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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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This content will become publicly available on May 1, 2026
Integrated structural model of the palladin–actin complex using XL ‐ MS , docking, NMR , and SAXS
Abstract Palladin is an actin‐binding protein that accelerates actin polymerization and is linked to the metastasis of several types of cancer. Previously, three lysine residues in an immunoglobulin‐like domain of palladin have been identified as essential for actin binding. However, it is still unknown where palladin binds to F‐actin. Evidence that palladin binds to the sides of actin filaments to facilitate branching is supported by our previous study showing that palladin was able to compensate for Arp2/3 in the formation ofListeriaactin comet tails. Here, we used chemical crosslinking to covalently link palladin and F‐actin residues based on spatial proximity. Samples were then enzymatically digested, separated by liquid chromatography, and analyzed by tandem mass spectrometry. Peptides containing the crosslinks and specific residues involved were then identified for input to the HADDOCK docking server to model the most likely binding conformation. Small‐angle x‐ray scattering was used to provide further insight into palladin flexibility and the binding interface, and NMR spectra identified potential interactions between palladin's Ig domains. Our final structural model of the F‐actin:palladin complex revealed how palladin interacts with and stabilizes F‐actin at the interface between two actin monomers. Three actin residues that were identified in this study also appear commonly in the actin‐binding interface with other proteins such as myotilin, myosin, and tropomodulin. An accurate structural representation of the complex between palladin and actin extends our understanding of palladin's role in promoting cancer metastasis through the regulation of actin dynamics.
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- Award ID(s):
- 2216453
- PAR ID:
- 10650781
- Editor(s):
- Cortajarena, Aitziber L
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 34
- Issue:
- 5
- ISSN:
- 0961-8368
- Page Range / eLocation ID:
- e70122
- Subject(s) / Keyword(s):
- actin complex, crosslinking mass spectrometry, docking, Ig domain, NMR, SAXS
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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