Shape and Subunit Organisation of the DNA Methyltransferase M.AhdI by Small-angle Neutron Scattering

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 Å and 190 Å, respectively) are close of those of the entire MTase (51 Å and 190 Å). In contrast, the S subunits in situ have experimentally determined values of Rg = 35 Å and Dmax = 110 Å, 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.

HsdR Subunit of the Type I Restriction-Modification Enzyme EcoR124I: Biophysical Characterisation and Structural Modelling

“…A dynamic structure of HsdR agrees with the predicted intrinsic disorder of its C-terminal region and relatively flexible connection between individual domains. These models may serve as a useful platform for the design of experiments aiming at the determination of points of possible interactions between the individual domains within the HsdR subunit, as well as between HsdR and the MTase core; for instance, using residue-specific cross-linking, 50 labelling and fluorescence resonance energy transfer (FRET) or electron paramagnetic resonance (EPR) experiments, 51 or SANS experiments on the multisubunit REase using selective subunit deuteration techniques 10 …”

Structure and operation of the DNA-translocating type I DNA restriction enzymes

“…The structures show EcoKI bound to a DNA duplex or to a DNA mimic anti-restriction protein, and EcoR124I in the absence and presence of a DNA duplex or an antirestriction protein. With the aid of numerous biochemical constraints, known crystallographic structures of type I RM subunits, the location of the HsdS and HsdM within the MTase at low resolution (Callow et al 2007;Taylor et al 2010), and our recent reconstruction of the core MTase structure (Kennaway et al 2009), we can construct a unique arrangement of the subunits within the EM structures, rationalize all previous knowledge about these complex machines, and reveal an evolutionary link between type I and type II RM systems.…”

New sources and instrumentation for neutrons in biology

“…This natural contrast difference between biomaterials is therefore exploited to locate individual components in functional biological structures or assemblies, such as in protein-nucleic acid complexes (see for example [19][20][21][22]). The addition of specific isotope labelling of biological macromolecules further opened the technique to protein-protein complexes that would normally lack contrast between the subunits [23,24].…”

“…For multi-component systems in which the scattering length densities are all similar (e.g. a multi-subunit protein system), selective deuteration allows effective modelling of individual components [17,19,20,23,77,78]. Similar approaches are equally powerful when studying biological membranes with NR [69].…”

“…The impact is also evident in the scientific press. In SANS, two recent examples in which labelling of selected sub-units has allowed quaternary structure to be determined have been published by King et al [85], and by Callow et al [23]. In the former case, selective deuteration of a full set of ternary complexes of troponin has been used to study the large structural change that occurs upon calcium binding and to identify the sub-unit in which this change principally takes place.…”