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.
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
Sulan
S
Luo
Kalyana Bharati
KB
Akondi
Dongting
D
Zhangsun
Yong
Y
Wu
Xiaopeng
X
Zhu
Yuanyan
Y
Hu
Sean
S
Christensen
Cheryl
C
Dowell
Norelle L
NL
Daly
David J
DJ
Craik
Ching-I Anderson
CI
Wang
Richard J
RJ
Lewis
Paul F
PF
Alewood
J
J
Michael McIntosh
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