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Structure of pvu II DNA-(cytosine N4) methyltransferase, an example of domain permutation and protein fold assignment
W Gong1, M O'Gara, R M Blumenthal
1W.M.Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
Abstract:
We have determined the structure of Pvu II methyltransferase (M. Pvu II) complexed with S -adenosyl-L-methionine (AdoMet) by multiwavelength anomalous diffraction, using a crystal of the selenomethionine-substituted protein. M. Pvu II catalyzes transfer of the methyl group from AdoMet to the exocyclic amino (N4) nitrogen of the central cytosine in its recognition sequence 5'-CAGCTG-3'. The protein is dominated by an open alpha/beta-sheet structure with a prominent V-shaped cleft: AdoMet and catalytic amino acids are located at the bottom of this cleft. The size and the basic nature of the cleft are consistent with duplex DNA binding. The target (methylatable) cytosine, if flipped out of the double helical DNA as seen for DNA methyltransferases that generate 5-methylcytosine, would fit into the concave active site next to the AdoMet. This M. Pvu IIalpha/beta-sheet structure is very similar to those of M. Hha I (a cytosine C5 methyltransferase) and M. Taq I (an adenine N6 methyltransferase), consistent with a model predicting that DNA methyltransferases share a common structural fold while having the major functional regions permuted into three distinct linear orders. The main feature of the common fold is a seven-stranded beta-sheet (6 7 5 4 1 2 3) formed by five parallel beta-strands and an antiparallel beta-hairpin. The beta-sheet is flanked by six parallel alpha-helices, three on each side. The AdoMet binding site is located at the C-terminal ends of strands beta1 and beta2 and the active site is at the C-terminal ends of strands beta4 and beta5 and the N-terminal end of strand beta7. The AdoMet-protein interactions are almost identical among M. Pvu II, M. Hha I and M. Taq I, as well as in an RNA methyltransferase and at least one small molecule methyltransferase. The structural similarity among the active sites of M. Pvu II, M. Taq I and M. Hha I reveals that catalytic amino acids essential for cytosine N4 and adenine N6 methylation coincide spatially with those for cytosine C5 methylation, suggesting a mechanism for amino methylation.
Insights
The Pvu II methyltransferase structure reveals a common fold among DNA methyltransferases, suggesting a shared mechanism for amino methylation. This finding aids in understanding enzyme function and evolution.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- DNA methyltransferases are crucial enzymes involved in gene regulation and DNA repair.
- Understanding the structural basis of methyltransferase activity is key to deciphering their biological roles.
Purpose of the Study:
- To determine the three-dimensional structure of Pvu II methyltransferase (M. Pvu II) in complex with S-adenosyl-L-methionine (AdoMet).
- To elucidate the structural basis for M. Pvu II's catalytic mechanism and its relationship to other methyltransferases.
Main Methods:
- Multi-wavelength anomalous diffraction (MAD) was employed.
- Crystals of selenomethionine-substituted M. Pvu II were utilized for structure determination.
Main Results:
- The structure of M. Pvu II complexed with AdoMet was determined, revealing an alpha/beta-sheet fold with a V-shaped cleft.
- AdoMet and catalytic residues are positioned within the cleft, suitable for DNA binding and cytosine methylation.
- The M. Pvu II structure shares significant similarity with other DNA methyltransferases (M. Hha I, M. Taq I), indicating a common structural framework.
Conclusions:
- M. Pvu II methylates the N4 position of cytosine within the 5'-CAGCTG-3' sequence.
- The conserved structural fold and active site organization suggest a common evolutionary origin and functional mechanism for DNA methyltransferases.
- The findings support a model where DNA methyltransferases share a common fold with permuted functional regions, and catalytic residues for different methylation types spatially coincide.