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 con
Trang 1M I N I R E V I E W
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, CCXmCXnC, where loop 1 comprises four residues (m ¼ 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, CCXmCXnC (Xm and Xn
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 toD Craik, Institute for Molecular Bioscience,
University of Queensland, Brisbane, QLD, 4072, Australia
Fax: + 61 73346 2029, Tel.: + 61 73346 2019,
E-mail: d.craik@imb.uq.edu.au
Abbreviation: nAChR, nicotinic acetylcholine receptor
(Received 22 January 2004, revised 19 March 2004,
accepted 6 April 2004)
Trang 2expected, 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/H2O and 30% acetonitrile/H2O [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 activea-conotoxins *Refers to the amidated C-terminus IC50relates
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
PnIA GCCSLPPCAANNPDYC* 4/7 a7, a3b2a 252, 9.6 [47] X-ray 1.1
PnIB GCCSLPPCALSNPDYC* 4/7 a7, a3b2a 61.3, 1970 [47] X-ray 1.1
EpI GCCSDPRCNMNNPDYC* 4/7 a3b2, a3b4a 1.6c[55] X-ray 1.1
GID IRDcCCSNPACRVNNOHVC 4/7 a7, a3b2, a4b2a 5, 3, 150 [16] NMR 0.34
PIA RDPCCSNPVCTVHNPQIC* 4/7 a6b2b3, a3b2a 0.95, 74.2 [31] – –
AnIB GGCCSHPACAANNQDYC* 4/7 a3b2, a7a 0.3, 76 [30] – –
AuIA GCCSYPPCFATNSDYC* 4/6 a3b4a >750 [56] – –
AuIC GCCSYPPCFATNSGYC* 4/6 a3b4a >750 [56] – –
arat nAChR subunits,bhuman nAChR subunits,cACh-evoked currents in parasympathetic neurons from rat intracardiac ganglia
Trang 3Structure-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 activea-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
Trang 4identified 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 activea-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
nAChR subtype IC50relative to Native (nM)
Structural comparisons with native structures
[W10F]ImI GCCSDPRC FR C* a7a no significant change [42] –
[R11E]ImI GCCSDPRCAWE C* a7b no significant change [46] similar
[R7L]ImI GCCSDPL AWR C* a7b
[R7A]ImI GCCSDPA AWR C* a7c
Monodisulfide ImI GCCSDPRCAWR C* a7c no significant change [21] relatively disordered
[N11S]PnIA GCCSLPPCAASNPDYC* a7, a3b2a
[D1–4]GID CCSNPACRVNNOHVC a7, a3b2, a4b2a fl a4b2, no change
for others [16]
– LaaMII LGCCSNPVCHLEHSNLC* a3b2d no significant change [53] minimal change
from native MII 5LaaMII GCCSLPVCHLEHSNLC* a3b2d
a
rat nAChR subunits,bhuman nAChR subunits,cHEK cells used in competitive binding assay,dACh-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, R7 and W10, are shown in pink
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]
Trang 5[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 a7 vs 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 a7 activity has yet to be
determined As the side chain length increases the ratio
between a3b2 and a7 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 a7 and a3b2 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 a4b2 and a7
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 ofa-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
Trang 6CysI-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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