AlphaFold-Multimer Modelling of Linked nAChR Subunits Challenges Concatemer Design Assumptions

Abstract

Nicotinic acetylcholine receptors (nAChRs) are well described in vertebrates, yet less studied in arthropods, and the subunit stoichiometry of heteromeric arthropod nAChRs remains unresolved. This study combined a computational and experimental approach to predict and validate the stoichiometries of two heteromeric nAChRs from the parasitic arthropod Lepeophtheirus salmonis. AlphaFold2- and AlphaFold-Multimer-based modelling, supported by multiple sequence alignment and functional expression in Xenopus laevis oocytes, identified the most likely stoichiometries for two compositions of subunits previously described, named Lsa-nAChR1 and Lsa-nAChR2. For receptor Lsa-nAChR1, the highest scoring stoichiometry was 1b122b2. Lsa-nAChR2 exhibited three possible stoichiometries, confirmed by both computational modelling and experiments. These are 3{beta}23{beta}1{beta}2, 3{beta}13{beta}1{beta}2, and 333{beta}1{beta}2. All stoichiometries are written from a counterclockwise, extracellular orientation. Strikingly, structural modelling also suggested that linker flexibility in concatemer constructs may allow a novel conformation with a different subunit between the linked subunits, referred to here as "wedging". These results indicate that the use of flexible linker sequences does not reliably enforce subunit position or assembly directionality, as shown here for nAChRs. These findings challenge the assumption that linked concatemers unambiguously dictate receptor stoichiometry. Thus, interpretations of concatemer-based studies, on nAChRs and other receptor systems, may warrant careful reevaluation.