Atypical alpha-conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor.
Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. alpha-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of alpha-conotoxins may serve as a platform for development of new therapies. Previously characterized alpha-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel alpha-conotoxin LtIA, which lacks this highly conserved motif but potently blocked alpha3beta2 nAChRs with a 9.8 nm IC(50) value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-block of alpha3beta2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR beta subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for alpha3beta2 versus alpha3beta4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC(50) value on alpha3beta2(F119Q) versus wild type nAChRs, whereas T59K and V111I beta2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha3beta2 nAChR that includes beta2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.
Luo
Sulan
S
Key Laboratory of Tropical Biological Resources, Ministry of Education, Ocean College, College of Materials and Chemical Engineering, Center for Experimental Biotechnology, Hainan University, Haikou Hainan 570228, China. luosulan2003@163.com
Akondi
Kalyana Bharati
KB
Zhangsun
Dongting
D
Wu
Yong
Y
Zhu
Xiaopeng
X
Hu
Yuanyan
Y
Christensen
Sean
S
Dowell
Cheryl
C
Daly
Norelle L
NL
Craik
David J
DJ
Wang
Ching-I Anderson
CI
Lewis
Richard J
RJ
Alewood
Paul F
PF
Michael McIntosh
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D020916:Conotoxins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000494:pharmacology*; D052078:Conus Snail / Q000235:genetics; D005260:Female; D066298:In Vitro Techniques; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018733:Nicotinic Antagonists / Q000737:chemistry / Q000494:pharmacology; D019906:Nuclear Magnetic Resonance, Biomolecular; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction; D017510:Protein Folding; D051381:Rats; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D014981:Xenopus
D020916:Conotoxins; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; C419362:nicotinic receptor alpha3beta2