Defining Nicotinic Agonist Binding Surfaces through Photoaffinity Labeling

Tomizawa, M.; Maltby, David; Medzihradszky, Katalin F.; Zhang, Nanjing; Durkin, Kathleen A.; Presley, Jack M.; Talley, Todd T.; Taylor, Palmer; Burlingame, and Alma L.; Casida, John E.

doi:10.1021/bi700667v

Cited by 49 publications

(75 citation statements)

References 53 publications

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“…This relationship between Ls-and Ac-AChBPs in neonicotinoid selectivity is clearly interpretable to that for vertebrate and insect nAChR subtypes. Finally, in nicotinoids, one fundamental bound conformation is conserved for all AChBP and nAChR subtypes (7)(8)(9)(10)23). The same agonist molecule can also adopt different binding orientations at other Cys-loop receptors, depending on the nature and position of aromatic amino acid side chains, that is, serotonin (5-HT) at 5-HT 3 versus MOD-1 receptors and ␥-aminobutyric acid (GABA) at GABA A versus GABA C receptors (24,25).…”

Section: Discussionmentioning

confidence: 99%

“…The neonicotinoids and nicotinoids including five photoaffinity probes were available from our previous studies (9,10) Biology. Ls-and Ac-AChBP subtypes were expressed in HEK-293 cells by using cDNAs chemically synthesized from oligonucleotides engineered for mammalian codon usage and were purified as described in refs.…”

Section: Methodsmentioning

confidence: 99%

“…Understanding drugnAChR interactions was greatly facilitated by the discovery and crystallization of mollusk homopentameric ACh-binding proteins (AChBPs) as structural surrogates for the extracellular ligand-binding domain of the nAChR (6)(7)(8). Photoaffinity labeling combined with mass spectrometry (MS) technology provides a direct and physiologically relevant chemical biology method for three-dimensional structural investigation of drugreceptor interactions (9,10). In the present study, two chemotypes of nicotinic agonists (neonicotinoids and nicotinoids) with distinct pharmacophores are used as photoaffinity probes [supporting information (SI) Fig.…”

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confidence: 99%

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Atypical nicotinic agonist bound conformations conferring subtype selectivity

Tomizawa

Maltby

Talley

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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116

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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 subunit combinations confer differential selectivity for nicotinic drugs. We investigated the subtype selectivity of nAChR agonists by comparing two ACh-binding proteins (AChBPs) as structural surrogates with distinct pharmacological profiles [i.e., Lymnaea stagnalis (Ls) AChBP of low neonicotinoid and high nicotinoid sensitivities and Aplysia californica (Ac) AChBP of high neonicotinoid sensitivity] mimicking vertebrate and insect nAChR subtypes, respectively. The structural basis of subtype selectivity was examined here by photoaffinity labeling. Two azidoneonicotinoid probes in the Ls-AChBP surprisingly modified two distinct and distant subunit 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. Intriguingly, for the Ls-AChBP, the neonicotinoids have two bound conformations 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 selectivity is based on two disparate bound conformations of nicotinic agonists, thereby establishing an atypical concept for neonicotinoid versus nicotinoid selectivity between insect and vertebrate nAChRs.acetylcholine-binding protein ͉ imidacloprid ͉ neonicotinoids ͉ nicotinic receptor ͉ photoaffinity labeling T he nicotinic acetylcholine (ACh) receptor (nAChR) is critically important in synaptic neurotransmission and the target 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 vertebrate 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 structural 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 drugnAChR interactions was greatly facilitated by the discovery and crystallization of mollusk homopentamer...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Atypical nicotinic agonist bound conformations conferring subtype selectivity

Tomizawa

Maltby

Talley

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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116

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“…Their azido derivatives as photoaffinity probes showed the same positioning of the chloropyridinyl moiety for neonicotinoids and nicotinoids. 20,21) Molecular modeling revealed an inverted pharmacophore relationship of neonicotinoids and nicotinoids, as shown in Fig. 4.…”

Section: Achbp As a Structural Model For Neonicotinoid And Nicotinoidmentioning

confidence: 98%

Insecticide interactions with .GAMMA.-aminobutyric acid and nicotinic receptors: predictive aspects of structural models

Casida¹,

Tomizawa²

2008

J. Pestic. Sci.

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An insecticide binding site may be blocked by widely diverse chemicals, as with the g-aminobutyric acid receptor (GABAR), or accept only closely related compounds, as illustrated by the nicotinic acetylcholine (ACh) receptor (nAChR). The human GABAR b3-homopentamer resembles the insect receptor in sensitivity and specificity for noncompetitive antagonists (NCAs), prompting exhaustive site-directed mutagenesis (cysteine scanning), which pinpointed the critical active site as pore-facing residues Ala-2Ј, Thr-6Ј and Leu-9Ј and led to a binding site model which fits several classes of insecticidal NCAs. The nAChR agonist site was approached with an ACh binding protein (AChBP) by photoaffinity labeling and neonicotinoid docking. Chloropyridinyl chlorine contacts Ile-106/Met-116 and nitrogen is directed to Ile-118/Trp-147 via a solvent bridge(s). The guanidine/amidine plane undergoes p-stacking with Tyr-188, and the nitro/cyano pharmacophore interacts with Ser-189/Cys-190, thereby defining the binding pocket that models AChBP and possibly nAChR potency and selectivity.

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“…Utilizing a photoreactive analogue of epibatidine (azidoepibatidine; Fig. 1) and mass spectrometry, Tomizawa et al (21) identified photolabeled amino acids in the Aplysia AChBP (Tyr 195 in Segment C and Met 116 in Segment E), establishing an orientation for bound azidoepibatidine consistent with the orientation of epibatidine in an AChBP crystal structure (12).…”

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confidence: 97%

[3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors

Srivastava¹,

Hamouda²,

Pandhare³

et al. 2009

Journal of Biological Chemistry

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Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo ␣ 2 ␤␥␦ and expressed human ␣4␤2

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Defining Nicotinic Agonist Binding Surfaces through Photoaffinity Labeling

Cited by 49 publications

References 53 publications

Atypical nicotinic agonist bound conformations conferring subtype selectivity

Atypical nicotinic agonist bound conformations conferring subtype selectivity

Insecticide interactions with .GAMMA.-aminobutyric acid and nicotinic receptors: predictive aspects of structural models

[3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors

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