Cryo-EM structures of the MnmE-MnmG complex reveal large conformational changes and provide new insights into the mechanism of tRNA modification.

MnmE and MnmG form a conserved protein complex responsible for the addition of a 5-carboxymethylaminomethyl (cmnm5) group onto the wobble uridine of several transfer RNAs (tRNAs). Within this complex, both proteins collaborate intensively to catalyze a tRNA modification reaction that involves glycine as a substrate in addition to three different cofactors, with FAD and NADH binding to MnmG and methylenetetrahydrofolate (5,10-CH2-THF) to MnmE. Without structures of the MnmEG complex, it remained enigmatic how these substrates and co-factors can be brought together in a concerted manner. Prior small angle X-ray scattering data suggested that the MnmE (α2) and MnmG (β2) homo-dimers can adopt either an α2β2 or α4β2 complex, depending on the nucleotide state of MnmE. Here, we report the cryo-EM structures of the MnmEG complex in the α2β2 and α4β2 oligomeric states. These structures reveal that MnmE undergoes large conformational changes upon interaction with MnmG, resulting in an asymmetric MnmE dimer. In particular, the functionally important C-terminal helix of MnmE relocates from the 5,10-CH2-THF-binding pocket of MnmE to the FAD-binding pocket of MnmG, thus suggesting a mechanism for the transfer of an activated methylene group from one active site to the other. Together, these findings provide crucial new insights into the MnmEG-catalyzed reaction.