Journal
BIOCHEMISTRY
Volume 46, Issue 30, Pages 8798-8806Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi700667v
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Funding
- NCRR NIH HHS [RR001614, S10 RR019934, P41 RR001614, RR015084, RR019934] Funding Source: Medline
- NIEHS NIH HHS [R01 ES08424, R01 ES008424] Funding Source: Medline
- NIGMS NIH HHS [R37 GM018360, R37-GM18360] Funding Source: Medline
- NINDS NIH HHS [F32 NS043063, U0-1 NS05846, F32 NS043063-01] Funding Source: Medline
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Nicotinic acetylcholine (ACh) receptor (nAChR) agonists are potential therapeutic agents for neurological dysfunction. In the present study, the homopentameric mollusk ACh binding protein (AChBP), used as a surrogate for the extracellular ligand-binding domain of the nAChR, was specifically derivatized by the highly potent agonist azidoepibatidine (AzEPI) prepared as a photoaffinity probe and radioligand. One EPI-nitrene photoactivated molecule was incorporated in each subunit interface binding site based on analysis of the intact derivatized protein. Tryptic fragments of the modified AChBP were analyzed by collision-induced dissociation and Edman sequencing of radiolabeled peptides. Each specific EPI-nitrene-modified site involved either Tyr195 of loop C on the principal or (+)-face or Met116 of loop E on the complementary or (-)-face. The two derivatization sites were observed in similar frequency, providing evidence of the reactivity of the azido/nitrene probe substituent and close proximity to both residues. [H-3]AzEPI binds to the alpha 4 beta 2 nAChR at a single high-affinity site and photoaffinity-labels only the alpha 4 subunit, presumably modifying Tyr225 spatially corresponding to Tyr195 of AChBP. Phe137 of the beta 2 nAChR subunit, equivalent to Met116 of AChBP, conceivably lacks sufficient reactivity with the nitrene generated from the probe. The present photoaffinity labeling in a physiologically relevant condition combined with the crystal structure of AChBP allows development of precise structural models for the AzEPI interactions with AChBP and alpha 4 beta 2 nAChR. These findings enabled us to use AChBP as a structural surrogate to define the nAChR agonist site.
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