in N-methylpyrrolidone in the presence of
N,N
-dicyclohexylcarbodiimide/1-hydroxybenzotriazole. The
following side chain protections were used: t-butyl ether for Ser and Thr; t-butyl ester for Glu and Asp; trityl for
Cys, His, Asn, and Gln; 2,2,5,7,8-pentamethylchromane-6-sulfonyl for Arg; and t-butoxycarbonyl for Lys and Trp. The peptide was
simultaneously cleaved from the resin and deprotected by treatment (500 mg of resin/50 ml) with 90% trifluoroacetic acid, 5% H2O,
5% triisopropylsilane for 1.5 h at room temperature. The resin
was filtered, and the solution was precipitated with
methyl-t-butylether, washed three times with ether,
dissolved in 20% acetic acid, and lyophilized.
The crude peptides (0.1 mg/ml) were oxidized in 50 mM
phosphate buffer, pH 7.8, containing 5 × 103
M reduced and 5 × 10
4 M
oxidized glutathione. Oxidation was complete after 1 h at room temperature. The solution was then acidified to pH 3.0 with acetic acid
and directly applied to a Vydac C18 column (25 × 1 cm) for purification. The peptides were eluted with an acetonitrile gradient containing 0.1% trifluoroacetic acid. The fractions containing the
oxidized peptides were analyzed by analytical HPLC. The adequately oxidized components were identified by mass spectrometry. The yields in
purified and oxidized toxins ranged from 20 to 40%.
Disulfide assignments were achieved by peptide mapping using the Lys-C endoprotease (1:20 by mass) in 20 mM Tricine buffer, containing 1 mM EDTA, pH 7.6, for 7 h at 35 °C. Digests were submitted to reverse phase-HPLC on a Vydac C18 column (25 × 0.46 cm), and the resulting fragments were collected, lyophilized, and analyzed by electrospray mass spectrometry. The mass of the fragments unambiguously indicated that the pairings were Cys2-Cys37, Cys11-Cys30, and Cys20-Cys34.
Amino acid analyses were performed on an Applied Biosystems model 130A automatic analyzer, equipped with an on-line model 420A derivatizer, and mass analysis on a Nermag R10-10 mass spectrometer, coupled to an Analitica of Branford electrospray source. Amino acid compositions and mass analyses agreed with the expected theoretical values.
Peptide concentrations were determined by monitoring absorbances at 280 mm, using a molar extinction coefficient of 8100, calculated on the basis of the known amino acid content (11). For analogs where Trp or Tyr were deleted, the peptide concentrations were determined on the basis of quantitative amino acid analyses.
Circular dichroism spectra were recorded using a Jobin-Yvon CD6
dichrograph, driven by an IBM PC operating with a CD6 data acquisition
and manipulation program. Spectra in the range 180-250 nm were run at
20 °C in 5 mM sodium phosphate, pH 7.0, with a 0.1 cm
quartz cell and a protein concentration of 1.5-2.0 × 105 M.
Synthetic BgK (9.8 mg) was dissolved in 440 µl of solvent, leading to a final concentration of 4.9 mM. Solvents were either a mixture of 400 µl of water and
40 µl of D2O, or 440 µl of D2O. pH was
fixed at 3.7, and chemical shifts were measured relative to
TSP-d4. All NMR experiments were performed at
600 MHz (AMX600 Brüker spectrometer) at 20 and 30 °C, in order
to resolve assignment ambiguities. At each temperature and in each
solvent, a DQF-COSY spectrum (12), a nuclear Overhauser spectroscopy
spectrum (300 ms mixing time) (13), and a total correlation
spectroscopy spectrum (14, 15) were recorded. Quadrature detection was
performed using the States method (16) in the indirect dimension and
using simultaneous mode acquisition in the directly detected dimension. The water signal was suppressed by low power irradiation at all times
except during t1 and t2.
The spectra were recorded with 128 (t1) × 1024 (t2) (96 × 1024 in D2O) and
with a sweep width of 7812 Hz (6578 Hz in D2O). The
3JNH-H (respectively
3JH
-H
) coupling constants were measured
from the high resolution DQF-COSY in H2O (respectively
D2O).
All data were
transformed using FELIX software (17). Prior to Fourier transformation,
spectra were weighted with a 18° shifted sine bell (90° for the
spectra to be integrated) and were zero-filled in the
t1 dimension to yield 1000 × 1000 matrices
after reduction (1000 × 4000 for the DQF-COSY). Each NOE was
integrated with FELIX, using a 2.48 Å distance between the and the
protons of Tyr26 for calibration. A range of ±25% of
the distance value was used to define the upper and the lower bounds of
the restraints. Structures were obtained by molecular modeling using
successively DIANA (18) for preliminary structure calculation and
X-PLOR for simulated annealing (19, 20). After having solved assignment
ambiguities with the help of DIANA, 50 structures were calculated using
this software, and the 30 structures with a target function below 10 were further refined by simulated annealing in X-PLOR 3.1 (with files
parallhdg.pro and topallhdg.pro). The 15 structures with the lowest
energy were kept for analysis. Within these 15 structures, all but
three distance violations are no larger than 0.3 Å and all dihedral
restraints violations are lower than 5°.
Competition experiments
were made on rat brain synaptosomes using 125I-labeled
-dendrotoxin, as described previously (21).
BgK was isolated from B. granulifera (11), and its amino acid sequence (see Fig.
1) has been recently corrected.2 BgK, like a
number of scorpion toxins such as charybdotoxin, (i) binds to potassium
channels, (ii) contains 37 residues, and (iii) possesses 6 half-cystines. However, our attempts to align the amino acid sequences
of BgK and scorpion toxins have failed, suggesting that BgK adopts a
structure that differs from the well-known /
fold of scorpion
toxins (22). We therefore decided to elucidate its structure by NMR and
molecular modeling.
Structural Assignment
Three unique amino acids having specific spin systems were used as starting points for sequential assignments. These are Trp5, His13, and Gly18. The unique tyrosine (Tyr26) was also used to assign the C-terminal part of the amino acid sequence of BgK. As no proline was present in the protein, these four starting points as well as the existence of all but one sequential NH-NH connectivities allowed us to achieve the complete assignment of the amino acid sequence of BgK (Table I).
|
A total of 767 distance restraints deriving from NOEs
(316 intra-residual correlations, 172 sequential, 155 short range
(|i j|
4), 124 long range
(|i
j| > 4) and 12 dihedral
restraints (7 from
angles, 5 from
1 angles) deriving from
coupling constants was used. This large set of constraints (21 restraints by residue) yielded a well defined structure, as reflected
by the mean r.m.s.d. value of its backbone, which was as low as
0.8 ± 0.2 Å between two structures. The calculated structures
were consistent with both experimental data and the standard covalent
geometry. The structures had no distance violations larger than 0.32 Å, and only three distance violations were larger than 0.3 Å. All
dihedral restraint violations were lower than 5°. The covalent
geometry was respected, as revealed by the low
r.m.s.d.
values of
the bond length (0.003 Å) and the valence angles (1.52°).
The existence of two helices,
running from residues 9 to 16 and residues 24 to 31, was clearly
indicated by the presence of numerous short-range NOEs between
h(i) and hn(i + 3) protons and between
h
(i) and h
(i + 3) protons (Fig. 1). No
other regular secondary structure emerged from these data. Fig.
2a shows the 15 best structures of BgK, which
result from transformation of NMR data and molecular modeling. The two
helical stretches are well defined with r.m.s.d. values of 0.4 ± 0.1 Å and 0.5 ± 0.2 Å for the position 9-16 and 24-31
stretches, respectively. Though to a lesser extent, the other parts of
the toxin structures were also relatively well defined.
Panels b and c of Fig. 2 show,
respectively, the overall fold of BgK and the spatial organization of
its secondary elements. The N- and C-terminal regions are maintained in
spatial proximity by the disulfide 2-37, whereas the third disulfide
20-34 brings the loops 16-24 and 31-37 inside the center of the
molecule, providing the toxin with a globular shape. The 11-30
disulfide, which links the two helices, adopts a unique conformation,
which is almost left-handed (23). It is centrally located in the
structure, consistently with a tight organization of a local
hydrophobic core. The other disulfide bonds adopt either two
conformations (disulfide 20-34) or no prevailing conformation
(disulfide 2-33).
Mutational Analysis
To identify functional residues of BgK,
we submitted the toxin to an alanine-scanning experiment, producing all
single point variants by chemical synthesis, as described under
"Materials and Methods." Competition experiments were performed
between the variants and radiolabeled -dendrotoxin on membrane from
rat brain synaptosomes. Twenty-five variants have been synthesized and
investigated regarding their effects on dendrotoxin binding. Fig.
3 shows five inhibition curves obtained with native BgK
and four variants, which display a substantially lower competition
activity, as compared with native BgK. Except for the variant T33A, all
inhibition curves are parallel to that observed with the native toxin.
Similar parallel inhibition curves were obtained for other variants,
except K25A, for which no inhibition was observed even in the presence
of 3 × 10
7 M protein. In the presence
of the variant F6A, a 10% increase in binding of labeled dendrotoxin
was observed. Although we are not able to explain this observation, as
yet, one should recall that different subtypes of potassium channels
are present in brain and are composed of heterooligomeric mixtures of
different protein subunits. Experiments with subtype-specific
antibodies reveal that most of the channels in the brain contain
Kv1.2 (~80%) or Kv1.1 (~50%) subunits
(24).
-Dendrotoxin blocks Kv1.1 and Kv1.2 channels with almost equal affinity (IC50 values ~ 20 nM), and has greater than 10 times less affinity for
other cloned channels (25). Therefore, in view of such complexity, the
present competition data have to be considered with caution. From
curves similar to those shown in Fig. 3, we deduced IC50
values for all variants. These values are compiled in Table
II. The correlation coefficient for fitting the data
points was calculated from the Hill equation, y = Rmax/[1 + (X/IC50)P]. As can be seen from Table
II, a value close to 1 was obtained in all cases. Moreover, preliminary
competition experiments performed with cloned Kv1.2
channels and a number of the variants exhibiting a lower inhibitory
capacity, as compared to native BgK, nicely paralleled those reported
in Table II.3 Therefore, we anticipate that
data obtained with brain synaptosomes mostly reflect the functional
importance of residues implicated in the capacity of BgK to bind to
Kv1.2 potassium channels. Nevertheless, in order not to
overinterpret our data, we considered only the relative inhibitory
capacity of the variants, without any attempt to deduce their binding
affinities.
|
Competition experiments, shown in Table II, revealed that mutation of
lysine 25 into alanine is associated with the largest affinity
decrease. This mutation, however, caused no significant change in the
secondary structure of the toxin, as inferred from the circular
dichroism spectra of the variant which is quite similar to the spectrum
of the native toxin (Fig. 4). We conclude, therefore, that the low competition ability of this mutant is not due to distortions in the toxin structure but to the absence of the lysine side chain and possibly to the loss of its positive charge. Upon mutations of three other residues, Phe6, Tyr26,
and Thr33, BgK was a less potent inhibitor, since its
competition capacity decreased by factors of 46, 38, and 19, respectively. However, while the circular dichroism spectra of the
first two variants were indistinguishable from that of the native
toxin, the spectrum of the T33A variant showed some distortions (data
not shown), which could not be readily interpreted but which might
reflect some structural perturbations. Additionally, the inhibition
curve with the T33A variant (see Fig. 3) was not quite parallel to
those observed with the native BgK or other variants. In addition,
synthesis of this variant was particularly difficult to achieve,
leading to a relatively low yield of recovery. Therefore, all these
observations suggest that introduction of an alanine at position 33 may
be associated with structural perturbations, which might account for
the substantial decrease in inhibitory activity of BgK. As a result,
one cannot safely propose that the side chain of residue 33 is
implicated in the binding of the toxin to the BgK target. Therefore, in
the absence of further data, only the side chains of Lys25,
Phe6, and Tyr26 are concluded to be involved in
the functional site of BgK. Mutations at two positions,
Ser23 and His13, also induced approximately 8- and 6-fold decrease in competition capacity. Although relatively low,
these values might also reflect involvement of these residues in the
binding site of BgK. Mutations at the other positions either had no
effect on the affinity of BgK or caused changes in inhibitory capacity
that are lower than 3-fold. These side chains are not considered,
therefore, as actors in the recognition capacity of BgK for
dendrotoxin-sensitive binding sites. In summary, alanine-scanning-based
experiments indicated that three, and perhaps five, residues of BgK
belong to the surface by which the toxin interacts with the
channels.
Potassium channels constitute a major target for various toxins produced by venomous animals from distinct phyla, including cnidaria (26), arthropods (10), and reptiles (27). Thus, a number of toxins, found in venoms of (i) sea anemones such as BgK (11) or ShK (28), (ii) scorpions like charybdotoxin (29) or noxiustoxin (30), and (iii) snakes like dendrotoxin (27), bind to dendrotoxin-sensitive K+ channels (31, 32). However, it is unclear as to whether these proteins share any structural or chemical elements that could account for their common capacity to recognize similar channels.
In this paper, we first provided evidence that BgK from the sea anemone
B. granulifera is structurally unrelated to scorpion toxins,
although these two groups of toxins share a similar number of residues,
the same number of disulfides, and comparable functional properties.
The present structural analysis revealed that the fold adopted by BgK
contains two nearly perpendicular stretches of helices, with no
additional canonical secondary structures (Fig. 2). The globular
architecture of the toxin is stabilized by the three disulfides, one of
them linking the two helices. This structure is similar to that of ShK,
as indicated by a paper that was published during the review of our
manuscript (33). ShK, from the Stichodactyla helianthus sea
anemone, is a 35-residue toxin that also binds to potassium channels
(28). As in BgK, the structure of ShK includes two helices running from
residues 14-19 and 21-24. However, these two stretches are not
located at identical corresponding positions in both toxins. In fact, the absence of four consecutive residues in the amino acid sequence of
ShK, as compared to BgK (residues 12-15 using BgK numbering), leads to
a different organization of the N-terminal part of the toxin. More
precisely, the N-terminal part of BgK is successively composed of an
extended strand (1-8), an helix 1 (9-16), and a loop (17-23),
whereas in ShK, it is composed of an extended strand (1-8), a loop
(9-13), and helix 1 (14-19). The structures of the C-terminal parts
of the two toxins are more similar in the two toxins. Therefore, BgK
and ShK adopt the same type of fold with, however, a number of marked
structural differences located mostly in the N-terminal regions. Quite
distinctly, charybdotoxin (ChTX), a typical scorpion toxin that blocks
potassium channels (29), adopts a different fold with a short helix
linked by two disulfides to a three-stranded -sheet (22). No
-sheet structure was found in BgK.
Although BgK and ChTX have different structures, they both inhibit the binding of labeled dendrotoxin to potassium channels (11, 31), suggesting that they bind to regions of the channels that are recognized by dendrotoxin. In agreement with this view, previous observations reported that the sites recognized by ChTX (34, 35) and dendrotoxin (36, 37) commonly include the very conserved P-region of the pore of the channels. The channel region that is recognized by BgK is unknown; however, the toxin binds to several subtypes of Kv1 channels (i.e. Kv1.1, Kv1.2, and Kv1.3) with almost equal affinity,2 suggesting that the target of BgK also includes a highly conserved region of the channels, possibly the P-region. In the absence of further data on channel regions that are recognized by these toxins, one way to understand the molecular basis associated with their common capacity to inhibit the binding of dendrotoxin consists in comparing the sites by which these toxins recognize their targets.
With a view toward identifying the functionally important residues of
BgK, we submitted the toxin to an alanine-scanning experiment and
compared the ability of all the synthetic variants to inhibit the
binding of dendrotoxin membranes from rat brain synaptosomes. Twenty-five positions out of 37 have been modified. Therefore, if one
excepts the six half-cystines, which are likely to play a structural
role, nearly 80% of the positions of BgK have been individually
explored regarding their possible involvement in the binding of the
toxin to dendrotoxin-sensitive sites in rat brain synaptosomes. Our
data showed that introduction of an alanine at five positions,
i.e. Lys25, Phe6, and
Tyr26, and, to a lesser extent, at His13 and
Ser23, caused a decrease in the ability of the toxin to
compete with dendrotoxin for its specific binding sites, without
changing the secondary structure of the toxin. Moreover, preliminary
and unpublished data performed with cloned Kv1.2 channels
and a number of BgK variants parallel those obtained with rat brain
synaptosomes, strongly supporting the view that the residues
Lys25, Phe6, Tyr26, and perhaps
His13 and Ser23, are involved in the functional
site of BgK. Of these residues, however, mutation at Lys25
most dramatically affected the capacity of BgK to inhibit dendrotoxin binding, suggesting that this particular lysine is the major binding actor of BgK. This finding agrees with recent observations made with
ShK, a homologous K+ channel-blocking toxin from the sea
anemone S. helianthus whose lysine 22, which corresponds to
Lys25 in BgK, plays a critical binding role toward the same
channels (38). Previously, ChTX was submitted to site-directed
mutagenesis, and the residues by which the toxin binds to the
voltage-sensitive Shaker K+ channel were
identified (8, 39). Clearly, Lys27 was the most critical
residue, with four neighboring amino acids (Tyr36,
Met29, Asn30, and Arg34) whose
mutation also affected the affinity of ChTX to the channel. Thus, the
functional residues of ChTX form a homogeneous area located on the flat
-sheet face that is exposed to solvent (8, 39) and that covers
approximately 200 Å2 (18 × 12 Å). Although BgK has
no
-sheet structure, it nevertheless possesses a flat surface of
similar size (21 × 9 Å), which is formed by the edge of the
9-16 helix and its two flanking loops. Strikingly, this surface
harbors the functional residues of BgK (see Fig. 5A).
Therefore, BgK and ChTX possess comparable flat surfaces, which include
a similar small number of energetically important residues, among which
a lysine is the major binding actor (Fig.
5B).
If one assumes that these lysines play a similar binding role in BgK
and ChTX, their superimposition can be readily associated with
superimposition of Tyr26 in BgK with Tyr36 in
ChTX, two aromatic residues that play an important binding function in
the toxins (Fig. 6, top). The distances that
separate the C of lysines from the center of the benzene rings of
the tyrosines are 6.6 ± 1.0 Å. The common capacity of the two
toxins to recognize potassium channels is therefore associated with the conservation of a similar diad of functional residues, a lysine and a
close aromatic residue. The other functional residues are different in
the two toxins and do not form any evident superimposable pattern.
Remarkably, however, if one rotates ChTX 90° around a central axis
localized near the crucial lysine, Phe6 of BgK superimposes
with Tyr36 in ChTX (Fig. 6, bottom). Thus, by
virtue of the four-fold symmetrical organization of the channel (40),
Phe6 in BgK is likely to interact with the same aromatic
binding site as Tyr26, but on another monomer of the
channel.
Similar diads are present in toxins that are homologous to BgK and
ChTX. Thus, the diad Lys25-Tyr26 is conserved
in the three known K+ channel-blocking toxins from sea
anemones (Fig. 7). The situation is slightly more
complex with K+ channel-blocking scorpion toxins. Thirteen
K+ channel blocking scorpion toxins are presently known.
They have been previously divided into four subfamilies (41). Three of these subfamilies possess the same Lys27-Tyr36
diad as in charybdotoxin. In contrast, all toxins forming the fourth
subfamily, i.e. the two kaliotoxins and the three agitoxins (AgTX1-3), have a threonine at position 36. Strikingly, all these toxins uniquely possess a phenylalanine at position 24 or 25, located on the exposed face of the -sheet. Mutational studies of a
member of this family (AgTX2) showed that this
phenylalanine is involved in the binding to the Shaker
potassium channel (42). Interestingly, the diad
Phe25-Lys27 in AgTX2 can be
superimposed on the diad Lys27-Tyr36 in ChTX
provided the
-sheets of the two toxins form a 90° angle. We
suggest, therefore, that Tyr36 in toxins of three subgroups
of scorpion toxins and Phe25 in the remaining toxins occupy
the same binding site on the four-fold symmetrical channel, but on
different monomers. Thus, the common capacity of sea anemone and
scorpion toxins to recognize K+ channels is associated with
the conservation of a functional diad, composed of an essential lysine
assisted by a 6.6 ± 1.0 Å distant aromatic residue whose precise
nature (Tyr or Phe) and location may differ from one toxin to
another.
Why is such a diad conserved among two families of functionally similar but structurally unrelated proteins? In scorpion toxins, the positively ammonium group of the lysine of the diad may mimic K+ ions entering the pore, occluding the ion pathway (39, 43, 44). A similar role may be associated with the functional lysine of the toxins from sea anemones. The role played by the aromatic residue of the diad is less obvious. Recent bimutational analyses (42) showed that Phe25 in AgTX2 mainly interacts in the Shaker with the hydrophobic Met448, a position that is conserved or conservatively mutated (Ile, Val) in other K+ channels (45). Then, the conserved aromatic residues in the diads provide the toxin with two unique features. First, they offer the possibility for local hydrophobic interactions to take place between toxins and channels. Second, since their rings are somewhat parallel to the plane defined by the flat surface of the toxin, they allow the crucial lysine to protrude outside the toxin surface, thus being in an appropriate position to plug into the pore. Evidently, residues other than those of the diads also play some functional role in the toxins. These additional residues clearly differ from one toxin to another. Therefore, we suggest that the diads constitute a conserved functional core around which less conserved residues provide the different toxins with a proper binding specificity, which varies from one toxin to another. A similar situation that has also been found with snake curaremimetic toxins (5, 46).
In conclusion, two families of toxins with distinct folding patterns display a conserved functional diad. Each diad contains a critical lysine which should possess environmental and/or structural features that other lysines do not share and which allow it to exert a predominating recognition role toward potassium channels. Its location on a flat surface, its protruding position and the constant close proximity of an aromatic ring probably contribute to give a unique role to the functional lysine. Whether similar criteria are shared by other structurally unrelated but functionally similar toxins remains to be investigated.