Tài liệu Báo cáo khoa học: Structure-activity relationships of a-conotoxins targeting neuronal nicotinic acetylcholine receptors ppt

MINIREVIEW
Structure-activity relationships of a-conotoxins targeting neuronal
nicotinic acetylcholine receptors
Emma L. Millard, Norelle L. Daly and David J. Craik
Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia
a-Conotoxins that target the neuronal nicotinic acetylcho-
line receptor have a range of potential therapeutic applica-
tions and are valuable probes for examining receptor
subtype selectivity. The three-dimensional structures of
about half of the known neuronal specific a-conotoxins have
now been determined and have a consensus fold containing a
helical region braced by two conserved disulfide bonds.
These disulfide bonds define the two-loop framework char-
acteristic for a-conotoxins, CCX
m
CX
n
C, where loop 1
comprisesfourresidues(m¼ 4) and loop 2 between three
and seven residues (n ¼ 3, 6 or 7). Structural studies, par-
ticularly using NMR spectroscopy have provided an insight
into the role and spatial location of residues implicated in
receptor binding and biological activity.
Keywords: NMR; peptide; X-ray crystallography.
Introduction
As outlined in other articles in this series, the a-conotoxins
have a range of potential therapeutic applications and have
proved to be valuable pharmacological tools based on their
ability to selectively inhibit the nicotinic acetylcholine
receptor (nAChR) [1–3]. The focus of this review is on
the three-dimensional structures of a-conotoxins and the

progress made towards dissecting the features involved in
receptor subtype selectivity. In particular, a-conotoxins
targeting neuronal rather than muscle nAChRs will be
discussed. Muscle specific a-conotoxins have been covered
in other more general reviews [4–6]. There is much current
interest in various neuronal receptor subtypes implicated in
diverse neurological disorders such as Alzheimer’s disease
and epilepsy [7–9], and in the regulation of small-cell lung
carcinoma [10,11].
The sequences, subtype selectivity and potency of
a-conotoxins targeting neuronal nAChRs are given in
Table 1, together with information on their structural
characterization. The cysteine residues and disulfide con-
nectivity are invariant throughout these sequences and
define a two-loop framework, CCX
m
CX
n
C(X
m
and X
n
refer to the number of noncysteine residues), where the
loops correspond to the residues between successive cysteine
residues. The number of residues in the two loops (m/n) is
used to group the a-conotoxins into different frameworks.
ImI and ImII have a 4/3 framework and the other peptides
in Table 1 contain either a 4/6 or 4/7 framework. It is
interesting to note that although the majority of 4/6 and 4/7
a-conotoxins are selective for neuronal nAChRs, conotoxin

EI contains a 4/7 framework but binds to the muscle-type
nAChR [12].
The sequence conservation of the a-conotoxins extends
beyond the cysteine residues, with a Ser and Pro in loop 1
being highly conserved. However, there is a significant degree
of sequence variation in the remaining residues, particularly
in loop 2. It is this sequence diversity that provides the
exquisite selectivity that a-conotoxins display for various
nAChR subtypes (Table 1). Structures of neuronally active
a-conotoxins, in conjunction with activity studies, have
provided clues to understanding the complexity involved in
binding to the nAChR. A summary of this structural
information and the insights into structure-activity relation-
ships of a-conotoxins is presented in this review.
Structural features of a-conotoxins
The three-dimensional structures of a-conotoxins have
been determined, primarily using NMR spectroscopy. It
is unusual for such small peptides to crystallize but a few
a-conotoxins have been amenable to analysis with X-ray
crystallography. To date no neuronally active conotoxins
have been structurally characterized using both techniques,
however, the neuromuscularly active conotoxin GI has been
studied using both methods and the structures overlay very
closely [13–15].
Despite the small size of a-conotoxins they have well-
defined structures with a characteristic overall fold. With the
structures of more than half of the known neuronally active
a-conotoxins determined it is possible to determine the
consensus structural features. These features involve
restraints imposed by the conserved disulfide connectivity

and a helical region centred around Cys III. The helix
typically encompasses residues 5–12. A comparison of the
known structures is given in Fig. 1 with the three framework
classes presented separately for clarity. It is clear that the
backbone fold of loop 1 is highly conserved, including the
first turn of the helix. The major differences, as might be
Correspondence to D. Craik, Institute for Molecular Bioscience,
University of Queensland, Brisbane, QLD, 4072, Australia.
Fax: + 61 73346 2029, Tel.: + 61 73346 2019,
E-mail:
Abbreviation: nAChR, nicotinic acetylcholine receptor.
(Received 22 January 2004, revised 19 March 2004,
accepted 6 April 2004)
Eur. J. Biochem. 271, 2320–2326 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04148.x
expected, occur in loop 2, a direct reflection of the differing
number of residues seen in this loop. However, even when
the residue numbers are the same, as in the overlay of 4/7
a-conotoxins, loop 1 superimposes better than loop 2. In
this case the structural differences are related to the sequence
diversity rather than the number of residues.
Analysis of the surface features of a-conotoxins reveals
the fact that the cysteine residues are significantly more
buried than the other residues, although the small size of the
molecules prevents complete burial. As a consequence of
this burial, a small surface exposed hydrophobic patch
is present in the a-conotoxins [16]. The extent of this
hydrophobic patch varies amongst the different conotoxins
but generally involves residues adjacent or close in sequence
to Cys III. The charge distribution also varies amongst the
a-conotoxins and this may influence the differences

observed in potency and specificity. Indeed, it was originally
thought that the net overall charge was related to the
nAChR specificity [17], with a-conotoxins that target
muscle subtypes having net positive charges and those
targeting neuronal being either negative or neutral. ImI is an
exception [18] as is the recently discovered ImII [19].
The overall quality of the structures determined is very
good and appears to be not only related to the compact
disulfide connectivities but also to a network of hydrogen
bonds. As expected, an analysis of the structures reveals that
hydrogen bonds are associated with the a-helical region, but
other parts of the structure also contain hydrogen bonds.
Several structures have been determined independently
by different groups and the structures appear to be in good
agreement. The structure of ImI in solution has been
determined in five studies, all in aqueous solution [18,20–23].
They are all similar and show the basic backbone fold seen
in all a-conotoxins. These structures were determined at
various pH values between 3.0 and 6.0 and all were of high
precision, with backbone rmsd values between 0.34 A
˚
and
0.78 A
˚
. Two different structures of MII have also been
published. One of the structures was determined in aqueous
buffer at pH 3.3 [24] and the second was determined in
aqueous solution (pH 3.9) as well as in 30% trifluoro-
ethanol/H
2

O and 30% acetonitrile/H
2
O [25]. Both struc-
tures showed the same fold and were well-defined with
backbone rmsd values of 0.76 and 0.07 A
˚
, respectively. Two
reports also exist for the structure of AuIB [26,27]. Both
were determined in aqueous solution with similar pH values
and backbone rmsd values of 0.27 and 0.36 A
˚
. The second
study extended the investigation to examine the role of
different disulfide bond isomers in determining structures.
The structures of PnIA, PnIB and the desulfated form of
native EpI ([Y15]EpI) were determined using X-ray crys-
tallography [17,28,29].
Recently discovered a-conotoxins, ImII [19], GID [16],
AnIB [30] and PIA [31], demonstrate the fact that the
diversity of conotoxin primary structures will probably
increase as more are discovered. In ImII, a highly conserved
proline residue present in all other a-conotoxins is not
present, while in GID, AnIB and PIA an N-terminal
extension, or tail, not seen in any other a-conotoxin is
present. A novel a-conotoxin (Vc1.1) that displays signifi-
cant sequence variation in loop 2 compared to previously
characterized a-conotoxins has also recently been identified
by gene sequencing of Conus victoriae [32].
The absence of Pro6 in ImII may have implications for
its mechanism of action as this residue is thought to be

important for activity in ImI. This is supported by the fact
that although ImII is still active at the a7 nAChR, it appears
to act at a different binding site from ImI [19]. The structure
of ImII has not yet been determined, but may provide
further information on the importance of the proline
substitution.
GID incorporates an N-terminal tail that contains four
residues prior to the first cysteine residue [16]. This is the
largest of the a-conotoxins reported to date. GID also
contains post-translational modifications not previously
reported for a-conotoxins. A c-carboxyglutamic acid is
present at position 4 and a hydroxyproline at position 16.
Both of these modifications are common in other classes
of conotoxins [33–39]. Interestingly, the post-translational
modification most commonly found in a-conotoxins,
namely an amidated C-terminus, is not present in GID.
Table 1. Sequence, receptor specificity and structural information on neuronally active a-conotoxins. *Refers to the amidated C-terminus. IC
50
relates
to receptors expressed in Xenopus oocytes. rmsd/resolution: rmsd is relevant for the NMR structures, resolution refers to those structures completed
by X-ray crystallography. The cysteine residues are highlighted in bold.
Conotoxin Sequence m/n nAChR subtype IC
50
(n
M
) Method rmsd/resolution (A
˚
)
MII
GCCSNPVCHLEHSNLC* 4/7 a3b2

a
0.5 [54] NMR 0.07
PnIA
GCCSLPPCAANNPDYC* 4/7 a7, a3b2
a
252, 9.6 [47] X-ray 1.1
PnIB
GCCSLPPCALSNPDYC* 4/7 a7, a3b2
a
61.3, 1970 [47] X-ray 1.1
EpI
GCCSDPRCNMNNPDYC* 4/7 a3b2, a3b4
a
1.6
c
[55] X-ray 1.1
GIC GCCSHPACAGNNQHIC* 4/7 a3b2
b
1.1 [51] – –
GID
IRDcCCSNPACRVNNOHVC 4/7 a7, a3b2, a4b2
a
5, 3, 150 [16] NMR 0.34
PIA
RDPCCSNPVCTVHNPQIC* 4/7 a6b2b3, a3b2
a
0.95, 74.2 [31] – –
AnIB
GGCCSHPACAANNQDYC* 4/7 a3b2, a7
a

0.3, 76 [30] – –
AuIA
GCCSYPPCFATNSDYC* 4/6 a3b4
a
>750 [56] – –
AuIC
GCCSYPPCFATNSGYC* 4/6 a3b4
a
>750 [56] – –
AuIB
GCCSYPPCFATNPD-C* 4/6 a3b4
a
750 [56] NMR 0.27
ImI
GCCSDPRCAWR C* 4/3 a7
a
132 [19] NMR 0.34
ImII
ACCSDRRCRWR C* 4/3 a7
a
441 [19] – –
a
rat nAChR subunits,
b
human nAChR subunits,
c
ACh-evoked currents in parasympathetic neurons from rat intracardiac ganglia.
Ó FEBS 2004 Structures of neuronally active a-conotoxins (Eur. J. Biochem. 271) 2321
Structure-activity relationships
of a-conotoxins

Although there is a strong conservation of certain residues
in a-conotoxins, the natural sequence variation has enabled
the conotoxins to be used as pharmacological tools in
helping us to understand the specificity of both neuronal
and neuromuscular nAChRs. Mutational analyses have
also been valuable in elucidating factors important for
activity. Table 2 lists the most informative mutations that
have been made in a range of a-conotoxins. In addition,
an alanine scan of the non-cysteine residues has been
performed on PnIB [38].
Extensive mutational analysis has been carried out on
ImI, PnIA, PnIB and to a lesser extent on GID and MII.
Analysis of ImI has revealed that Asp5-Pro6-Arg7 and
Trp10 are important for biological activity [40–43] at the
neuronal a7 nACh receptor. Many studies have involved
residue substitution with Ala, however, replacements with
other residues have allowed fine details to be discerned. For
example, substitution of Trp10 with Tyr or Phe had little or
no effect on the binding of ImI, indicating that an aromatic
residue was required at this position for activity [42–45].
Further point mutations of ImI were performed by Rogers
et al. ([R11E]ImI, [R7L]ImI and [D5N]ImI) and Lamthanh
et al. ([R7A]ImI) and the three-dimensional structures of
the mutants were determined [21,46]. It was noted that very
small conformational changes in ImI, especially for the side
chains involved in binding, are associated with a loss of
activity.
Analysis of the molecular surfaces reveals that the side
chains of the active residues in ImI are on a solvent
accessible face of the molecule. Figure 2 shows surface

representations of ImI, detailing the position of the residues
Fig. 1. Consensus structural features of neuronally active a-conotoxins. (A) On the left is an overlay of the three-dimensional structures of the 4/7
framework a-conotoxins, PnIA (dark blue), PnIB (green), EpI (light blue), MII (red) and GID (gold) superimposed over residues 3–10 for all except
GID, which was superimposed over the corresponding residues 6–13. This comparison highlights the conservation of the helical region in these
peptides and the variation observed in loop 2. The backbone atoms are shown in stick format and the N- and C-termini are labeled. A schematic
representation of the conserved residues in the 4/7 a-conotoxin sequences is shown on the right. Disulfide connectivities are shown by connections
between the conserved cysteine residues and amino acids that are conserved throughout most of the neuronally active a-conotoxins are indicated by
blue shaded circles and single letter amino acid codes. Red circles indicate the residues that have been associated with biological activity in one or
more conotoxin. White circles represent regions in the molecules where there is variability in the residue type and in the case of loop 2 in both the
type and number of residues. The N- and C-termini are labeled. (B) On the left is the three-dimensional structure of the 4/6 framework a-conotoxin,
AuIB shown in stick format with the N- and C-termini labeled. On the right is the corresponding schematic representation of the conserved residues
in the 4/6 a-conotoxin sequences. (C) On the left is the three-dimensional structure of the 4/3 framework a-conotoxin, ImI shown in stick format.
On the right is the corresponding schematic representation of the conserved residues in the 4/3 a-conotoxin sequences. Some conserved residues
have been reported to be associated with biological activity and these are represented with blue outlined red circles.
2322 E. L. Millard et al.(Eur. J. Biochem. 271) Ó FEBS 2004
identified as important for biological activity at the a7
neuronal nAChR. It has been proposed that Asp5 and Pro6
contribute to receptor binding because of their structural
role rather than through direct interaction at the binding site
[46]. This is based on the empirical preference for Asp
residues to be in the N-cap position of helices and the
tendency for Pro to also be near the capping position. By
capping (or initiating) the helical element these residues may
play a vital role in correctly orienting key binding residues.
Arg7 was suggested to have a functional role in binding as
the structure of R7A only differs from the native in the side
chain position of residue 7 but the analogue is not active
[21,46].
Mutagenesis studies on PnIA have shown that substitu-
tion of residues 10 and 11 has significant effects on the

binding affinity of PnIA for receptors on native tissues
[29,47–49]. The point mutations performed were
[A10L]PnIA and [N11S]PnIA. These substitutions were
chosen as they represent the residues that differ between
PnIA and PnIB. This study showed that [A10L]PnIA had
increased affinity for the a7 receptor and [N11S]PnIA has
affinity decreased by  30-fold. The increase in potency of
Table 2. Sequence and activity data for modified neuronally active a-conotoxins. D Refers to the truncated residues from the N-terminal tail of GID,
L refers to lipoamino acid (Laa), the residues in blue are the mutated residues, the cysteines involved in disulfide bonds are in red, * refers to the
amidated C-terminus.
m/n Conotoxin Sequence
nAChR
subtype IC
50
relative to Native (n
M
)
Structural comparisons
with native structures
4/3 [W10Y]ImI
GCCSDPRCAYR C* a7
a
no significant change [43] –
[W10F]ImI
GCCSDPRCAFR C* a7
a
no significant change [42] –
[R11E]ImI
GCCSDPRCAWE C* a7
b

no significant change [46] similar
[R7L]ImI GCCSDPLCAWR C* a7
b
fl [46] similar
[D5N]ImI
GCCSNPRCAWR C* a7
b
fl [46] similar
[R7A]ImI
GCCSDPACAWR C* a7
c
fl [21] similar
Monodisulfide ImI
GCCSDPRCAWR C* a7
c
no significant change [21] relatively disordered
4/6 AuIB ribbon
GCCSYPPCFATNPD-C* a3b4
d
› [27] disordered
4/7 [A10L]PnIA
GCCSLPPCALNNPDYC* a7, a3b2
a
›, fl [47] –
[N11S]PnIA
GCCSLPPCAASNPDYC* a7, a3b2
a
fl [47] –
[R12A]GID
IRDc CCSNPACAVNNOHVC a7, a3b2, a4b2

a
fl [16] –
[D1–4]GID
CCSNPACRVNNOHVC a7, a3b2, a4b2
a
fl a4b2, no change
for others [16]
–
LaaMII
LGCCSNPVCHLEHSNLC* a3b2
d
no significant change [53] minimal change
from native MII
5LaaMII
GCCSLPVCHLEHSNLC* a3b2
d
fl [53] a-helix disrupted
a
rat nAChR subunits,
b
human nAChR subunits,
c
HEK cells used in competitive binding assay,
d
ACh-evoked currents in parasympathetic
neurons from rat intracardiac ganglia.
Fig. 2. Surface representations of ImI and
PnIA. (A) The three-dimensional structure of
ImI. The heavy atoms are shown in stick for-
mat and the residues reported as important for

a7-subtype activity at the neuronal nAChR,
i.e.D5,P6,R7andW10,areshowninpink.
The N- and C-termini are labeled. The two
views are rotated by 180° about the y-axis. (B)
The surface diagram of the three-dimensional
structure of PnIA. The two residues reported
to have significant influence on specificity and
potency, A10 and N11, are shown in pink and
labeled. Residues 4–7 and 9–10 in the closely
related PnIB have also been shown to influ-
ence activity [38].
Ó FEBS 2004 Structures of neuronally active a-conotoxins (Eur. J. Biochem. 271) 2323
[A10L]PnIAis thought to be associated with a slower rate of
dissociation of the conotoxin from the a7 receptor [50]. The
mutant studies carried out on PnIA suggest that both
positions 10 and 11 have a significant influence on selectivity
for the a7 subunit of the nAChR. NMR chemical shift
analysis of the mutants provides a very sensitive method of
assessing structural differences and in this case clearly shows
that the changes in potency and selectivity are not related to
structural changes in the backbone.
A surface diagram of PnIA showing the residues
important for binding is shown in Fig. 2. The increase in
potency for [A10L]PnIA has been attributed to the longer
side chain of the leucine at position 10. In general, for the
4/7 a-conotoxins there appears to be a correlation between
the length of the aliphatic side chain in position 10
(numbering based on PnIA) and greater a7vs.a3b2
selectivity. As the length of the side chain at position 10
increases the conotoxins become more a7-selective and less

a3b2-selective [16]. GIC has the smallest side chain possible,
a glycine, and is a potent a3b2 inhibitor [51] although
its selectivity with respect to a7activityhasyettobe
determined. As the side chain length increases the ratio
between a3b2anda7 increases up to > 100 for EpI
containing a methionine at this position.
The recent discovery of GID has added to knowledge
of the structure-activity relationships of a-conotoxins by
revealing that the highly charged N-terminal tail contributes
to a4b2 activity. This was determined by analysis of a
truncated analogue of GID ([D1–4]GID) that displayed no
significant change in activity for the a7anda3b2 receptor
subtypes [16], but a4b2 activity was significantly decreased.
A predefined structure of this tail region is not present in
solution and the three-dimensional structures indicate that
this region is disordered. However, a particular conforma-
tion may be present upon binding to the receptor. Further-
more, the uncommon feature of an arginine residue at
position 12 (Table 1) appears to contribute to a4b2anda7
subtype activity but not a3b2 activity. A decrease in a4b2
and a7 subtype activity but not a3b2 activity was observed
in the [R12A]GID mutant [16].
An alternative approach to developing structure-activity
relationships of a-conotoxins has been to examine the
effects of re-engineering the disulfide bonds. A minimal
scaffold has been found for ImI in which the Cys3 to Cys12
disulfide bond has been deleted [21]. Loss of this single
disulfide bond had no effect on the binding affinity, and the
overall structure is quite similar to the native although the
peptide appeared to be more flexible than the native form.

The structure was less well defined with an overall backbone
rmsd value of 1.49 compared to 0.78 A
˚
for native ImI.
Molecules with non-native disulfide connections have also
been produced. This approach was first demonstrated for
the muscle specific conotoxin GI, where all three possible
disulfide bond isomers (globular, ribbon and beads, Fig. 3)
were studied [52]. In this case the non-native (ribbon and
beads) forms were less active and more flexible than the
native globular isomer. The ribbon connectivity of AuIB
has been synthesized and although the overall structure
appears to be more disordered the biological activity was
unexpectedly increased [27]. The structures of the native
globular AuIB and ribbon AuIB are shown in Fig. 3, where
the disruption in the helix is apparent in the latter. Given the
importance of this element of structure in all of the native
a-conotoxins it seems surprising that increased activity is
observed when it is disrupted. However this may be
rationalized by the fact that the ribbon form is more flexible
than the native isomer and this may allow the molecule to
better complement the binding surface of the receptor.
However, the degree of flexibility is clearly important as too
much flexibility leads to entropic losses in binding energy
and potentially to decreased activity, as was noted for the
non-native isomers of a-conotoxin GI.
In common with other peptides, conotoxins have limita-
tions on their use as therapeutic agents as they have poor
bioavailability. Structural studies have provided insight
into approaches aimed at improving the bioavailability of

conotoxins. In particular, two lipophilic analogues of the
conotoxin MII were recently developed [53], by adding a
lipidic group (2-amino-
D
,
L
-dodecanoic acid, Laa) to either
the N-terminus (LaaMII) or to Asn5 (5LaaMII). The
N-terminal LaaMII was shown to have a tertiary structure
similar to that of the native conotoxin and maintained the
activity for the a3b2 subtype activity associated with the
native peptide. However the 5LaaMII peptide did not adopt
the helical structure seen in all the a-conotoxins and did not
show any activity. This indicates a greater tolerance for
modification at the N-terminal of MII than at residue 5. The
active LaaMII was found to have improved permeability
across Caco-2 cell monolayers compared to native MII and
thus is considered to have potential for further in vivo
biodistribution experiments [53].
Concluding remarks
A conserved framework is evident in the three-dimensional
structures of a-conotoxins, with the major element of
secondary structure being an a-helix. The fold is largely
determined by the conserved disulfide connectivity between
Fig. 3. Disulfide bond isomers of a-conotoxins. (A) A schematic rep-
resentation of the globular, ribbon and beads isomers possible in any
of the a-conotoxins. (B) The three-dimensional structure of native
AuIB with the globular disulfide connectivity, CysI-CysIII and CysII-
CysIV. The disulfide bonds are shown in blue. (C) A three-dimensional
structure of the ribbon disulfide bond isomer of AuIB, where the

connectivities are CysI-CysIV and CysII-CysIII, with the disulfide
bonds shown in blue.
2324 E. L. Millard et al.(Eur. J. Biochem. 271) Ó FEBS 2004
CysI-CysIII and CysII-CysIV that braces the structure.
Altering the disulfide bonds has highlighted their important
structural influence, as less defined structures are obtained
when the connectivity is altered or when a single disulfide
bond is removed. Interestingly, studies of non-native
disulfide-bonded forms have also indicated that structural
flexibility can influence the biological effects observed for
a-conotoxins, either in a positive or negative way. Muta-
tional analysis has indicated residues that are important for
selectivity and potency, and structural analyses of such
mutants have suggested that what appear to be only minor
changes in the overall fold can have dramatic effects on
receptor activity. Recently discovered a-conotoxins suggest
that the diversity of conotoxin primary structures is likely to
increase, and this will aid in the elucidation of structure-
activity relationships and in the characterization of the
nAChR subtypes.
Acknowledgements
Work on conotoxins in our laboratory is supported by a grant from the
Australian Research Council. We thank Annette Nicke for helpful
discussions.
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