Cryo-EM structure of detergent-bound <i>Vibrio cholerae</i> MakA captures a candidate intermediate between the soluble form and mature assembly of an α-pore-forming toxin

Abstract

<jats:title>Abstract</jats:title> <jats:p> The tripartite toxin MakA/MakB/MakE from <jats:italic>Vibrio cholerae</jats:italic> belongs to a family of α-pore-forming toxins that undergo extensive conformational rearrangements upon interacting with membrane. While soluble structures of MakA, MakB and MakE have been determined, the structural basis of membrane association and pore assembly remains poorly understood. Here, we report a cryo-electron microscopy structure of a detergent-associated form of MakA at a global resolution of 3.13 Å, with focused refinement of a flexible region improving the local resolution to 2.68 Å. This structure consists of a trimer of MakA asymmetric homodimers, in which each subunit adopts a transmembrane α-helical conformation distinct from the previously reported soluble structure. The asymmetric homodimer closely resembles the building block observed in the previously reported helical MakA assembly and is structurally similar to the homodimeric building blocks of the <jats:italic>Aeromonas hydrophila</jats:italic> AhlB and <jats:italic>Serratia marcescens</jats:italic> SmhA pores, and the heterodimeric building blocks of the <jats:italic>Xenorhabdus nematophila</jats:italic> XaxAB and <jats:italic>Yersinia enterocolitica</jats:italic> YaxAB pores, suggesting a conserved assembly principle among distantly related α-pore-forming toxins. Complementary hydropathy analysis, AlphaFold2 modeling, and liposome-based assays reveal distinct roles for the three components, with MakA acting as the primary membrane-inserting subunit, MakE exhibiting low but consistent membrane-association, and MakB alone remaining largely soluble. Together, these results support a model in which the MakA asymmetric homodimer represents a plausible early membrane-insertion unit and building block for pore assembly, while the trimer of dimers represents an early assembly intermediate. </jats:p>