STRUCTURE OF PVUII DNA (CYTOSINE N4) METHYLTRANSFERASE, AN EXAMPLE OFDOMAIN PERMUTATION AND PROTEIN FOLD ASSIGNMENT

We have determined the structure of PvuII methyltransferase (M.PvuII) complexed with S-adenosyl-L-methionine (AdoMet) by multiwavelength ano malous diffraction, using a crystal of the selenomethionine-substitute d protein. M.PvuII catalyzes transfer of the methyl group from AdoMet to the exocyclic amino (N4) nitrogen of the central cytosine in ifs re cognition sequence 5'-CAGCTG-3'. The protein is dominated by an open a lpha/beta-sheet structure with a prominent V-shaped cleft: AdoMet and catalytic amino acids are Located at the bottom of this cleft. The siz e and the basic nature of the cleft are consistent with duplex DNA bin ding. The target (methylatable) cytosine, if flipped out of the double helical DNA as seen for DNA methyltransferases that generate 5-methyl cytosine, would fit into the concave active site next to the AdoMet. T his M.PvuII alpha/beta-sheet structure Is very similar to those of M.H haI (a cytosine C5 methyltransferase) and M.TaqI (an adenine N6 methyl transferase), consistent with a model predicting that DNA methyltransf erases share a common structural fold while having the major functiona l regions permuted into three distinct Linear orders, The main feature of the common fold is a seven-stranded beta-sheet (6 down arrow 7 up arrow 5 down arrow 4 down arrow 1 down arrow 2 down arrow 3 down arrow ) formed by five parallel beta-strands and an antiparallel beta-hairpi n, The beta-sheet is flanked by six parallel alpha-helices, three on e ach side. The AdoMet binding site is located at the C-terminal ends of strands beta 1 and beta 2 and the active site is at the C-terminal en ds of strands beta 4 and beta 5 and the N-terminal end of strand beta 7., The AdoMet-protein interactions are almost identical among M.PvuII , M.HhaI and M.TaqI, as well as in an RNA methyltransferase and at lea st one small molecule methyltransferase. The structural similarity amo ng the active sites of M.PvuII, M.TaqI and M.HhaI reveals that catalyt ic amino acids essential for cytosine N4 and adenine N6 methylation co incide spatially with those for cytosine C5 methylation, suggesting a mechanism for amino methylation.