Dynamic inter-domain transformations mediate the allosteric regulation of human 5, 10-methylenetetrahydrofolate reductase

One-carbon metabolism drives an extensive network of signalling and biosynthetic pathways essential for cell growth and redox regulation. In all domains of life, 5,10-methylenetetrahydrofolate reductase (MTHFR) signals the commitment of folate-derived one-carbon units into biosynthesis of the global donor of trans-methylation, S-adenosyl-L-methionine (SAM). Eukaryotic MTHFR appends to its conserved TIM-barrel catalytic domain (CD) a unique regulatory domain (RD) that confers feedback inhibition by SAM. We determined, through cryo-electron microscopy, multiple structures of human MTHFR bound to SAM, and its demethylated product S-adenosyl-L-homocysteine (SAH) that relieves SAM inhibition. In the active SAH-bound state, the CD is flexible and its active site accessible for catalysis. SAM binding in the inhibited state substantially rearranges the inter-domain linker, which: remodels the RD pocket, reorients the CD, inserts a loop from the linker into the CD blocking active site access, and mobilizes Tyr404 to lock onto the flavin adenine dinucleotide cofactor. The switch between the active and inhibited states is facilitated by binding of either one SAH or two SAM molecules to the RD, respectively. Occupation of both SAM sites is required for inhibition, but the second site is occluded in the presence of SAH. Together our data explain the long-distance inhibitory mechanism of MTHFR employed by dual SAM binding, demonstrate how a single bound SAH confers a dis-inhibitory signal, and pave the way for rational design of novel therapeutics targeting MTHFR.