Search for: All records

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. 

We examine changes in the picosecond structural dynamics with irreversible photobleaching of red fluorescent proteins (RFP) mCherry, mOrange2 and TagRFP-T. Measurements of the protein dynamical transition using terahertz time-domain spectroscopy show in all cases an increase in the turn-on temperature in the bleached state. The result is surprising given that there is little change in the protein surface, and thus, the solvent dynamics held responsible for the transition should not change. A spectral analysis of the measurements guided by quasiharmonic calculations of the protein absorbance reveals that indeed the solvent dynamical turn-on temperature is independent of the thermal stability/photostate however the protein dynamical turn-on temperature shifts to higher temperatures. This is the first demonstration of switching the protein dynamical turn-on temperature with protein functional state. The observed shift in protein dynamical turn-on temperature relative to the solvent indicates an increase in the required mobile waters necessary for the protein picosecond motions, that is, these motions are more collective. Melting-point measurements reveal that the photobleached state is more thermally stable, and structural analysis of related RFP’s shows that there is an increase in internal water channels as well as a more uniform atomic root mean squared displacement. These observations are consistent with previous suggestions that water channels form with extended light excitation providing O2 access to the chromophore and subsequent fluorescence loss. We report that these same channels increase internal coupling enhancing thermal stability and collectivity of the picosecond protein motions. The terahertz spectroscopic characterization of the protein and solvent dynamical onsets can be applied generally to measure changes in collectivity of protein motions.