Atypical nicotinic agonist bound conformations
conferring subtype selectivity
Motohiro Tomizawa*, David Maltby†, Todd T. Talley‡, Kathleen A. Durkin§, Katalin F. Medzihradszky†,
Alma L. Burlingame†, Palmer Taylor‡, and John E. Casida*¶
*Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, CA
94720-3112;†Mass Spectrometry Facility, University of California, San Francisco, CA 94143-2240;‡Department of Pharmacology, Skaggs School of Pharmacy
and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093-0650; and§Molecular Graphics and Computation Facility, College of
Chemistry, University of California, Berkeley, CA 94720-1460
Contributed by John E. Casida, December 12, 2007 (sent for review November 14, 2007)
The nicotinic acetylcholine (ACh) receptor (nAChR) plays a crucial
role in excitatory neurotransmission and is an important target for
drugs and insecticides. Diverse nAChR subtypes with various sub-
unit combinations confer differential selectivity for nicotinic drugs.
We investigated the subtype selectivity of nAChR agonists by
comparing two ACh-binding proteins (AChBPs) as structural sur-
rogates with distinct pharmacological profiles [i.e., Lymnaea stag-
nalis (Ls) AChBP of low neonicotinoid and high nicotinoid sensi-
tivities and Aplysia californica (Ac) AChBP of high neonicotinoid
sensitivity] mimicking vertebrate and insect nAChR subtypes, re-
here by photoaffinity labeling. Two azidoneonicotinoid probes in
the Ls-AChBP surprisingly modified two distinct and distant sub-
unit interface sites: loop F Y164 of the complementary or (?)-face
subunit and loop C Y192 of the principal or (?)-face subunit,
whereas three azidonicotinoid probes derivatized only Y192. Both
the neonicotinoid and nicotinoid probes labeled Ac-AChBP at only
one position at the interface between loop C Y195 and loop E
M116. These findings were used to establish structural models of
the two AChBP subtypes. In the Ac-AChBP, the neonicotinoids and
nicotinoids are nestled in similar bound conformations. Intrigu-
ingly, for the Ls-AChBP, the neonicotinoids have two bound con-
formations that are inverted relative to each other, whereas
nicotinoids appear buried in only one conserved conformation as
seen for the Ac-AChBP subtype. Accordingly, the subtype selectiv-
ity is based on two disparate bound conformations of nicotinic
agonists, thereby establishing an atypical concept for neonicotin-
oid versus nicotinoid selectivity between insect and vertebrate
acetylcholine-binding protein ? imidacloprid ? neonicotinoids ?
nicotinic receptor ? photoaffinity labeling
of potential therapeutic agents for neurological dysfunction and
of major neonicotinoid insecticides for crop protection and
animal health. The drug-binding sites are localized at subunit
interfaces of the nAChR pentameric structure. Specific verte-
brate and insect subunit combinations make up diverse nAChR
subtypes that differ in pharmacological profiles (1, 2). Highly
subtype-selective nicotinic agents are required for development
of therapeutics and insecticides. A family of peptide antagonists,
the ?-conotoxins, serve as important probes for studying struc-
tural determinants of subtype selectivity (3–5). However, the
molecular mechanism of selectivity for small agonist molecules
is not resolved because most of the key amino acids in the
nAChR-binding pocket are conserved in all of the receptor
subtypes and species and the binding region for antagonists
extends over a large interfacial surface. Understanding drug–
nAChR interactions was greatly facilitated by the discovery and
crystallization of mollusk homopentameric ACh-binding pro-
teins (AChBPs) as structural surrogates for the extracellular
he nicotinic acetylcholine (ACh) receptor (nAChR) is crit-
ically important in synaptic neurotransmission and the target
ligand-binding domain of the nAChR (6–8). Photoaffinity la-
beling combined with mass spectrometry (MS) technology pro-
vides a direct and physiologically relevant chemical biology
method for three-dimensional structural investigation of drug–
receptor interactions (9, 10). In the present study, two chemo-
types of nicotinic agonists (neonicotinoids and nicotinoids) with
distinct pharmacophores are used as photoaffinity probes [sup-
porting information (SI) Fig. 4] precisely capturing unique
bound conformations (Fig. 1) at two AChBP subtypes that differ
in pharmacological profiles, thereby defining an intriguing
mechanistic basis for subtype-selective agonist action at insect
and vertebrate nAChRs.
Subtype Selectivity. Neonicotinoids such as imidacloprid (IMI)
or cyano pharmacophore, respectively, are not protonated at
physiological pH and are selective agonists for the insect recep-
tor subtype, whereas four nicotinoids [epibatidine (EPI), des-
cyanothiacloprid (DCTHIA), desnitroimidacloprid (DNIMI),
and nicotine (NIC)] with a cationic functionality preferentially
act on the vertebrate subtype (2). AChBP from the freshwater
snail Lymnaea stagnalis (Ls) (6, 7) has higher affinity for
Author contributions: M.T. and J.E.C. designed research; M.T., D.M., and T.T.T. performed
research; M.T., D.M., T.T.T., K.A.D., K.F.M., A.L.B., P.T., and J.E.C. analyzed data; and M.T.
and J.E.C. wrote the paper.
The authors declare no conflict of interest.
¶To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
insecticide IMI, azido (N3) in the photoaffinity probe, or nitrene (N) after
photoactivation, capturing normal and inverted bound conformations. As-
terisk designates the nitro tip oxygen. Other neonicotinoids and nicotinoids
studied are shown in SI Fig. 4.
Structures of IMI and derivatives. R designates position of H in the
February 5, 2008 ?
vol. 105 ?
nicotinoids than neonicotinoids (Table 1), pharmacology rem-
iniscent of the vertebrate nAChR subtype. Another AChBP
from the saltwater mollusk Aplysia californica (Ac) (8) has
similar affinity for both neonicotinoids and nicotinoids and may
therefore serve as a structural surrogate for interactions of both
neonicotinoids with the insect receptor subtype and nicotinoids
with the vertebrate subtype (9, 10). Thus, two AChBPs from
mollusks have distinct pharmacology suggestive of the nAChRs
from species as divergent as mammals and insects. Ac-AChBP
has Y55 on loop D in contrast to tryptophan at the equivalent
position (W53) with Ls-AChBP and all of the nAChR subtypes.
Interestingly, the Y55W mutant of the Ac-AChBP gives a similar
neonicotinoid affinity profile to that of the wild type (WT)
(except for IMI with 5- to 14-fold enhanced potency), whereas
Y55W has fundamentally higher affinity for nicotinoids than the
WT (SI Table 3).
Photoaffinity Labeling. Azidoneonicotinoid and azidonicotinoid
photoaffinity probes (acting as photoactivated nitrenes) ade-
quately and specifically modified Ls-AChBP with up to one
agonist molecule for each subunit based on analysis of the intact
derivatized protein (SI Fig. 5 and SI Table 4). MS/MS analysis
for three nicotinoid probes precisely pinpointed one and only
one derivatized site at Y192 on loop C of the (?)-face subunit
(SI Table 5). Stunningly, the two neonicotinoid nitrene mole-
cules captured two distinct sites: the expected Y192 and the
distant Y164 on loop F of the (?)-face subunit established by
unambiguous adduct fragments (Fig. 2, SI Fig. 6, and SI Table
5). In the Ac-AChBP, both neonicotinoid and nicotinoid probes
photoaffinity labeled loop C Y195 and loop E M116, residues
that presumably are spatial neighbors to the azido substituent of
the probes (Table 2) (9, 10).
Multiple Bound Conformations on AChBP. Docking, conformational
searches, and molecular dynamics (MD) simulations using the
AChBP crystal structures gave models for binding site interac-
tions consistent with the observed ligand potency and photola-
beling. For neonicotinoids with a nitro or cyano substituent, the
Ls-AChBP subtype binds the ligands in two distinct bound
conformations with similar energy and yield (Fig. 3 and SI Fig.
7). One neonicotinoid-bound conformation is completely in-
verted compared with the common conformation. In this in-
verted form, loop F Y164 from the (?)-face subunit is spatially
positioned to be modified by the photoreactive nitrene of the
neonicotinoid [MD distance between putative nitrene position
and OH oxygen of Y164 is ?4 Å (nuclei to nuclei)]. The IMI
nitro tip oxygen H bonds with the backbone M114 HN (2.1 Å)
of loop E on the complementary subunit and/or possibly under-
goes water-bridging to L102 and M114 (2–4 Å). The chlorine
atom is proximal to the Y164 side chain (?4 Å). Imidazolidine
hydrogens on the C4 and C5 carbons are near the C187 and C188
Table 1. Comparison of pharmacological profiles for [3H]EPI
binding site in two AChBP subtypes
Ki? SEM, nM, n ? 3–4*
80 ? 8
219 ? 7
970 ? 57
1,180 ? 66
8,800 ? 1,500
0.3 ? 0.1¶
16 ? 1.5
18 ? 3
100 ? 5
*The average Hill coefficient of test compounds is essentially 1.0. Kivalues of
representative photoaffinity probes are: for N3-THIAolf with ?3H?EPI, 100 ?
0.05 and 10 for Ls- and Ac-AChBPs, respectively (9).
†Chemical structures are given in SI Fig. 4.
‡Data from Tomizawa et al. (9, 10).
§Estimated dissociation constant for ?3H?acetamiprid is 1,130 nM in Ls-AChBP.
¶Dissociation constants determined by direct radioligand titration method.
(given as representative data). The site of modification is indicated unambiguously as Y164 by the modified immonium ion and the appearance of the
appropriately shifted C-terminal (y) and N-terminal (b) fragment ions. The immonium ion and the first modified ions in the y and b series are printed in red.
Collision-induced dissociation spectrum of m/z 946.38(3?) corresponding to triply charged IMI nitrene-labeled peptide (E149 to R170) from Ls-AChBP
Tomizawa et al.
February 5, 2008 ?
vol. 105 ?
no. 5 ?
sulfurs and the Y192 OH (2–4 Å) on loop C from the (?)-face
subunit. Similar interactions are also observed for THIA with
cyano nitrogen in place of nitro oxygen (SI Fig. 7). In the normal
bound conformation with the Ls-AChBP subtype, chlorine faces
the backbone carbonyl oxygens of loop E L102 and L112 for van
der Waals contacts (3.0 and 4.6 Å, respectively) and the pyridine
nitrogen is expected to H bond with the backbone carbonyl
oxygens of M114 and W143 (3.1 and 3.8 Å, respectively) possibly
by solvent bridge(s). The loop C Y192 OH oxygen from the
(?)-face subunit is also suitably positioned for photoderivatiza-
tion by the nitrene (?4 Å). The nitro or cyano tip atom contacts
loop C C187 HN and the backbone of S186 (not displayed) (2–4
These interactions in the normal bound conformation are the
same as those in Ac-AChBP (10). Each of three nicotinoids with
a cationic functionality is nestled with one conserved confor-
mation in the agonist-binding site of Ls-AChBP (SI Fig. 8): that
is, the chloropyridine moiety interacts with loop E and a part of
loop B for van der Waals contacts and H bondings, whereas the
ammonium/iminium moiety primarily H bonds with the back-
cation-? interaction with the side chain of W143. Aromatic
residues Y89, Y185, Y192, and W53 surround this region.
nAChR Structural Models. Molecular models of Ls- and Ac-
AChBPs described above serve as the basis to develop nAChR
Table 2. Photoaffinity probes and modified site(s) in AChBPs
*Results from refs. 9 and 10. Ac-AChBP Y195 and M116 are spatial neighbors
to the azido substituent of the probes. Ls-AChBP Y192 corresponds to Ac-
†The same results were obtained with each of two neonicotinoid probes
(N3-IMI and N3-THIAolf) and each of three nicotinoid probes (N3-EPI, N3-
DNIMI, and N3-DCTHIAolf) (see chemical structures given in SI Fig. 4).
?2?1 nAChR subtypes established with 1UW6 and 2BYQ, respectively, as the scaffolds. The right and center four IMI-bound structures are in the normal position
and the left two are inverted (see also Fig. 1). Amino acids in green are from the (?)-face or ?-subunit, and in brown are from the (?)-face or ?-subunit.
THIA-docked AChBP and nAChR subtypes and the EPI-bound structure of Ls-AChBP based on the present photoaffinity-labeling results are shown in SI Figs. 7
and 8, respectively.
Structural models for IMI-binding site interactions with AChBP and nAChR subtypes. (Upper) For AChBP subtypes, IMI is docked onto the Lymnaea
www.pnas.org?cgi?doi?10.1073?pnas.0711724105Tomizawa et al.
structural templates describing the interactions of subtype-
selective nicotinic agonists. Two models for interfacial agonist-
binding domains of nAChR subtypes of chick ?4?2 (low neo-
nicotinoid sensitivity) and an insect (Myzus) nAChR (high
neonicotinoid sensitivity) are established based on crystal struc-
tures from Ls-AChBP (1UW6) and Ac-AChBP (2BYQ), respec-
tively (Fig. 3 and SI Fig. 7). As with Ls-AChBP, the chick ?4?2
receptor yields two bound conformations for IMI or THIA that
are once again inverted relative to each other. In the inverted
IMI/THIA-bound conformation, the nitro or cyano tip oxygen/
nitrogen H bonds with loop E L139 HN (1.8 Å) and possibly
makes water bridges involving L139 and N127. Chlorine forms
van der Waals contacts with loop F D189 carboxyl oxygen (3.7
Å). The alternative normal conformation of IMI/THIA is also
consistent with that in the Ls-AChBP. The aphid Myzus persicae
nAChR subtype (11–13) was selected for modeling because it is
highly neonicotinoid sensitive and, in contrast to many other
insects of defined genomic sequence, it is a major target world-
wide in neonicotinoid use. In the Myzus ?2?1 interface, IMI and
THIA occupy the binding site in the normal bound conforma-
tion defined by the Ac-AChBP subtype (10) and the normal one
in Ls-AChBP. In addition, variable loop E residues among the
AChBPs and nAChRs are spatially and functionally consistent in
each case. Nicotinoids (EPI, DNIMI, and DCTHIA) yield one
common bound conformation (images not shown) as with those
in Ac-AChBP (8–10).
Neonicotinoid-Binding Site Interactions and Selectivity. Neonicoti-
noids are selective agonists for the insect versus vertebrate
nAChR subtypes. The distinct neonicotinoid nitro or cyano
in the insect subtype (14–16). Sequence alignments of the
nAChR ?-subunits from diverse species of insects and verte-
brates suggest a purported site for interaction with the nitro/
cyano electronegative tip, that is, a basic residue (arginine or
lysine) adjacent to the loop D tryptophan in various insect
?-subunits. Pentameric stoichiometries of the various insect
nAChRs have not been resolved, because they can be examined
functionally only as recombinant hybrids consisting of various
insect ?-subunits and a vertebrate ?2-subunit (17–21). A mutant
of Drosophila ?2/chick ?2 hybrid nAChR with a basic residue
(T77R or K) on the ?2-subunit attenuates IMI-elicited agonist
responses, but the influence of the mutation is modest (21)
compared with that in WT vertebrate ?2-containing hybrid
receptors (18, 20); therefore, this residue may play an alternative
or supplemental role associated with conformational rearrange-
ments of the binding pocket after ligand binding (10). Target-site
resistance to some neonicotinoids in the brown planthopper
results from a nAChR mutation in which a tyrosine on loop B
of the Nl?1 or Nl?3 subunit is substituted by serine (T151S),
a structural surrogate, provides a suitable template for delin-
eating the determinants of neonicotinoid binding to the insect
receptor (10). The neonicotinoid nitro or cyano pharmacophore
interacts primarily with loop C C190 and S189. In contrast, the
cationic functionality of three nicotinoids (EPI, DNIMI, and
DCTHIA) faces in the opposite direction contacting loop B
W147 for H bonding and cation-? interaction (8–10).
Atypical Concept for Subtype Selectivity. The functional amino
AChBPs and diverse nAChR subtypes, yet there is considerable
neonicotinoid selectivity. This suggests a limitation for the
pharmacological approach combined with site-directed mu-
tagenesis. In the present investigation, we discovered for two
AChBP subtypes that one has low affinity (Ls-AChBP) and the
other high affinity (Ac-AChBP) for neonicotinoids. Thus, the
present direct comparison of the two AChBPs in neonicotinoid-
binding site interactions structurally defines the mechanism of
subtype selectivity, consequently rationalizing the high and low
affinities of the insect and vertebrate nAChR subtypes, respec-
tively. Neonicotinoid photoaffinity labeling of Ls-AChBP in
physiologically relevant media yields two distant modification
sites at loop F Y164 and loop C Y192. However, only one site
Y192 is assigned in nicotinoid photolabeling. In the Ac-AChBP,
both chemotypes of probes bind at one interfacial position
between Y195 and M116 on loops C and E, respectively (9, 10).
Our results lead to three conclusions. First, a mixture of two very
disparate bound ligand conformations at the Ls-AChBP subtype
reveals the inferior affinity of neonicotinoids at the site. Second,
to its high neonicotinoid sensitivity. This relationship between
Ls- and Ac-AChBPs in neonicotinoid selectivity is clearly inter-
pretable to that for vertebrate and insect nAChR subtypes.
Finally, in nicotinoids, one fundamental bound conformation is
conserved for all AChBP and nAChR subtypes (7–10, 23). The
same agonist molecule can also adopt different binding orien-
tations at other Cys-loop receptors, depending on the nature and
position of aromatic amino acid side chains, that is, serotonin
(5-HT) at 5-HT3versus MOD-1 receptors and ?-aminobutyric
acid (GABA) at GABAAversus GABACreceptors (24, 25).
In summary, this investigation examining nicotinic agonist
labeling of AChBP subtypes establishes that multiple bound
ligand conformations may contribute to the binding constant,
which therefore reflects a weighted average of a multiplicity of
binding orientations, rather than the ligand residing in a single
bound state or conformation.
Materials and Methods
Chemicals. The neonicotinoids and nicotinoids including five photoaffinity
probes were available from our previous studies (9, 10) except for [3H]EPI
(?)-EPI, and (?)-NIC obtained from Amersham Biosciences, TOCRIS, and
Biology. Ls- and Ac-AChBP subtypes were expressed in HEK-293 cells by using
to the AChBP were determined by a scintillation proximity assay (9, 10).
Photoaffinity-labeling experiments and MS measurements of derivatized in-
tact AChBP subunit and MS/MS analyses of tryptic fragments pinpointing the
sites of modifications were performed according to our earlier methodology
Calculations. Docking calculations were performed by using the AutoDock 4
suite (27, 28). The receptor was treated as rigid whereas flexible ligands were
docked in a 15-Å cubic grid centered on the active site. In each case, 200
Lamarkian Genetic Algorithm searches were carried out. Good-quality hits
were those with binding energies below ?7 kcal/mol. A 1,000-step low mode
Monte Carlo (MC) conformational search was performed on IMI and THIA in
the Ls-AChBP 1UW6 (7) active site. THIA was then minimized in 1UW6 along
1UW6. MD simulations involved NIC and IMI in the 1UW6 active site with
varying numbers of explicit waters. These were equilibrated for 100 ps and
using Macromodel 9.1 in Maestro 7.5 (Schro ¨dinger) (29) with the OPLS2005
forcefield and a water continuum model (30). Unless otherwise specified, all
minimizations also used Macromodel with these parameters.
The Myzus ?2?1 interface homology model was built by the Swiss-Model
server (31) by using Ac-AChBP 2BYQ (chain A) (8) as a template based on
sequence alignments made by using the CLUSTAL W web server at the Euro-
then used as a scaffold to assemble the individual chains Myzus ?2 and ?1,
respectively. The model side chains were optimized by using the Swiss PDB
viewer. The model was further refined with Macromodel including minimi-
Tomizawa et al.
February 5, 2008 ?
vol. 105 ?
no. 5 ?
zation of all side chains within 15 Å of the binding pocket with the remainder Download full-text
of the structure fixed. The chick ?4?2 interface homology model was estab-
lished with 1UW6 as a scaffold according to our earlier procedure (9).
ACKNOWLEDGMENTS. We thank Dennis Dougherty, Bruce Hammock, and
Jeffrey Scott for important suggestions. This work was supported by the
National Institute of Environmental Health Sciences Grant R01 ES08424 (to
M.T. and J.E.C.), National Center for Research Resources Grants RR015084,
RR001614, and RR019934 (to D.M., K.F.M., and A.L.B.), National Institutes of
Health Grants R37-GM18360 and UO1-NS05846 (to T.T.T. and P.T.), and Na-
tional Science Foundation Grant CHE-0233882 (to K.A.D.).
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