Active site dynamics of the HhaI methyltransferase: insights from computer simulation

A molecular dynamics study was performed on the DNA methyltransferase M. Hha I in a ternary complex with DNA and AdoMet in solution. Methylation involves addition of the Cys81 sulfhydryl anion to the 6-position of Cyt18, followed by a nucleophilic attack of the resultant carbanion at C5 on the AdoMet methyl group. It was found in this simulation that the distances between the sulfhydryl group (SG) of Cys81 to the C6 of Cyt18 (SG-C6) and methyl carbon (CH3) of AdoMet to the C5 of cytosine (CH3-C5) are dependent on the dihedral angle chi (O4'-C1'-N1-C2) of the nucleotide. When the chi angle of Cyt18 is low (< -80 degrees), the SG-C6 and CH3-C5 distances are large. A high chi angle (> -80 degrees) for the target cytosine residue reduces the distances for both SG-C6 and CH3-C5, and the angles formed between the cytosine ring and AdoMet correspond well to values for the transition state structures formed during methylation of cytosine from ab initio calculations. Two possible proton sources for protonation of N3 of the cytosine residue upon formation of the covalent intermediate were found in the simulation. The protonated amine group of AdoMet could provide a proton via a water bridge, or Arg163 could also be the source of the proton for N3 via a water bridge. The simulation provides insights into how the H5 of cytosine could go from the active site into solvent. Conserved residues Asn304 and Gln82 stabilize a water network within the active site of M. Hha I which provides a route for H5 to diffuse into bulk solvent. An initially distant water molecule was able to diffuse into the active site of the enzyme and replace a position of a crystallographic water molecule in close proximity to the C5 of cytosine. The movement of this water molecule showed that a channel exists between Gln82 and the AdoMet in M. Hha I which allows both water and protons to easily gain access to the active site of the enzyme.