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Abstract N6-adenine methylation occurs in both DNA and RNA (referred to as 6mA and m6A, respectively). As an extensively characterized epi-transcriptomic mark found in virtually all eukaryotes, m6A in mRNA is deposited by METTL3-METTL14 complex. As a transcription-associated epigenetic mark abundantly present in many unicellular eukaryotes, 6mA is coordinately maintained by two AMT1 complexes, distinguished by their mutually exclusive subunits, AMT6 and AMT7. These are all members of MT-A70 family methyltransferases (MTases). Despite their functional importance, no structure for holo-complexes with cognate DNA/RNA substrate has been resolved. Here, we employ AlphaFold3 (AF3) and molecular dynamics (MD) simulations for structural modeling ofTetrahymenaAMT1 complexes, with emphasis on ternary holo-complexes with double-stranded DNA (dsDNA) substrate and cofactor. Key structural features observed in these models are validated by mutagenesis and various other biophysical and biochemical approaches. Our analysis reveals the structural basis for DNA substrate recognition, base flipping, and catalysis in the prototypical eukaryotic DNA 6mA-MTase. It also allows us to delineate the reaction pathway for processive DNA methylation involving translocation of the closed form AMT1 complex along dsDNA. As the active site is highly conserved across MT-A70 family of eukaryotic 6mA/m6A-MTases, the structural insight will facilitate rational design of small molecule inhibitors, especially for METTL3-METTL14, a promising target in cancer therapeutics.
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This content will become publicly available on September 1, 2026
Structural insight into the substrate binding of the AMT complex via an inhibitor‐trapped state
Abstract N6‐adenine (6mA) DNA methylation plays an important role in gene regulation and genome stability. The 6mA methylation inTetrahymena thermophilais mainly mediated by the AMT complex, comprised of the AMT1, AMT7, AMTP1, and AMTP2 subunits. To date, how this complex assembles on the DNA substrate remains elusive. Here we report the structure of the AMT complex bound to the OCR protein from bacteriophage T7, mimicking the AMT–DNA encounter complex. The AMT1–AMT7 heterodimer approaches OCR from one side, while the AMTP1 N‐terminal domain, assuming a homeodomain fold, binds to OCR from the other side, resulting in a saddle‐shaped architecture reminiscent of what was observed for prokaryotic 6mA writers. Mutation of the AMT1, AMT7, and AMTP1 residues on the OCR‐contact points led to impaired DNA methylation activity to various extents, supporting a role for these residues in DNA binding. Furthermore, structural comparison of the AMT1–AMT7 subunits with the evolutionarily related METTL3–METTL14 and AMT1–AMT6 complexes reveals sequence conservation and divergence in the region corresponding to the OCR‐binding site, shedding light on the substrate binding of the latter two complexes. Together, this study supports a model in which the AMT complex undergoes a substrate binding‐induced open‐to‐closed conformational transition, with implications in its substrate binding and processive 6mA methylation.
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- Award ID(s):
- 2435178
- PAR ID:
- 10653714
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Protein science
- Volume:
- 34
- Issue:
- 9
- ISSN:
- 0961-8368
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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