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Physica B 385–386 (2006) 853–855

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Preliminary neutron scattering studies of the Type I restriction-modification enzyme M.Ahdl

Philip Callowa,�, Peter Timminsb, Geoff Knealec

aDeuteration Laboratory, ILL, Grenoble, France/Keele University, UKbLarge Scale Structures Group, ILL, Grenoble, FrancecBiophysics Laboratories, Portsmouth University, UK

Abstract

Type I restriction-modification (R-M) systems encode multisubunit/multidomain enzymes. Two genes (M and S) are required to form

the 160 kDa methyltransferase that methylates a specific base within the recognition sequence and protects DNA from cleavage by the

endonuclease. Small angle neutron scattering (SANS) revealed an unusually large structural change in the EcoR124I methyltransferase

following DNA binding; this involves a major repositioning of the subunits of the enzyme, resulting in a 60 A reduction in the dimensions

of the enzyme on forming a complex with DNA. The related methyltransferase M.Ahdl, with stoichiometry M2S2 has been prepared in

two protonated/deuterated states (S and M subunits protonated, S deuterated and M protonated) for which SANS data have been

collected in a number of H:D solvent contrasts. The contrast match point of the selectively deuterated enzyme confirms the successful

reconstitution of the enzyme with deuterated S subunits. Ab initio shape determination using this contrast matched data is in progress to

determine the subunit organization of M.Ahdl and the large change in structure that occurs on DNA binding.

r 2006 Elsevier B.V. All rights reserved.

PACS: 61.12.Ex

Keywords: SANS; Restriction modification; Contrast variation; Selective deuteration

1. Introduction

Type I restriction-modification (R–M) systems encodemultisubunit/multidomain enzymes that recognise anasymmetric bipartite DNA sequence [1,2]. Type I systemscomprise 3 genes, one for each of the subunits (S, M and R)that are responsible for specificity, methylation andrestriction, respectively. Two genes (M and S) are requiredto form the trimeric 160 kDa methyltransferase (MTase),M2S, that methylates a specific base within the recognitionsequence and protects the DNA from cleavage by theendonuclease [3,4]. Sequence specificity is conferred by thetwo target recognition domains (TRDs) of the S subunit,each binding a half-site within the DNA recognitionsequence [5,6]. The corresponding endonuclease is a

e front matter r 2006 Elsevier B.V. All rights reserved.

ysb.2006.05.124

ng author.

ss: callow@ill.fr (P. Callow).

pentameric enzyme, formed from the MTase by theaddition of two R subunits to form a complex ofstoichiometry R2M2S with a typical Mr of around400 kDa [7–9]. Small angle X-ray scattering revealed anunusually large structural change in the EcoR124I methyl-transferase following DNA binding [10] resulting in a 60 Areduction in the dimensions of the enzyme (and a decreasein Rg from 56 to 40 A) on forming a complex with DNA.The related MTase, M.Ahdl, differs from a typical Type

I enzyme in having the TRDs on separate subunits [11],with the stoichiometry M2S2. As both the M and S subunitsare soluble in this system, this allows reconstitution of theenzyme from separately expressed subunits, which cantherefore be differentially deuterated.In order to determine the arrangement of the subunits of

the methyltransferase (and their rearrangement on DNAbinding), M.Ahdl has been prepared in two hydrogenated/deuterated states for small angle neutron scattering

ARTICLE IN PRESSP. Callow et al. / Physica B 385–386 (2006) 853–855854

(SANS) experiments, the first as a fully hydrogenatedenzyme, the second with the M subunit hydrogenated andthe S subunit perdeuterated. By varying the H:D content ofthe solvent the selectively labelled subunits can be contrastmatched and scattering data collected for the individualsubunits in situ in the methyltransferase complex.

Here we compare the scattering length densities, andthus the contrast match point, for the fully hydrogenatedand selectively perdeuterated M.Ahdl enzyme.

2. Materials and methods

The M and S subunits of the M.Ahdl were overexpressedin BL21(DE3) grown on Enfors minimal medium using anInfors fermentation system at 30 1C. Glycerol was used asthe carbon source, for the hydrogenated protein h8-glycerol was used and d8-glycerol was used for theexpression of the perdeuterated S subunit. H2O was alsoreplaced with D2O (provided by the ILL) for theperdeuteration of the protein. Purification was performedas described by Marks et al. [11]. Data was collected usingthe D22 diffractometer at the ILL using a range ofD2O:H2O solvent contrasts.

3. Results

The scattering curves collected for both the fullyhydrogenated and deuterated enzymes were fitted usingthe Guinier approximation [12] giving a radius of gyrationand intensity at zero angle (I0). A plot of scatteringamplitude (scattering amplitude ¼ O(I0/tcl); where I0 isintensity at zero angle; t is transmission of sample; c isconcentration (mg/ml) and l ¼ path length (mm)) againstthe percentage of D2O present in the sample buffer givesthe contrast match point for the hydrogenated andselectively perdeuterated M.Ahdl where scattering ampli-tude is zero (see Fig. 1).

For the fully hydrogenated M.Ahdl enzyme the contrastmatch point is where the D2O:H2O ratio is 41% and for the

Fig. 1. A plot of scattering amplitude against the percentage of D2O in the

solvent. } ¼ data for hydrogenated M.Ahdl; J ¼ data for selectively

perdeuterated M.Ahdl. The solid and dashed line represent the best fit to

the experimental data.

selectively perdeuterated M.Ahdl complex the value is56%. These values correspond to a scattering lengthdensity (r) of 2.3� 10�6 A�2 for the hydrogenated proteinand 3.3� 10�6 A�2 for the selectively perdeuteratedsample.

4. Discussion

For the selectively labelled M.Ahdl the S subunit isperdeuterated, which makes up approximately 30% of thetotal mass of the protein. A scattering length density valueof 3.3� 10�6 A�2 shows that the deuterated S subunit has ascattering length density of 5.6� 10�6 A�2 [ ¼ (2.3�10�6� 0.7)+(5.6� 10�6� 0.3)]. This means that aD2O:H2O ratio of 89% in the solvent buffer will contrastmatch the deuterated S subunit, allowing data to becollected for the hydrogenated M subunits only.It is clear from the experimental data that scattering

curves measured where there is 41% D2O in the solvent willonly record scattering resulting from the perdeuterated Ssubunit of the M.Ahdl methyltransferase. Through ab inito

shape determination methods [13] this is allowing us todetermine the position of the S subunit, with respect to theM subunits, within the M.Ahdl methyltransferase enzyme.These results are providing novel information about the

relationship between the S and M subunits of thisimportant R–M system. The modelling work currently inprogress using these new results indicate that the S subunitis located at the centre of the R–M complex (as has beensuggested by other biophysical studies [14]) and appears toact as a ‘hinge’ for the repositioning of the M subunits,thus explaining the large change in dimensions of thecomplex, on DNA binding.

Acknowledgements

The authors gratefully acknowledge the assistance ofAndrey Sukhodub during sample preparation, the ILL foruse of the D22 diffractometer and the EPSRC andWellcome Trust for funding.

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