Structural insights into the heterotrimeric alternatively spliced P2X7 receptors

Summary AlphaFold2-Multimer was used to generate structures of the heterotrimeric P2X7 receptors composed of wild-type P2X7A subunits and alternatively spliced subunits (P2X7B, P2X7E, P2X7J, and P2X7L) that have been confirmed in humans. The study supports laboratory research by providing insight into the structure and flexibility of the heterotrimeric alternatively spliced receptors in a simulated environment and may thereby aid structure-guided drug design. Abstract P2X7 receptors (P2X7Rs) are membrane-bound ATP-gated ion channels that are composed of three subunits. Different subunit structures may be expressed due to alternative splicing of the P2RX7 gene, altering the receptor’s function when combined with the wild-type P2X7A subunits. In this study, the application of the deep-learning method, AlphaFold2-Multimer (AF2M), for the generation of trimeric P2X7Rs was first validated by comparing an AF2M-generated rat wild-type P2X7A receptor with a structure determined by cryogenic electron microscopy (Protein Data Bank Identification: 6U9V). The results suggested AF2M could firstly, accurately predict the structures of P2X7Rs and secondly, accurately identify the highest quality model through the ranking system. Subsequently, AF2M was used to generate models of heterotrimeric alternatively spliced P2X7Rs consisting of one or two wild-type P2X7A subunits in combination with one or two P2X7B, P2X7E, P2X7J, and P2X7L splice variant subunits. The top-ranking models were deemed valid based on AF2M’s confidence measures, stability in molecular dynamics simulations, and consistent flexibility of the conserved regions between the models. Visual analysis of the heterotrimeric receptors identified missing residues in the ATP binding sites of the P2X7E, P2X7J, and P2X7L splice variants, likely translating into dysfunctional binding sites. Overall, the models produced in this study (available as supplementary material) unlock the possibility of structure-based studies into the heterotrimeric P2X7Rs.