Shape and subunit organisation of the DNA methyltransferase M.AhdI by small-angle neutron scattering.
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 M2S2and 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 of1H: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.
Callow
P
P
EPSAM and ISTM Research Institutes, Keele University, Staffordshire ST5 5BG, UK; ILL-EMBL Deuteration Laboratory, Partnership for Structural Biology, Institut Laue Langevin, 38042 Grenoble Cedex 9, Grenoble, France.
Sukhodub
A
A
Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, PO1 2DT, UK.
Taylor
James E N
JEN
Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, PO1 2DT, UK.
Kneale
G G
GG
Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, PO1 2DT, UK. Electronic address: geoff.kneale@port.ac.uk.
D016980:Aeromonas hydrophila / Q000201:enzymology*; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D015254:DNA Modification Methylases / Q000737:chemistry*; D006865:Hydrogenation; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D033363:Neutron Diffraction; D021122:Protein Subunits / Q000737:chemistry*; D053838:Scattering, Small Angle; D016415:Sequence Alignment
D021122:Protein Subunits; D015254:DNA Modification Methylases