Structural Mechanism and Regulation of the Phospholipid Methylase Cho2

Abstract Phosphatidylethanolamine (PE) methylation is a conserved eukaryotic pathway that maintains phospholipid homeostasis and influences diverse methylation-dependent processes by regulating the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio and cellular methylation capacity. In fungi, the first and rate-limiting step of this pathway is catalyzed by Cho2, an unusual membrane methyltransferase containing two pseudo-symmetrical catalytic domains and a C-terminal soluble domain (C Sol ). Here, we report four cryo-electron microscopy structures of fungal Cho2 at 3.26–2.56 Å resolution, revealing an elongated homodimer in which each protomer contains two homologous six-transmembrane catalytic domains. The structures define the molecular basis for phospholipid recognition and reveal a mechanism for feedback inhibition by phosphatidylcholine. Biochemical analyses further show that the two catalytic domains are functionally coupled, while structural modeling identifies a communication helix that likely coordinates conformational changes associated with cofactor binding and catalysis. In addition, analyses of a C Sol -truncation mutant and an inhibitory nanobody demonstrate that the C Sol domain positively regulates enzymatic activity by promoting SAM binding. Together, these findings establish the molecular basis of Cho2 function and uncover multiple regulatory mechanisms governing the first committed step of fungal PE methylation.