Precise mapping of a snake venom phospholipase A2 interaction with a human nicotinic acetylcholine receptor.

The identification of selective molecules that interact with specific human nicotinic acetylcholine receptor (nAChR) subtypes is essential for elucidating their structure and function. Animal venoms, particularly snake venoms, offer a diverse repertoire of bioactive molecules that may selectively bind to human nAChR subtypes. In this study, we cloned transcripts encoding human nAChR subunits and successfully expressed their extracellular domains (ECDs) in yeast cells. We further examined the interactions between nAChR subunits and snake venom toxins, identifying that snake Asp49-phospholipase A2 (Asp49-1) strongly interacts with the human β2 and β4 subunits while exhibiting weaker interactions with α subunits. To delineate the specific regions of Asp49-1 involved in binding to nAChR β2, we analyzed its three-dimensional structure and dissected it into six fragments. Our results indicate that the N-terminal region of Asp49-1, comprising 29 amino acids, exhibits strong binding affinity to nAChR β2, whereas the C-terminal region interacts weakly. Similarly, to determine the interaction sites on the nAChR β2 subunit, we segmented its ECD into four fragments, expressed them in yeast, and identified the proximal N-terminal region as the primary binding site for Asp49-1. Based on these interaction regions, we proposed the three-dimensional complex structure of Asp49-1 and α4β2 nAChR subtype. These findings contribute to a deeper understanding of nAChR subtype-specific interactions and have significant implications for the development of novel therapeutic strategies targeting the α4β2 nAChR subtype, which plays a critical role in various pathological conditions, including neurodegenerative disorders.