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Comparative Study
. 2000 Oct 15;28(20):3950-61.
doi: 10.1093/nar/28.20.3950.

Structure of RsrI methyltransferase, a member of the N6-adenine beta class of DNA methyltransferases

R D Scavetta  1 , C B Thomas, M A Walsh, S Szegedi, A Joachimiak, R I Gumport, M E Churchill
Affiliations
Comparative Study

Structure of RsrI methyltransferase, a member of the N6-adenine beta class of DNA methyltransferases

R D Scavetta et al. Nucleic Acids Res. .

Abstract

DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction-modification systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction endonucleases afforded by the bacterial methyltransferases. The first structure of an N:6-adenine methyltransferase belonging to the beta class of bacterial methyltransferases is described here. The structure of M. RSR:I from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 A resolution using X-ray crystallography. Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The catalytic core most closely resembles the PVU:II methyltransferase, a cytosine amino methyltransferase of the same beta group. The larger nucleotide binding pocket observed in M. RSR:I is expected because it methylates adenine. However, the most striking difference between the RSR:I methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.

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Figures

Figure 1
Figure 1
Proposed catalytic mechanism of amino methylation by DNA methyltransferases.
Figure 2
Figure 2
Stereo view of a representative section of the experimental electron density map contoured at 1.4σ at 1.75 Å resolution. The model in the catalytic region near the DPPY motif and the DPPY motif itself are superimposed on the map. Phase information from the MAD SeMet M·RsrI data was used to extend the phases to the resolution of the 1.75 Å native data. This figure was generated using SETOR (56) and Photoshop (Adobe).
Figure 3
Figure 3
Ribbon diagram of M·RsrI. The catalytic domain containing the methylase fold is to the left of the central V-shaped cleft and the TRD domain is to the right. Rainbow coloring from blue through green to red indicates the N- to C-terminal position of the residues in the model. The helices are lettered and β-strands numbered in white and other residue positions are labeled in black. The catalytic DPPY motif is labeled in blue, indicating its position at the base of the loop that contains helix D1. The diagram was made using MOLSCRIPT (57), Raster3D (58) and Photoshop (Adobe).
Figure 4
Figure 4
Diagram of secondary structure profiles of six methyltransferase structures, M·RsrI, M·PvuI, M·DpnM, M·TaqI and M·HhaI/M·HaeIII, aligned based on the common structural elements. M·HhaI and M·HaeIII can be represented by one diagram. All six MTases have a core structure similar to that represented by M·HhaI, with the motif positions illustrated at the top of the diagram. Variations among the MTases include additional secondary structural elements located in loop regions. The catalytic X1PPX2 location is denoted by labeled amino acids. Dashed lines represent regions of the structures that were not modeled. The starred positions in the M·RsrI schematic diagram represent the locations of mutations L72P and D173A. This diagram was made using ShowCase.
Figure 5
Figure 5
The 5′-MTA contacts within the M·RsrI active site. Stereo view of the 5′-MTA (navy) in an Fo – Fc electron density map (red) contoured at 1.0 σ, which was produced by omitting the 5′-MTA from the model during simulated annealing refinement and map calculations. The surrounding residues contact the 5′-MTA and apparent hydrogen bonds are shown as green lines. This figure was generated using SETOR (56) and Photoshop (Adobe).
Figure 6
Figure 6
Electrostatic surface potential of the four amino methyltransferases. This view of the V-shaped cleft separating the catalytic domain (on the right) from the TRD (on the left) is rotated about the vertical axis by ∼180° and tilted toward the viewer by ∼90° with respect to the view in Figure 3. The structures have been aligned as described in Materials and Methods. Cofactors are included in several of the diagrams. Purple/bue shading represents positive potential and red negative potential, based on surface potentials calculated using GRASP (51).
Figure 7
Figure 7
Comparison of M·RsrI and M·PvuII. (A) Alignment of M·RsrI and M·PvuII sequences on the basis of common structural elements. The secondary structure designations of α for α-helix, β for β-strand and 3 for 310-helix indicate the positions of these secondary structure elements in M·RsrI. The M·RsrI sequence numbering is directly below its sequence. The regions that differ significantly between M·RsrI and M·PvuII are underlined and those that are not represented in the structures are also italicized. The detailed structural assignments and loop assignments are available as Supplementary Material (Tables S1 and S2). (B) Stereo diagram of the superimposed M·PvuII (magenta) and M·RsrI (aqua) structures. The catalytic DPPY motif of M·RsrI is illustrated in yellow and numbering in black for M·RsrI and magenta for M·PvuII indicates particular regions discussed in the text. The view is similar to that in Figure 3. (C) Close-up view of the X1PPX2 motif aligned only on X1PPX2, using the same color scheme. The diagrams were made using MOLSCRIPT (57), Raster3D (58) and Photoshop (Adobe).
Figure 8
Figure 8
Diagram of M·RsrI showing the interactions of residues affecting DNA binding and catalytic activity of M·RsrI. (A) The loop from residue 50 to 90 is shown with relevant side chains. Leu72 is located at the tip of this loop that contains the catalytic residues, DPPY. (B) The large protruding arm is shown with side chains in a view similar to Figure 3. Hydrogen bonding/ion pair interactions are shown by the gray lines. Asp173 is involved in a network of electrostatic interactions that appear to stabilize the observed structure of the protruding arm. The diagrams were made using MOLSCRIPT (57), Raster3D (58) and Photoshop (Adobe).
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