| Summary: | The enzyme methionine synthase (MTR) is a modular five-domain protein that utilises cobalamin (Cbl) as a cofactor to perform a methyl transfer from methyltetrahydrofolate (MeTHF) to homocysteine (Hcy), producing tetrahydrofolate (THF) and methionine (Met). The eventual oxidation of Cbl leads to the inactivation of MTR, which requires an electron from its partner protein methionine synthase reductase (MTRR) and a methyl group from S-adenosylmethionine (SAM) for its recovery. Deficiencies in MTR are related to developmental disorders, megaloblastic anaemia, and other health consequences in humans. Most of the structural and biophysical data on MTR are derived from its bacterial orthologue, which shows that it is a very dynamic protein, with different conformations acquired throughout its catalytic and recovery cycle. To address the lack of structural data on human MTR, and on studies focusing on MTR interactions, cryo-EM, biophysical assays, and AlphaFold were employed in this study. Using a recombinant full-length human MTR, novel high-resolution structures of the protein were acquired using cryo-EM. The structures of <em>apo</em> and Cbl-loaded MTR revealed the position of the conserved residue Tyr1177 and provided novel insights into the reactivation cycle of the human protein, revealing differences from its bacterial orthologue. Furthermore, with the use of AlphaFold Multimer coupled with biophysical assays, the complex formation of MTR with MTRR and MMADHC, which delivers Cbl to <em>apo</em> MTR, was studied. The models revealed a novel interaction interface in the MTR-MTRR complex, which supports the data suggesting the formation of a higher-order complex. The results presented herein provide answers to unsolved questions on the structural and interactional aspects of MTR, broadening the knowledge of the biology of this protein.
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