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. 2007 May 25;369(1):177-185.
doi: 10.1016/j.jmb.2007.03.012. Epub 2007 Mar 14.

Shape and subunit organisation of the DNA methyltransferase M.AhdI by small-angle neutron scattering

P Callow  1 , A Sukhodub  2 , James E N Taylor  2 , G G Kneale  3
Affiliations

Shape and subunit organisation of the DNA methyltransferase M.AhdI by small-angle neutron scattering

P Callow et al. J Mol Biol. .

Abstract

Type I restriction-modification (R-M) systems encode multisubunit/multidomain enzymes. Two genes (M and S) are required to form the methyltransferase (MTase) that methylates a specific base within the recognition sequence and protects DNA from cleavage by the endonuclease. The DNA methyltransferase M.AhdI is a 170 kDa tetramer with the stoichiometry M2S2 and has properties typical of a type I MTase. The M.AhdI enzyme has been prepared with deuterated S subunits, to allow contrast variation using small-angle neutron scattering (SANS) methods. The SANS data were collected in a number of 1H:2H solvent contrasts to allow matching of one or other of the subunits in the multisubunit enzyme. The radius of gyration (Rg) and maximum dimensions (Dmax) of the M subunits in situ in the multisubunit enzyme (50 A and 190 A, respectively) are close of those of the entire MTase (51 A and 190 A). In contrast, the S subunits in situ have experimentally determined values of Rg=35 A and Dmax=110 A, indicating their more central location in the enzyme. Ab initio reconstruction methods yield a low-resolution structural model of the shape and subunit organization of M.AhdI, in which the Z-shaped structure of the S subunit dimer can be discerned. In contrast, the M subunits form a much more elongated and extended structure. The core of the MTase comprises the two S subunits and the globular regions of the two M subunits, with the extended portion of the M subunits most probably forming highly mobile regions at the outer extremities, which collapse around the DNA when the MTase binds.

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Figures

Figure 1
Figure 1
(a) SANS data for hydrogenated M.AhdI in 100% 2H2O (green), and for M.AhdI with the S subunit deuterated/M subunit hydrogenated in 100% 2H2O (blue) and in 40% 2H2O (red). The continuous lines are the model fits resulting from ab initio shape determination using DAMMIN. (b) Distance distribution functions calculated for the SANS data using GNOM.
Figure 2
Figure 2
Low-resolution ab initio models for M.AhdI and for its subunits in situ derived from contrast variation SANS experiments. Views for each structure correspond to 90° rotations about the vertical axis.
Figure 3
Figure 3
Comparison of ab initio models for M.AhdI subunits in situ with structures of related subunits determined by X-ray crystallography. (a) Dimer of the M subunits of AhdI (pale blue) and EcoKI (dark blue where structures overlap). (b) Dimer of the S subunits of AhdI (pale red) and the monomeric S subunit of M. jannaschii (dark red where structures overlap).
Figure 4
Figure 4
Sequence alignments of the M subunits of AhdI and EcoKI, and the S subunits of AhdI and M. jannaschii. (a) Alignment of the M subunits of AhdI and EcoKI. (b) Alignment of a dimer of AhdI S subunits and a monomer of the S subunit of M. jannaschii. The coiled-coil regions corresponding to the central conserved region (CCR, dark green) and distal conserved region (DCR, light green) are indicated, based on the crystal structure of the M. jannaschii S subunit. Amino acid sequences in both cases were aligned using Blast2seq, and identical or similar amino acid residues are colored according to the ClustalX color scheme using Jalview.
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