Structural and functional analysis of LarC, a CTP-dependent cyclometallase required for nickel-pincer nucleotide cofactor biosynthesis

Biosynthesis of the nickel-pincer mononucleotide metallocofactor requires a CTP-dependent nickel insertion reaction catalyzed by LarC, whose mechanism of C-Ni bond formation is not fully understood. Here, we report the first cryo–electron microscopy structures of full-length LarC from Moorella thermoacetica with and without a mimic of the CMPylated reaction intermediate. LarC assembles as a hexamer comprising a central LarC2 domain core and peripheral LarC1 domain trimers connected by long, flexible interdomain linkers. The LarC1 domains contain a conserved histidine-rich region for nickel binding and an adjacent conserved acidic pocket, both essential for activity. Structural modeling suggests that the intermediate binds within the acidic pocket adjacent to the putative nickel-binding site, while cryo-EM density for an intermediate analog identifies an interdomain cleft near the LarC2 CTP-binding site as a likely transfer site. Based on these findings, we propose that the intermediate is transferred through the interdomain cleft from LarC2, where it is CMPylated, to LarC1 for nickel insertion.