Defining nicotinic agonist binding surfaces through photoaffinity labeling.
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. [3H]AzEPI binds to the alpha4beta2 nAChR at a single high-affinity site and photoaffinity-labels only the alpha4 subunit, presumably modifying Tyr225 spatially corresponding to Tyr195 of AChBP. Phe137 of the beta2 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 alpha4beta2 nAChR. These findings enabled us to use AChBP as a structural surrogate to define the nAChR agonist site.
Tomizawa
Motohiro
M
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720-3112, USA.
Maltby
David
D
Medzihradszky
Katalin F
KF
Zhang
Nanjing
N
Durkin
Kathleen A
KA
Presley
Jack
J
Talley
Todd T
TT
Taylor
Palmer
P
Burlingame
Alma L
AL
Casida
John E
JE
D000109:Acetylcholine / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D000818:Animals; D001048:Aplysia / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001952:Bridged-Ring Compounds / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D007097:Imines / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D018722:Nicotinic Agonists / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019920:Photoaffinity Labels / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D014316:Tritium / Q000737:chemistry; D014357:Trypsin
C476378:5-azidoepibatidine; D001952:Bridged-Ring Compounds; D003432:Cross-Linking Reagents; D007097:Imines; D018722:Nicotinic Agonists; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011725:Pyridines; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; D014316:Tritium; C017621:phenylnitrene; D014357:Trypsin; D000109:Acetylcholine