AlphaFold-Multimer modelling of linked nAChR subunits challenges concatemer design assumptions

Nicotinic acetylcholine receptors (nAChRs) are well described in vertebrates, yet less studied in arthropods, and the subunit stoichiometry and configuration of heteromeric arthropod nAChRs remains unresolved. This study combined a computational and experimental approach to predict and validate the subunit configurations 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 subunit configurations for two compositions of subunits previously described, named Lsa-nAChR1 and Lsa-nAChR2. For receptor Lsa-nAChR1, the highest scoring configuration was α1β1α2α2β2. Lsa-nAChR2 exhibited three possible subunit configurations, confirmed by both computational modelling and experiments. These are α3β2α3β1β2, α3β1α3β1β2, and α3α3α3β1β2. All subunit configurations 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 configuration. Thus, interpretations of concatemer-based studies, on nAChRs and other receptor systems, may warrant careful reevaluation.