In addition to peptides isolated from venom new a-conotoxins have recently been identified by cDNA cloning from venomous glands and have been Keywords binding protein; acetylcholine-elic
Trang 1increased selectivity towards Torpedo californica and
some neuronal subtypes of nicotinic acetylcholine
receptors
Igor E Kasheverov1, Maxim N Zhmak1, Catherine A Vulfius2, Elena V Gorbacheva2,
Dmitry Y Mordvintsev1, Yuri N Utkin1, Rene´ van Elk3, August B Smit3and Victor I Tsetlin1
1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
2 Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Russia
3 Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Vrije Universiteit, Amsterdam, the Netherlands
a-Conotoxins are a group of relatively short peptides
(12–19 amino acid residues, two disulfide bridges) from
the venom of poisonous marine snails of the Conus
genus [1] In addition to peptides isolated from venom new a-conotoxins have recently been identified by cDNA cloning from venomous glands and have been
Keywords
binding protein;
acetylcholine-elicited Cl – current; a-conotoxin analogs;
identified Lymnaea neurons; nicotinic
acetylcholine receptor
Correspondence
V I Tsetlin, Shemyakin-Ovchinnikov
Institute of Bioorganic Chemistry, Russian
Academy of Sciences, Miklukho-Maklaya
str 16 ⁄ 10 Moscow, Russia
Tel ⁄ Fax: +7 495 335 57 33
E-mail: vits@ibch.ru
(Received 28 March 2006, revised 16 June
2006, accepted 4 August 2006)
doi:10.1111/j.1742-4658.2006.05453.x
a-Conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthe-sis of a-conotoxin analogs may yield novel antagonists of higher potency and selectivity We incorporated additional positive charges into a-conotox-ins and analyzed their binding to nAChRs Introduction of Arg or Lys res-idues instead of Ser12 in a-conotoxins GI and SI, or D12K substitution in a-conotoxin SIA increased the affinity for both the high- and low-affinity sites in membrane-bound Torpedo californica nAChR The effect was most pronounced for [D12K]SIA with 30- and 200-fold enhancement for the respective sites, resulting in the most potent a-conotoxin blocker of the Torpedo nAChR among those tested Similarly, D14K substitution in a-conotoxin [A10L]PnIA, a blocker of neuronal a7 nAChR, was previously shown to increase the affinity for this receptor and endowed [A10L,D14K]PnIA with the capacity to distinguish between acetylcholine-binding proteins from the mollusks Lymnaea stagnalis and Aplysia califor-nica We found that [A10L,D14K]PnIA also distinguishes two a7-like anion-selective nAChR subtypes present on identified neurons of L stag-nalis: [D14K] mutation affected only slightly the potency of [A10L]PnIA to block nAChRs on neurons with low sensitivity to a-conotoxin ImI, but gave a 50-fold enhancement of blocking activity in cells with high sensitiv-ity to ImI Therefore, the introduction of an additional positive charge
in the C-terminus of a-conotoxins targeting some muscle or neuronal nAChRs made them more discriminative towards the respective nAChR subtypes In the case of muscle-type a-conotoxin [D12K]SIA, the contribu-tion of the Lys12 positive charge to enhanced affinity towards Torpedo nAChR was rationalized with the aid of computer modeling
Abbreviations
ACh, acetylcholine; AChBP, acetylcholine-binding protein; IC 50 , ligand concentration at which 50% inhibition is achieved; nAChR, nicotinic acetylcholine receptor; nH, Hill coefficient.
Trang 2synthesized chemically [2–5] a-Conotoxins have become
widely used tools in studies on nicotinic acetylcholine
re-ceptors (nAChRs) [6,7] because they can distinguish
between different nicotinic acetylcholine receptor
(nAChR) subtypes For example, a-conotoxins GI, MI
and SIA selectively block muscle-type nAChRs, whereas
some others block distinct neuronal nAChRs, e.g
a-conotoxins ImI and ImII target homo-oligomeric a7
nAChR [8], whereas a-conotoxins MII, PnIA, GIC
block heteromeric nAChR containing a3, a6 and b2
subunits [6] A change in one or several residues of the
naturally occurring a-conotoxin might result in a change
in its nAChR subtype selectivity [9] For example, the
A10L substitution in a-conotoxin PnIA switched its
selectivity from the a3b2 to the a7 nAChR [10,11]
Synthesis of diverse a-conotoxin analogs, mutations
in nAChRs and pair-wise mutation analysis have
enabled the identification of specific a-conotoxin
and⁄ or nAChR residues taking part in ligand–receptor
interactions [12–15] The crystal structure of the
acet-ylcholine-binding protein (AChBP) from the mollusk
Lymnaea stagnalis, which provides a high-resolution
structure for the extracellular domains of nAChRs
[16,17], has been used to build models for a-conotoxin
binding to distinct nAChRs [18] Recently, crystal
structures have been solved for AChBP complexes
with two a-conotoxins: [A10L, D14K]PnIA, a double
mutant of a-conotoxin PnIA [19], and for a-conotoxin
ImI [20,21] These structures provide a solid basis for
modeling the spatial structures of a-conotoxins with
the cognate nAChRs Modeling may also be a
start-ing point for the rational design of new a-conotoxins
with higher affinity and better selectivity towards
nAChRs
D14K substitution increased the affinity of the
starting [A10L]PnIA for chicken a7 nAChR and
L stagnalis AChBP [19] X-Ray data on the
AChBP)a-conotoxin complex were the basis for
con-structing a model for a7 nAChR complexes with
[A10L]PnIA and [A10L, D14K]PnIA [19] We used the
X-ray data and cryoelectron microscopy structure of
Torpedo nAChR [22] to build a respective model for
a-conotoxin [D12K]SIA, wherein the Lys12 positive
charge gave the most dramatic increase in the affinity
for T californica nAChR
Anion-selective nAChRs in some identified neurons
of the fresh-water snail L stagnalis and marine
mol-lusk Aplysia californica were found to resemble the a7
nAChRs of vertebrates in terms of their
pharmacologi-cal profile and the response kinetics to acetylcholine
(ACh) [23,24] To further elucidate the significance of
a positive charge in the C-terminus of a-conotoxins
we compared the action of [A10L]PnIA and
[A10L,D14K]PnIA on a7-like nAChRs in identified Lymnaea neurons This is of interest in light of the recent cloning of a set of nAChR subunits from this species and electrophysiological analysis of several of them expressed in Xenopus oocytes [25,26]
Results and Discussion
Synthesis of a-conotoxins New analogs of a-conotoxins GI, SI and SIA with arginine, lysine and⁄ or aspartate introduced at position
12 (Table 1) were synthesized using a solid-phase method with the simultaneous formation of the two disulfides For a-conotoxin SIA, which has Asp12 in this position, an additional D12S analog was also synthesized A series of a-conotoxin MI analogs was similarly synthesized In this case, we employed Lys-scanning mutagenesis for the possibly complete set of
MI variants, excluding the substitutions of structurally important amino acid residues (Cys, Pro) As a result, three novel analogs of a-conotoxin MI with a lysine residue introduced at position 5, 7 or 11 were obtained Simultaneous formation of the disulfides decreases the number of stages and usually gives higher peptide yields, although this is sometimes accompanied by the production of incorrectly bridged isomers [27] When several isomers were formed, the peptide with correctly formed disulfide bridges was assumed to have a higher potency to bind to the membrane-bound T californica nAChR in the radioligand-binding assay (see below)
It is known that incorrect disulfide formation in a-conotoxins that target the muscle-type nAChRs entails a decrease in the affinity [28] However, the enhanced affinity of the incorrectly formed isomer of a-conotoxin AuIB, targeting one neuronal-type nAChR, was revealed previously [29] and makes the method less predictive Therefore, all new synthesized analogs were also characterized using CD spectroscopy
to detect secondary structure changes in the ‘incorrect’ isomers (see below) In 13 syntheses of muscle-type conotoxins we found the generation of isomers only in two cases – one additional minor peak for [S12D]GI and two for SIA (all peaks had correct molecular masses)
a-Conotoxin [A10L]PnIA, known to act on a7 nAChR [10,11] was obtained by solid-phase peptide synthesis using the simultaneous formation of two disulfides as described previously [30] In the case of [A10L,D14K]PnIA, orthogonal protecting groups were used for correct pair-wise closing of disulfides to exclude the formation of other isomers (see Experi-mental procedures) The structures of all synthesized
Trang 3peptides were verified by MALDI analysis (Table 1)
and purity by RP-HPLC (data not shown)
CD spectroscopy
CD spectra were obtained for aqueous solutions of
native a-conotoxins GI, MI, SI, SIA and their analogs
[S12R⁄ K ⁄ D]GI, [H5K]MI, [S12R]SI, [D12S ⁄ K]SIA, as
well as for one isomer of [S12D]GI and two isomers
of SIA, which were produced in noticeable quantities
during peptide syntheses As an example the spectra of
a-conotoxin GI analogs are presented in Fig 1 Amino
acid substitutions at position 12 did not result in any
noticeable alterations in peptide secondary structure
However, the second (minor) isomer of [S12D]GI
dis-played a remarkable change in spectral characteristics
(inset in Fig 1) Similarly, the spectra of the SI and
SIA analogs with substitution at position 12 (as well
as [H5K]MI) were identical to that of the respective
naturally occurring a-conotoxins However, both
minor isomers of SIA had spectra resembling that of
minor [S12D]GI isomer (data not shown)
The available literature data indicate that single
amino acid substitutions do not markedly change the
CD curves of a-conotoxins However, breaking the
Cys–Cys disulfide bonds in a-conotoxin ImI [31] or
x-conotoxin MVIIA [32], or changing the size of the
disulfide-confined peptide loops by introduction of
an additional amino acid residue in a-conotoxin ImI
[33], resulted in a remarkable change in CD spectra,
with shifting of the ellipticity minimum into the
195–200 nm region This shift resembles that seen for
minor isomers of both [S12D]GI and SIA (see curve 4a in the inset of Fig 1) Taken together, these results indicate that analysis of biological activities (Fig 2) has been carried out on a series of a-conotoxins with correctly closed disulfides
Binding of synthesized a-conotoxin analogs to membrane-bound Torpedo nAChR
The activity of analogs was evaluated in competition with radioiodinated a-conotoxins GI or MI for binding
Fig 1 CD spectra of a-conotoxins GI (1, solid line), [S12K]GI (2, dotted line), [S12R]GI (3, dash-dot line) and main isomer of [S12D]GI (4, dash line) in water Inset: CD spectra of two [S12D]GI isomers – the main (4, solid line) and minor (4a, dash line) ones.
Table 1 The structures of synthesized naturally occurring a-conotoxins and their analogs All a-conotoxins have amidated C-termini as well
as disulfide bridges Cys1–Cys3 and Cys2–Cys4 The substituted residues in the analogs are indicated in bold type.
Mol mass, MH+
a Described in Celie et al [19].
Trang 4to membrane-bound T californica nAChR (Fig 2).
Both tracers bound specifically to the Torpedo receptor
with equal high affinity: Kd values for 125I-labeled GI
and MI were 24 ± 3 and 28 ± 6 nm, respectively By
contrast to a-conotoxin GI and M1, a-conotoxin SI
has an equal potency to both sites in the Torpedo
nAChR [34], whereas a-conotoxin SIA binds to only
one site [35] as revealed by competition with125
I-labe-led a-bungarotoxin That is why we did not prepare the
radioactive forms of these peptides, and125I-labeled GI
was used as a tracer to test the SI and SIA analogs In
these experiments the synthetic a-conotoxins GI, SI,
SIA and MI were used as controls The respective
lig-and concentrations at which 50% inhibition is achieved
(IC50values) are presented in Table 2
The introduction of a positively charged amino acid
residue instead of a neutral one in position 12 of
a-conotoxins GI and SI resulted in a three- to
seven-fold increase in the affinity to both binding sites of
the Torpedo nAChR (Fig 2A,B; Table 2) The most
remarkable was the D12K mutation in a-conotoxin SIA: the binding efficiencies to the high- and low-affin-ity sites increased for the [D12K]SIA analog by 35 and
260 times, respectively (Fig 2C; Table 2) This increase was due mainly to removal of the negatively charged amino acid residue in this position, because substitu-tion with neutral Ser also resulted in affinity enhance-ment to both sites (25 and 65 times, respectively) Conversely, the introduction of a negative charge in position 12 of a-conotoxin GI caused a considerable decrease in the affinity for the receptor (Fig 2A; Table 2) However, the introduction of an additional positive charge (Lys) at position 11 of a-conotoxin MI (which corresponds spatially to residue 10 of a-cono-toxins GI, SI and SIA) affected the peptide activity only slightly, whereas H5K or A7K mutations wor-sened the binding characteristics of these analogs (Fig 2D; Table 2)
It should be noted that all three minor isomers (of [S12D]GI and SIA) showed more than tenfold
Fig 2 Inhibition of 125 I-labeled a-conotoxins GI (A–C) and MI (D) binding to membrane-bound Torpedo nAChR with indicated a-conotoxins and their analogs Final concentrations of the radioligand and toxin-binding sites of receptor were 280 and 230 n M , respectively The data shown are the averages of two independent experiments The inhibition curves were fitted using ORIGIN 6.1 (MicroCal Software Inc.) in the frames of a two-site competition model for all peptides (with one exception for [A7K]MI) The respective IC50 values are presented in Table 2.
Trang 5decreased efficacies, compared with the major
com-pounds, in competition with radiolabeled a-conotoxin
GI for the T californica nAChR binding (data not
shown)
Both PnIA variants at concentrations of up to
100 lm were inactive in competition with 125I-labeled
GI for binding to the membrane-bound Torpedo
nAChR (data not shown)
We synthesized mainly the modified a-conotoxins
targeting the muscle-type nAChR Literature data on
the role of charged residues in this group of
a-cono-toxins are in part contradictory Several researchers
have shown that charged groups at the N-termini of
a-conotoxins GI, MI and SI exert only a weak influence
on the activity [33,35–39] The important role of Arg9
in the interaction with a high-affinity
a-conotoxin-binding site on the Torpedo nAChR has been
convin-cingly demonstrated: R9P and R9A substitutions in
a-conotoxin GI resulted in a two to three order of
magnitude loss in the affinity for the a⁄ c site, whereas the reverse substitutions P9R and P9K in a-conotoxin
SI enhanced the affinity for this site [34,35,40] How-ever, when Ala or Pro residues in a-conotoxin MI were substituted for the Lys10, whose spatial disposition is close to that of Arg9 in a-conotoxin GI, the interac-tion with the high-affinity a⁄ c-binding site was affected
to a much less degree [38–40] In addition, acylation of Lys10 with azidobenzoyl or benzoylbenzoyl groups practically did not change the capacity of the respect-ive derivatrespect-ives to interact with the membrane-bound TorpedonAChR [41]
Of all known muscle-type a-conotoxins, only a-conotoxin SIA interacts exclusively with one a⁄ c site
on the Torpedo nAChR [35] Interestingly, this peptide contains a negatively charged residue (Asp12) in the C-terminal part of the molecule (Table 1) whose role has not been examined previously D12S substitution resulted in a 25- and 65-fold increase in the affinity for the high- and low-affinity binding sites, respectively (Fig 2C; Table 2) Introduction of a positive charge (Lys) at this position resulted in an additional fourfold increase in affinity for the low-affinity site (Fig 2C; Table 2) Substitution of Lys or Arg for Ser12 in a-conotoxins GI and SI gave a reliable enhancement (three- to sevenfold) of the affinity for both binding sites (Fig 2A,B; Table 2) By contrast, introduction of
a negative charge at this position (S12D) in
a-conotox-in GI brought about a marked decrease a-conotox-in the affinity (Fig 2A; Table 2) It is noteworthy that use of 125 I-labeled a-conotoxin GI in these experiments, instead
of the usual 125I-labeled a-bungarotoxin [34,35,39,40], revealed the differences in potency to two sites for a-conotoxin SI and made possible the detection of a low-affinity binding site for a-conotoxin SIA in the TorpedonAChR From literature data it is known that the affinities of muscle-type a-conotoxins to the Tor-pedo nAChRs binding sites (tested in competition with
125I-labeled a-bungarotoxin) vary from one to three orders of magnitude [12,34,35,39] The given explan-ation for this scatter is the influence of the receptor state, test conditions, etc It is therefore not surprising that using a different tracer in the radioligand assay may result in different binding parameters for a-cono-toxins This was shown previously for one a-conotoxin
GI analog on the Torpedo receptor [41] There is convincing evidence (the crystal structures of the a-cobratoxin and a-conotoxin complexes with acetyl-choline-binding proteins) [19–21,42] that the binding sites for these two groups of competitive antagonists overlap, but are not identical
Grafting positive charges on to other positions
of a-conotoxin amino acid sequence resulted in
Table 2 Activity of a-conotoxins and their analogs tested in
compe-tition binding assays Using the membrane-bound Torpedo nAChR,
the inhibitory activities of a-conotoxins GI, SI, SIA or MI and their
analogs were evaluated in competition with 125 I-labeled
a-conotox-ins GI (GI, SI, SIA and their analogs) or MI (MI and its analogs): see
respective inhibition curves presented in Fig 2 IC50 values were
calculated using ORIGIN 6.1 in the frames of both one- and two-site
models using the joint data from two or three independent
experi-ments for each a-conotoxin The choice was made in favor of the
model giving the minimal ‘reduced chi-squared’ parameter
comple-mented with reasonable SE values and taking into consideration
the Hill coefficients (n H ) For all muscle-type conotoxins (with one
exception), a two-site model was found the best In the case of
[A7K]MI analog the program failed to fit the data to a two-site
model, so the respective IC 50 value was generated in the frames of
one-site model and ascribed to both sites Both PnIA variants were
inactive in competition with 125 I-labeled a-conotoxins GI at 100 l M
(4 ± 2% of inhibition).
a-Conotoxin nH
IC 50 ,l M high affinity site low affinity site
[D12S]SIA 0.46 ± 0.03 0.13 ± 0.02 6.6 ± 0.6
[D12K]SIA 0.52 ± 0.05 0.10 ± 0.05 1.7 ± 0.7
Trang 6weakening of the binding capacity as seen for a series
of Lys-analogs of a-conotoxin MI (Fig 2D; Table 2)
Our experiments revealed a new site in the
muscle-type a-conotoxins, wherein the presence of a charge
considerably affects the efficiency of interaction with
the Torpedo nAChR: a positive charge in the
C-termi-nus increases the affinity for both binding sites,
whereas a negative charge drastically decreases it
In contrast to muscle-type a-conotoxins, negatively
charged amino acid residues can be found in the
C-ter-minus of many a-conotoxins acting on neuronal
nAChRs, namely in AuIA, AuIB, AnIA⁄ C, EpI,
Vc1.1, PnIA⁄ B [43] Substitutions in the neuronal
a-conotoxins were used to modify their selectivity [9–
11]: A10L mutation in PnIA enhanced its affinity for
rat and chicken a7 nAChR and weakened it for a3b2,
converting the parent peptide from a3b2-preferring to
a7-preferring [10,11,44] However, no studies on the
role of the above-mentioned negative charges in the
C-terminus were performed earlier Based on
[A10L]PnIA, we recently synthesized a new analog that
bears an additional D14K substitution [19] This
sub-stitution increased the affinity of the ‘double mutant’
for the chicken a7 nAChR and for L stagnalis
AChBP, but not for A californica AChBP [19] In this
study we show that [A10L,D14K]PnIA exhibits high
affinity for one subtype of a7-like nAChRs in L
stag-nalisneurons, discriminating two nAChRs
a-Conotoxin blockade of Cl–currents elicited by
ACh in identified neurons of L stagnalis
PnIA analogs were tested on the identified neurons
(LP1–3, RP2,3) from left and right parietal ganglia of
L stagnalis The responses to ACh of these neurons
result from an increase in only Cl– conductance, as
revealed by I–V relationship determination at various
Cl– concentrations in the internal solution (C.A
Vulf-ius et al unpublished results) The AChRs in parietal
Lymnaea neurons resemble a7 nAChRs of vertebrates
in terms of the efficacy of choline, cytisine, and
nico-tine (all of them are full agonists) and their high
sensi-tivity to a-conotoxin ImI [23] Two groups of cells
distinct in terms of desensitization kinetics and
sensi-tivity to ImI (IC50 288 ± 27 and 10.3 ± 1.3 nm,
respectively) have been recognized [23]
Both PnIA mutants inhibited the ACh-elicited
cur-rent but had a weaker potency than a-conotoxin ImI
(Fig 3), in contrast to their much higher affinity for
Lymnaea AChBP [19] The residual unblocked current
was of approximately the same amplitude in the
pres-ence of saturating concentrations of ImI or either of
the two PnIA mutants The relative potency of the
PnIA variants differed significantly in two types of neurons There was no large distinction between [A10L]PnIA and [A10L,D14K]PnIA on cells with low sensitivity to a-conotoxin ImI (Fig 3A; IC50 30 and
15 lm, respectively) However, in neurons with high sensitivity to ImI, the [D14K] mutation increased the affinity 50-fold (Fig 3B; IC50 400 nm compared with
20 lm for [A10L]-variant) Average IC50 ratios for ImI⁄ [A10L,D14K]PnIA ⁄ [A10L]PnIA were 1 : 60 : 120 and 1 : 13 : 670 in two groups of cells Thus, only those nAChRs which can be blocked by a-conotoxin ImI at very low concentrations discriminate between [A10L]PnIA and [A10L,D14K]PnIA These results support our previous suggestion about the existence of two distinct populations of ImI-sensitive nAChRs in the Lymnaea neurons [23]
Enhancement of the affinity of the [D14K] mutant for nAChRs in a group of cells with high sensitivity to a-conotoxin ImI is comparable with the increase in the affinity for Lymnaea AChBP and chicken a7 nAChR [19], but the effect in the case of Lymnaea nAChRs is much more pronounced In contrast, introduction of a positive charge at position 14 does not seem important for the interaction with nAChRs in neurons with low sensitivity to ImI just as for the interaction with Aply-sia AChBP [19] Thus, a positive charge seems to be important for the interaction with some but not all neuronal nAChRs
Twelve nAChR subunits (A–L) have recently been identified in the CNS of L stagnalis and three (A, B, and I) have been expressed in Xenopus laevis oocytes yielding functional homopentameric nAChRs [25,26]
It is interesting to compare the heterologously expressed nAChRs with nAChRs in the Lymnaea neurons differing in the affinity for a-conotoxins ImI and [A10L,D14K]PnIA Pharmacological profiles
of heterologously expressed nAChR-A and native nAChRs are very similar, but the A-homomer mediates cation conductance [25] Anion-selective nAChR-B can
be activated by choline, nicotine and cytisine (all three drugs being full agonists) [25], and blocked slightly by
100 nm a-conotoxin ImI (the maximal concentration used) Therefore, nAChR-B might be a candidate for native nAChR which has low sensitivity to
a-conotox-in ImI and does not discrima-conotox-inate two PnIA variants However, more probably, nAChRs with low or high sensitivity to ImI in parietal neurons may be formed with the participation of some other subunits Alter-natively, some unidentified factors can influence a-conotoxin pharmacology on oocyte-expressed nAChRs
as has been earlier suggested from the comparison of a-conotoxin EpI and AuIB effects on the recombinant and native a3- and a7-containing nAChRs [45]
Trang 7Modeling a-conotoxin complexes with
T californica nAChR
X-Ray structures of Aplysia AChBP in complexes with
[A10L,D14K]PnIA [19] and ImI [20,21] provide the
basis for modeling the a-conotoxin complexes with
those nAChRs that are blocked specifically by the
respective a-conotoxins Using these X-ray crystal
structures and the cryoelectron microscopy structure of
4 A˚ resolution for the Torpedo marmorata nAChR
[22], we performed computer modeling of the [D12K]SIA complex with the T californica nAChR (Fig 4) The aim was to envisage the structure of mus-cle a-conotoxins with their target receptors and to explain a dramatic increase in the affinity contributed
by the D12K substitution The NMR structure of a homologous a-conotoxin SI [46] was used for docking experiments We modeled only complexes with an a–c interface of the receptor because the structure of some fragments of the d subunit still remains unsolved and the reliability of the complexes of ligands bound to the a–d interface is lower
According to our calculations, the fold of the a-conotoxin analog remains practically unchanged when serine or lysine are substituted for D12 The averaged rmsd is 0.19–0.22 A˚ Increased flexibility in the N-ter-minus in [D12S]SIA and especially in [D12K]SIA, compared with native SIA, was detected The main dif-ference is seen in the C- and N-termini In the case of native toxin, the position of the C-terminus is stabil-ized by the ionic pair (salt bridge) between the N-ter-minus and the side chain of the aspartate D12 In the case of [D12S]SIA, the ionic link is changed to the H-bond, which provides slightly more flexibility to the N-terminus The introduced lysine side chain is orien-ted mainly to the C-terminus, forming H-bond with it, being also directed to the aromatic ring of Y1 How-ever, in general, the conformation of the mutant toxins
is very similar to that of the wild-type molecule Docking and fast molecular dynamics simulations demonstrated a similar position for SIA and its analogs
in the binding pocket We found that all a-conotoxins are kept in the binding pocket mostly by Van der Waals’ interactions and by stacking of their disulfide bridges with aromatic residues of the receptor (Table 3), similarly to what has been demonstrated for
Fig 3 Comparison of blocking activity of three a-conotoxins on Lymnaea neurons with low (A) and high (B) sensitivity to ImI The insets show the ACh-elicited currents recorded from 4 neurons in control (solid lines) and after 5 min pretreatment with a-conotoxins ImI, [A10L]PnIA or [A10L,D14K]PnIA (dotted lines) Concentrations
of a-conotoxins (in lM) are marked left to the corresponding traces Calibrations are the same for all oscillograms The plots are inhibi-tion curves for three a-conotoxins Uninhibited currents were nor-malized by the control response just before treatment with the a-conotoxin The points are either the mean ± SE from 3 to 9 experiments or the mean from duplicates The curves were fitted
to the Hill equation The IC 50 and Hill coefficient (n H ) values are 0.25 l M and 1.16 (n ¼ 9 cells) for ImI, 15 l M and 0.62 (n ¼ 6) for [A10L,D14K]PnIA, 30 l M and 0.64 (n ¼ 7) for [A10L]PnIA in cells with low sensitivity to ImI (A); 0.03 l M and 0.81 (n ¼ 7) for ImI, 0.4 l M and 0.49 (n ¼ 2) for [A10L,D14K]PnIA, 20 l M and 0.71 (n ¼ 5) for [A10L]PnIA in cells with high sensitivity to ImI (B).
Trang 8a-conotoxins [A10L,D14K]PnIA and ImI bound to AChBP [19–21] The main difference between SIA and its analogs was found for the mutated residue of the toxin The D12 side chain plays no role in binding, at least its side chain forms no bonds with the receptor residues In the case of [D12S]SIA, the abovementioned increased flexibility of the N-terminus permits [D12S]SIA to enter deeper into the pocket and to form closer and stronger contacts (mainly Van der Waals) with the receptor The [D12K]SIA occupies the position
in the nAChR pocket similar to that of [D12S]SIA, but
in addition a new ionic interaction is observed: K12 is directly interacting with E57 of the nAChR c-subunit, whereas several amino acid residues (Q59, Y117 and some other) facilitate the formation of this bond (Fig 4) The reason why both SIA mutants have identi-cal affinities for the a⁄ c site (Table 2) may be that, according to docking experiments, the [D12S] analog is entering the binding site somewhat deeper and is form-ing stronger Van der Waals’ contacts, which may give a potential energy gain comparable with that of the ionic bond formed by the [D12K] variant
In summary, our results show that introduction of a positive charge to the C-terminus of a-conotoxins gives new analogs of distinct selectivity whose mode of action, with the purpose of future design of novel antagonists, can be rationalized in the light of the available X-ray data
Experimental procedures
Materials
nAChR-enriched membranes from the electric organ of
T californica used in the radioligand assays [47] were kindly provided by Prof F Hucho (Free University of Berlin, Germany) All iodinations of conotoxins were per-formed using chloramine T (Serva, Heidelberg, Germany) and Na [125I] (Izotop, Moscow, Russia) Monoiodinated (3-[125I]iodotyrosyl54)-a-bungarotoxin (~ 2000 CiÆmmol)1) was from Amersham Biosciences (Little Chalfont, UK) a-Cobratoxin was purified from crude venom of Naja kaou-thiaas described previously [48]
Synthesis of a-conotoxin analogs
All peptides except for PnIA variants were synthesized on Rink-resin using Fmoc-strategy and trityl protection of the cysteine thiol groups Coupling of amino acids was carried out with the hydroxybenzotriazole–carbodiimide procedure Deprotection with simultaneous cleavage of the peptides from resin was achieved using a mixture of trifluoro-acetic acid, ethanedithiol, m-cresole, and dimethylsulfide
Table 3 Amino acid interactions between nAChR (a–c interface)
and bound a-conotoxins SIA, [D12S]SIA and [D12K]SIA The
addi-tional interactions for the analogs are placed in square brackets.
Designations by type: normal, direct Van der Waals interactions;
underlined, H-bond (toxin residue atom ID-receptor residue atom
ID); bold, ionic pair; in parentheses – doubtful or weak.
Toxin residue
Receptor residue
Cys2–Cys7 Tyr190, Tyr198
(Tyr190)
Ala6 Thr150, Asp152(O-N) Arg79(O-NH1 ⁄ 2)
Tyr111(NZ-OH)
Fig 4 A model for complexes of a-conotoxin SIA and its
[D12K]-analog with the Torpedo nAChR extracellular domain The
extracel-lular domains of a- (left) and c- (right) subunits are in pink and tan.
a-Conotoxin SIA and [D12K]SIA molecules are shown by green and
blue sticks (-C-S-S-C-bridges in yellow), respectively Aromatic
amino acid residues of the Torpedo nAChR forming its
ligand-bind-ing site at the a–c interface are colored with orange Some
resi-dues of the c-subunit close to the second loop of the toxin
molecule (Table 3) are numbered Ionic pair between analog Lys12
and c-subunit Glu57 side chains is in red The side chain of the
Lys9 residue and the N- and C-termini of toxins are marked in
green.
Trang 9(9 : 0.3 : 0.3 : 0.3 v⁄ v ⁄ v ⁄ v) for 40 min at 25 C The crude
linear peptides were dissolved in 50% isopropanol, titrated
to pH 9.0 with N-ethyldiisopropylamine, and left at 25C
[27] The oxidation process, as monitored by reaction with
Ellman’s reagent, was complete in 18 h, and then the pH
was decreased to 5.0 with acetic acid RP-HPLC on a
semi-preparative C18 column was used to purify one
predomin-ant peak or in some cases of several isomers; each of them
was characterized by CD spectra and tested for ability to
bind to the membrane-bound T californica nAChR (see
below)
[A10L]PnIA and [A10L,D14K]PnIA were synthesized on
a Rink polymer using the
O-benzothiazol-1-yl-N,N,N¢,N¢-tetramethyluronium tetrafluoroborate⁄
N,N-diisopropyleth-ylamine method for activation of Fmoc-amino acids In the
[A10L]PnIA synthesis, all thiols were protected by a Trt
group In the [A10L,D14K]PnIA synthesis, Trt was used for
Cys3 and Cys16, and tBu for Cys2 and Cys8 Deblocking of
peptides was carried out with trifluoroacetic acid as
des-cribed above Linear peptides were purified by RP-HPLC on
a Reprosil-Pur C18column (250· 10 mm) using an
acetonit-rile gradient from 10 to 40% in 30 min Two disulfide
brid-ges in [A10L]PnIA were closed simultaneously in 0.1 m
NH4CO3solution [30] The required product was isolated by
HPLC and characterized with the aid of MALDI MS When
synthesizing [A10L,D14K]PnIA, disulfide bridges were
formed selectively First, after removal of the peptide from
the polymer, oxidation on air at pH 8.5 in the
isopropa-nol⁄ water mixture was used to form a disulfide between
Cys3 and Cys16 Then, using a silyl chloride⁄ sulfoxide
method [49], tBu protection was removed from Cys2 and
Cys8 with simultaneous formation of the respective disulfide
MALDI-TOF analysis was carried out on a Reflex III
mass spectrometer (Bruker, Bremen, Germany) using
2,5-dihydroxybenzoic acid as a matrix
CD spectroscopy
CD spectra were recorded on a JASCO J-810
spectropola-rimeter (JASCO International Co., Tokyo, Japan) The
results were expressed as molar ellipticity, [Q] (degÆcm2
Æ dmol)1), determined as [Q] ¼ Q · 100 · MRW ⁄ (c · L),
where Q is the measured ellipticity in degrees at a
wave-length k, MRW is the mean amino acid residue weight
cal-culated for each a-conotoxin as the division of peptide
molecular mass by the number of amino acid residues, c is
the peptide concentration in mgÆmL)1, and L is the light
path length in cm The instrument was calibrated with
(+))10-camphorsulfonic acid, assuming [Q]291¼ 7820
degÆcm2Ædmol)1[50]
Radioligand assays
125I-Iodination of a-conotoxins GI and MI was carried out
by the chloramine T method as described previously [41]
For competition binding assays, suspensions of nAChR-rich membranes (230 nm a-bungarotoxin binding sites pre-pared in 50 mm Tris⁄ HCl buffer, pH 8.0, containing
1 mgÆmL)1 of BSA) were incubated for 1 h with various amounts of the a-conotoxin analogs, followed by an addi-tional 35 min incubation with 280 nm 125I-labeled a-cono-toxin GI or 125I-labeled a-conotoxin MI Nonspecific binding was determined by preincubation of the membranes with a 200-fold excess of a-cobratoxin The membrane sus-pensions were applied to glass GF⁄ F filters (Whatman, Maidstone, UK) presoaked in 0.25% polyethylenimine, and the unbound radioactivity was removed from the filter by washes (3· 3 mL) with 50 mm Tris ⁄ HCl buffer, pH 8.0 The inhibition curves obtained are presented in Fig 2, the
IC50values given in Table 2
Data analyses were performed using origin 6.1 (Micro-Cal Software Inc, Northampton, MA) The competition curves of 125I-labeled GI⁄ MI binding inhibition with a-conotoxin analogs were fit both to one-site or two-site models
Electrophysiology
Experiments were carried out on identified giant neurons (LP1–3, RP2,3) isolated from L stagnalis right or left pari-etal ganglia after mild enzymatic digestion (protease from Streptomyces griseus, Sigma, St Louis, MO, 2 mgÆmL)1,
50 min at room temperature) Neurons were internally per-fused and voltage-clamped at )60 mV The composition of the internal and external solutions, techniques of ACh application and cell incubation with the toxins were as des-cribed previously [23] ACh-induced currents were digitized and sampled online on a Pentium PC via a home-made operational amplifier supplying a virtual ground and a Digidata1200 B interface (Axon Instruments Inc., Foster City, CA) Acquisition and analysis of the data were made using pclamp6 (Axon Instruments Inc.) IC50 values were determined as the toxin concentration required to reduce
by half the current fraction sensitive to this toxin
Model building
The model of the extracellular domains of the T californica nAChR subunits was constructed using modeller 7v7 (http://www.salilab.org/modeller) with the sequence align-ment from LGIC database (http://www.ebi.ac.uk/compneur-srv/LGICdb/LGICdb.php) on the basis of the crystal structures of L stagnalis AChBP complexes with nicotine (1UW6) and carbamylcholine (1UV6), the X-ray crystal structure of the complex of A californica AChBP with a-conotoxin [A10L,D14K]PnIA [19] and the T marmorata nAChR cryo-electron microscopy structure (2BG9), as will
be published in more detail elsewhere
The models of the SIA, [D12S]SIA and [D12K]SIA were built using the X-ray crystal structure of a-conotoxin SI
Trang 10(1HJE) All crystal structures were from the Protein Data
Bank (http://www.rcsb.org/pdb) Point mutations were
introduced in the molecule with spdbviewer 3.7 sp5 (http://
swissmodel.expasy.org/spdbv/) mutation instrument The
structure verification was carried out with what_check
(http://swift.cmbi.kun.nl/swift/whatcheck/) Then the
struc-tures were relaxed (300 steps of steepest descent with cutoff
10 A˚) with tinker (http://dasher.wustl.edu/tinker/) using
AMBER¢99 force field [51] during minimization and
molecular dynamics simulations Rather short (100
pico-second) trajectories were calculated at the temperature
300 K and dielectric permittivity e¼ 1 Time step of
integ-ration procedures were taken as small as 1 femtosecond
Radius of truncation for Coulomb interactions was 20 A˚
No periodic boundaries were applied Lennard–Jones
inter-actions were calculated only up to 16 A˚ (at that, from 15
to 16 A˚ a polynomial switch function was applied)
Berend-sen thermostat was applied [52]
Docking simulations and selection of solutions
Docking simulations were performed under hex4.2b
(http://www.csd.abdn.ac.uk/hex/) Thus flexible ligand was
docked to the rigid receptor Visual analysis in the spdb
viewerfollowed to reject false-positive solutions The
posi-tion of the toxin in the binding pocket proposed by the
program was considered valid if there was a contact of
toxin Lys9 residue with cTyr111 found by the pair-wise
mutagenesis studies [40] Molecular dynamics procedures
were run over the solutions after this selection using the
same parameters as was described in the previous section
Acknowledgements
This research was supported by the Russian
Founda-tion for Basic Research (06-04-49198; 05-04-48932),
partially by the Civilian Research and Development
Foundation grant RB1-2028, and by a grant of
RFBR-NWO (047.015.016) to ABS and VIT, grant MCB
RAN to VIT We also express our thanks to Prof N
Unwin for providing the coordinates of the Torpedo
nAChR, Prof F Hucho for fruitful discussions, and
Dr Irina A Kudelina for help with CD measurements
References
1 Terlau H & Olivera BM (2004) Conus venoms: a rich
source of novel ion channel-targeted peptides Physiol
Rev 84, 41–68
2 McIntosh JM, Dowell C, Watkins M, Garrett JE,
Yoshikami D & Olivera BM (2002) a-Conotoxin GIC
from Conus geographus, a novel peptide antagonist of
nicotinic acetylcholine receptors J Biol Chem 277,
33610–33615
3 Ellison M, McIntosh JM & Olivera BM (2003) a-Cono-toxins ImI and ImII Similar a7 nicotinic receptor antagonists act at different sites J Biol Chem 278, 757– 764
4 Dowell C, Olivera BM, Garrett JE, Staheli ST, Watkins
M, Kuryatov A, Yoshikami D, Lindstrom JM & McIn-tosh JM (2003) a-Conotoxin PIA is selective for a6 subunit-containing nicotinic acetylcholine receptors
J Neurosci 23, 8445–8452
5 Azam L, Dowell C, Watkins M, Stitzel JA, Olivera BM
& McIntosh JM (2005) a-Conotoxin BuIA, a novel pep-tide from Conus bullatus, distinguishes among neuronal nicotinic acetylcholine receptors J Biol Chem 280, 80–87
6 Nicke A, Wonnacott S & Lewis RJ (2004) a-Conotoxins
as tools for the elucidation of structure and function of neuronal nicotinic acetylcholine receptor subtypes Eur
J Biochem 271, 2305–2319
7 Tsetlin VI & Hucho F (2004) Snake and snail toxins acting on nicotinic acetylcholine receptors: fundamental aspects and medical applications FEBS Lett 557, 9–13
8 Ellison M, Gao F, Wang HL, Sine SM, McIntosh JM
& Olivera BM (2004) a-Conotoxins ImI and ImII target distinct regions of the human a7 nicotinic acetylcholine receptor and distinguish human nicotinic receptor sub-types Biochemistry 43, 16019–16026
9 McIntosh JM, Azam L, Staheli S, Dowell C, Lindstrom
JM, Kuryatov A, Garrett JE, Marks MJ & Whiteaker P (2004) Analogs of a-conotoxin MII are selective for a6-containing nicotinic acetylcholine receptors Mol Pharmacol 65, 944–952
10 Luo S, Nguyen TA, Cartier GE, Olivera BM,
Yoshika-mi D & McIntosh JM (1999) Single-residue alteration in a-conotoxin PnIA switches its nAChR subtype selectiv-ity Biochemistry 38, 14542–14548
11 Hogg RC, Miranda LP, Craik DJ, Lewis RJ, Alewood
PF & Adams DJ (1999) Single amino acid substitutions
in a-conotoxin PnIA shift selectivity for subtypes of the mammalian neuronal nicotinic acetylcholine receptor
J Biol Chem 274, 36559–36564
12 Chiara DC, Xie Y & Cohen JB (1999) Structure of the agonist-binding sites of the Torpedo nicotinic acetylcho-line receptor: affinity-labeling and mutational analyses identify cTyr-111⁄ dArg-113 as antagonist affinity deter-minants Biochemistry 38, 6689–6698
13 Quiram PA, Jones JJ & Sine SM (1999) Pairwise inter-actions between neuronal a7 acetylcholine receptors and a-conotoxin ImI J Biol Chem 274, 19517–19524
14 Bren N & Sine SM (2000) Hydrophobic pairwise inter-actions stabilize a-conotoxin MI in the muscle acetyl-choline receptor binding site J Biol Chem 275, 12692– 12700
15 Quiram PA, McIntosh JM & Sine SM (2000) Pairwise interactions between neuronal a7 acetylcholine receptors and a-conotoxin PnIB J Biol Chem 275, 4889–4896