Structure of the bacteriophage T4 DNA adenine methyltransferase

Abstract

DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy. T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain consisting of a five-helix bundle and a beta-hairpin that is conserved in the family of GATC-related MTase orthologs. Unexpectedly, the sequence-specific T4Dam bound to DNA in a nonspecific mode that contained two Dam monomers per synthetic duplex, even though the DNA contains a single GATC site. The ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.

Figure 1

Structure-based sequence alignment of the selected Dam MTase orthologs. SWISSPROT database accession numbers are listed in parentheses: bacteriophage T4 (P04392) (T4Dam), Escherichia coli (P00475) (EcoDam), and restriction-modification MTases EcoRV (P04393) and DpnIIA (p04043). In addition, the following sequences are used to derive the invariant (white characters highlighted in black) and the conserved residues (light blue): Salmonella typhimurium (P55893), Serratia marcescens (P45454), Yersinia pestis (Q9F7U9), Vibrio cholerae (Q08318), Neisseria meningitides (Q9X3Y7), M.HindIV (P44431), M.MboIA (p34720), M.CviBI (Q01511) and M.MjaIII (Q58015). The secondary structure of T4Dam is indicated above the sequence with cylinders for helices and arrows for strands. The MTase catalytic domain is green, the TRD yellow and the β-hairpin red. The conserved sequence motifs characteristic of DNA-amino MTases are labeled with roman numerals according to Malone et al . Dashed lines show the unstructured regions: the loop between strands β6 and β7 is disordered in both the binary and ternary structures of T4Dam, as well as in the M.DpnII-AdoMet structure. T4Dam was susceptible to proteolysis near residue 250 within this loop (data not shown).

Figure 2

The binary structure of T4Dam–AdoHcy. (a) Ribbon representation uses the same color scheme and labels of helices and strands as in Figure 1; the AdoHcy is shown in stick model. (b) Two views of GRASP surface representation. The invariant residues (see Fig. 1) are green (catalytic domain), red (β-hairpin) and yellow (helix bundle). T2 and T4Dam differ at three residues: residues 20 (T4 serine, T2 proline) and 188 (T4 aspartate, T2 glutamate) are colored in blue, residue 26 (T4 asparagine, T2 aspartate) is on a different surface in both views. Single or pair-wise mutations (T4 S20P, T4 N26D or both) converted T4Dam to a T2Damlike enzyme with a higher k_cat, whereas T4 D188E showed no change in enzyme activity. (c) A stereo view of the AdoHcy binding site. The balland- stick representation of AdoHcy is superimposed with a simulated-annealing omit map (F_o – F_c) that contoured at 2.5σ. The hydrogen bonds are shown as dashed lines. (d) Closed pocket (T4Dam) and open pocket (M.DpnII) of the cofactor binding site. The bound AdoHcy and AdoMet are in ball-and-stick model with the sulfur atom in green. The region forms the cover in T4Dam, which includes residues 175–203 (green) after motif IV. The corresponding region in M.DpnII (amino acids 195–212 in red) has a five-residue insertion that is unstructured. (e) A stereo view of the superposition of the active site of T4Dam-AdoHcy (yellow) and M.TaqIDNA (blue) complexes. Two water molecules (red balls) form hydrogenbonds with Asp171 and the backbone carbonyl of Pro172 (wat1) and Tyr181 (wat2). The target adenine-N6 amino group occupies the site of wat1.

Figure 3

The loose ternary structure of T4Dam–DNA–AdoHcy. (a) The 12-bp DNA (one strand in blue and the other in red) is stacked head-to-end forming a pseudo long duplex along the crystal a axis with the length of ~40 Å. Molecule A binds to a single DNA molecule, whereas molecule B binds at the joint of two DNA molecules. (b) T4Dam-phosphate interactions. The same regions (residues 129–134) of molecules A and B are shown relative to a single DNA molecule. (c) Schematic diagram of the protein-phosphate interactions. Residues making interactions are black for molecule A and red for molecule B. The asterisks indicate residues from a neighboring symmetry-related molecule.