Defining the Structure-Function Relationship behind Allosteric Regulation of 5,10-Methylenetetrahydrofolate Reductase
Bibliographic record
Abstract
5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in one-carbon metabolism (OCM), bridging the folate and methionine cycles. The flavoprotein catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of 5,10-methylenetetrahydrofolate, directing folate-derived one-carbon units away from nucleotide biosynthesis toward the production of S-adenosylmethionine (SAM). SAM serves as a methyl donor in the epigenetic regulation of DNA, RNA, histones, and modulation of proteins, lipids, and neurotransmitters. The over 200 known transmethylation reactions are governed by the ratio between SAM and its byproduct, S-adenosylhomocysteine (SAH), known as cellular methylation potential. Dysregulated MTHFR may have a detrimental impact on human health. This is highlighted by severe MTHFR deficiency, a rare inborn error of metabolism, which causes a perturbed methylation potential, toxic metabolite accumulation and reduced circulating folate. The disorder typically presents in children with hypotonia, failure to thrive, and, if left untreated, neurological impairment. In contrast, upregulated MTHFR activity has been implicated in certain cancers, with studies showing reduced tumour growth due to antisense inhibition of MTHFR. To maintain OCM homeostasis, human MTHFR has evolved a feedback regulation in which SAM-mediated inhibition is counteracted by SAH dis-inhibition, with additional modulation by phosphorylation status and NADPH binding. The existing crystal structure of active MTHFR reveals a catalytic domain (CD) that is structurally independent of the regulatory domain (RD), with a single SAH molecule bound within its allosteric pocket. By displacing SAH, SAM is thought to initiate inhibition by clashing with the flexible interdomain linker connecting the CD and RD. However, the exact mechanism by which allosteric inactivation of MTHFR is achieved remains unknown. To investigate the molecular mechanism of allosteric MTHFR inhibition, cryo-electron microscopy was employed in combination with biochemical investigation of full-length human MTHFR. Our 2.9 Å SAM-bound model reveals a closed compact conformation facilitated by significant rearrangements of the linker. In this state, the CD is reoriented to face the RD, allowing the insertion of a hydrophobic plug within the CD, blocking the shared folate and NADPH binding site. This conformation is stabilised by binding two SAM molecules within the allosteric pocket, which suggests a robust multi-step commitment to inhibition rather than a simple on-and-off response. To further explore the fine-tuning of SAM-mediated inhibition, we employed a combinatory approach of in silico modelling with assessments of thermal stability, ligand binding, and enzymatic activity of recombinant MTHFR. This analysis revealed that phosphorylations, primarily located within the N-terminus of MTHFR, prime active MTHFR for inhibition by forming transient interactions with the linker and enhancing the mobility of the CD. Conversely, we found that NADPH counters the closed inhibited conformation by competing with the hydrophobic plug within the CD. Collectively further underscoring the dynamic response MTHFR required to maintain OCM homeostasis. Additional complementary studies presented in this thesis uncover sequence-function data on MTHFR underscoring potential regions involved in its allosteric inhibition mechanism and investigation of the MTHFR interactome revealing potential protein-protein interactions further influencing MTHFR activity. This research provides the structural-functional basis for the allosteric regulation of MTHFR, underscoring its dynamic yet robust response to the cellular methylation potential, phosphorylation and NADPH. These findings deepen our knowledge of the intricate interplay between MTHFR and OCM metabolism and further support therapeutic interventions targeting MTHFR.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".