Working models for operon-forming NfeD protease with the stomatin, prohibitin, flotillin, and HflK/C (SPFH) ring-shaped microdomain organizer

Various functions of eukaryotic membranes are often governed both temporally and spatially by lipid rafts or functional membrane microdomains (FMM). FMM are rich in scaffold proteins that belong to the stomatin, prohibitin, flotillin, and HflK/C (SPFH) superfamily. In prokaryotic genomes, stomatin ortholog and nodulation formation efficiency D (NfeD) genes are believed to form an operon. The NfeD protease PH1510 specifically cleaves the C-terminal hydrophobic region of the stomatin ortholog PH1511, suggesting that the NfeD protease functions in cooperation with the stomatin ortholog. The structural characteristics of the stomatin ortholog complex with the NfeD protease are unknown. It is also unclear how the active site of the NfeD protease is formed. In this study, the heterodimer (PH1510_PH1511) was successfully modeled using two independent methods, AlphaFold2 (AF2) and AlphaFold3 (AF3), and 18 copies of the heterodimer were assembled into two distinct complex models with a molecular weight of 1.4 MDa, which differed in their subunit interfaces. The two distinct models were referred to as "sphere-shape" and "bell-shape," respectively. Additionally, the molecular interactions between the NfeD-specific domain of PH1510 and the central domain of PH1511 were successfully detected in vitro. The working models shown in this study suggest that the inactive residues Lys138 and Ser97 hidden in the "bell-shape" model might become exposed on the surface in the "sphere-shape" model, thereby forming an active Lys138-Ser97 dyad at the domain interfaces.