Nuclear Magnetic Resonance Solution Conformation of -Conotoxin
AuIB, an
3
4
Subtype-selective Neuronal Nicotinic Acetylcholine
Receptor Antagonist*
(Received for publication, October 4, 1999, and in revised form, December 17, 1999)
Jee-Hyun Cho‡, K. Hun Mok‡, Baldomero M. Olivera§, J. Michael McIntosh§¶, Kyu-Hwan Park‡,
and Kyou-Hoon Han‡
From the ‡Protein Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-
600, Republic of Korea and Departments of §Biology and ¶Psychiatry, University of Utah, Salt Lake City, Utah 84112
The neuronal nicotinic acetylcholine receptors consti-
tute a highly diverse group, with subtypes consisting of
pentameric combinations of and subunits. -Cono-
toxins are a homologous series of small peptides that
antagonize these receptors. We present the three-dimen-
sional solution structure of -conotoxin AuIB, the first
15-residue -conotoxin known to selectively block the
3
4
nicotinic acetylcholine receptor subtype. The pair-
wise backbone and heavy-atom root mean square devi-
ation for an ensemble of 20 structures are 0.269 and 0.720
Å, respectively. The overall fold of -conotoxin AuIB
closely resembles that of the 4/7 subfamily -conotox-
ins. However, the absence of Tyr
15
, normally present in
other 4/7 members, results in tight bending of the back-
bone at the C terminus and effectively renders Asp
14
to
assume the spatial location of Tyr
15
present in other
neuronal 4/7 -conotoxins. Structural comparison of
-conotoxin AuIB with the
3
2
subtype-specific -cono-
toxin MII shows different electrostatic surface charge
distributions, which may be important in differential
receptor subtype recognition.
The -conotoxins are small neuropharmacologically active
peptides of Conus origin that antagonize the nicotinic acetyl-
choline receptor (nAChR)
1
(1). The natural diversity of biosyn-
thesized conotoxin peptides has led to the classification of a
wide spectrum of disulfide-bridged peptides, which attack var-
ious ligand and ion-gated channels and receptors (1). The nic-
otinic acetylcholine receptors exhibit considerable diversity in
their own right because of the different compositions found in
the pentameric subunits constituting each nAChR subtype (2).
Although the mammalian neuromuscular subtype comprises
(
1
)
2
or (
1
)
2
subunits, the neuronal subtypes are com-
paratively more diverse with their hetero- or homopentameric
combinations of (
2
9
) or (
2
4
) subunits (3, 4). The
general conotoxin strategy of diversification is “combinatorial”
(5), in which amino acid residues are varied within a given
disulfide framework to specifically and selectively bind various
subtypes of the target channel or receptor. For the case of
-conotoxins, target selectivity is essentially defined depending
on which subunit interface of the nAChR (e.g.
1
/,
1
/, and
3
/
2
) each individual -conotoxin preferentially binds to (1).
Highly selective -conotoxins that permit differential blocking
of diverse nAChR subtypes have served as effective tools in
studying these receptors (1).
Of recent particular interest are the -conotoxins that act on
neuronal nAChRs. For example, -conotoxin AuIB specifically
targets the
3
4
subtype (6), whereas -conotoxin MII selec-
tively blocks the
3
2
subtype (7). On the other hand, -cono-
toxin ImI, the smallest of all -conotoxins and distinct because
of its 4/3 disulfide framework
2
, is a specific antagonist of the
homomeric
7
subtype (8). In addition, other neuronal -cono-
toxins such as PnIA (9), PnIB (9), and EpI (10) that are less
selective to a particular nAChR subtype have also been iden-
tified (Table I).
As shown in Table I, most neuronal -conotoxins belong to
the 4/7 subfamily, in which the amino acid sequence varies
within disulfide bridge-enclosed loops of 4 and 7 residues. In-
terestingly, -conotoxin EI, unique in its specificity for the
1
/
subunit interface in Torpedo neuromuscular nAChR, belongs to
the same 4/7 subfamily (11). The recent increase in the un-
derstanding of these conotoxins has been attributable to the
advances in the structural characterization of these molecules.
High-resolution three-dimensional structures of 4/7 subfam-
ily -conotoxins PnIA (12), PnIB (13), MII (14, 15), and
[Tyr
15
]EpI (16) show that, when backbone-superimposed, their
backbone fold is extremely similar. The recently solved NMR
structure of -conotoxin EI also has the same overall molecular
fold as the 4/7 neuronal -conotoxins
3
despite the variation in
its sequence both within the disulfide loops and at the N ter-
minus (Table I). These observations reaffirm that conotoxins
are based on common three-dimensional scaffolds and that
their subtype selectivity is conferred through sequence varia-
tion of a selected number of residues (5).
Through structural elucidation of these highly selective
-conotoxins and their analogs (17, 18), we have been using the
reverse-mapping approach first to identify receptor subtype
specific determinants in the ligands and second to indirectly
probe the regions in nAChR responsible for binding agonists
* This research was supported in part by Grant NB0980 from the
Ministry of Science and Technology, Korea. The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 1DG2) have been
deposited in the Protein Data Bank, Research Collaboratory for Struc-
tural Bioinformatics, Rutgers University, New Brunswick, NJ
(http:wwwresb.org1).
To whom correspondence should be addressed: Protein Engineering
Laboratory, Korea Research Institute of Bioscience and Biotechnology,
Yusong P.O. Box 115, Taejon 305-600, Republic of Korea. Tel.: 82-42-
860-4250; Fax: 82-42-860-4259; E-mail: khhan@biotech5.kribb.re.kr.
1
The abbreviations used are: nAChR, nicotinic acetylcholine recep-
tor; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser ef-
fect spectroscopy; COSY, correlation spectroscopy; RMSD, root mean
square deviation.
2
The -conotoxins are additionally grouped according to the number
of amino acid residues enclosed within each disulfide loop. Loop sizes of
four residues in the first loop and seven in the second are denoted as
4/7. Other subfamilies include 3/5 and 4/3.
3
K.-H. Park, J. E. Suk, R. Jacobsen, W. R. Gray, J. M. McIntosh,
B. M. Olivera, and K. Han, manuscript in preparation.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 12, Issue of March 24, pp. 8680 –8685, 2000
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
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