2ibs
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Crystal structure of the adenine-specific DNA methyltransferase M.TaqI complexed with the cofactor analog AETA and a 10 bp DNA containing 2-aminopurine at the target position
OverviewOverview
We report the crystal structure of the DNA adenine-N6 methyltransferase, M.TaqI, complexed with DNA, showing the fluorescent adenine analog, 2-aminopurine, flipped out of the DNA helix and occupying virtually the, same position in the active site as the natural target adenine., Time-resolved fluorescence spectroscopy of the crystalline complex, faithfully reports this state: base flipping is accompanied by the loss of, the very short ( approximately 50 ps) lifetime component associated with, fully base-stacked 2-aminopurine in DNA, and 2-aminopurine is subject to, considerable quenching by pi-stacking interactions with Tyr108 in the, catalytic motif IV (NPPY). This proves 2-aminopurine to be an excellent, probe for studying base flipping by M.TaqI and suggests similar quenching, in the active sites of DNA and RNA adenine-N6 as well as DNA cytosine-N4, methyltransferases sharing the conserved motif IV. In solution, the same, distinctive fluorescence response confirms complete destacking from DNA, and is also observed when the proposed key residue for base flipping by, M.TaqI, the target base partner thymine, is substituted by an abasic site, analog. The corresponding cocrystal structure shows 2-aminopurine in the, active site of M.TaqI, demonstrating that the partner thymine is not, essential for base flipping. However, in this structure, a shift of the 3', neighbor of the target base into the vacancy left after base flipping is, observed, apparently replicating a stabilizing role of the missing partner, thymine. Time-resolved fluorescence and acrylamide quenching measurements, of M.TaqI complexes in solution provide evidence for an alternative, binding site for the extra-helical target base within M.TaqI and suggest, that the partner thymine assists in delivering the target base into the, active site.
About this StructureAbout this Structure
2IBS is a Single protein structure of sequence from Thermus aquaticus with NEA as ligand. Active as Site-specific DNA-methyltransferase (adenine-specific), with EC number 2.1.1.72 Full crystallographic information is available from OCA.
ReferenceReference
2-Aminopurine Flipped into the Active Site of the Adenine-Specific DNA Methyltransferase M.TaqI: Crystal Structures and Time-Resolved Fluorescence., Lenz T, Bonnist EY, Pljevaljcic G, Neely RK, Dryden DT, Scheidig AJ, Jones AC, Weinhold E, J Am Chem Soc. 2007 May 16;129(19):6240-6248. Epub 2007 Apr 25. PMID:17455934
Page seeded by OCA on Wed Nov 21 12:14:10 2007