HsdR subunit of the type I restriction-modification enzyme EcoR124I: biophysical characterisation and structural modelling

Abstract

Type I restriction-modification (RM) systems are large, multifunctional enzymes composed of three different subunits. HsdS and HsdM form a complex in which HsdS recognizes the target DNA sequence, and HsdM carries out methylation of adenosine residues. The HsdR subunit, when associated with the HsdS-HsdM complex, translocates DNA in an ATP-dependent process and cleaves unmethylated DNA at a distance of several thousand base-pairs from the recognition site. The molecular mechanism by which these enzymes translocate the DNA is not fully understood, in part because of the absence of crystal structures. To date, crystal structures have been determined for the individual HsdS and HsdM subunits and models have been built for the HsdM-HsdS complex with the DNA. However, no structure is available for the HsdR subunit. In this work, the gene coding for the HsdR subunit of EcoR124I was re-sequenced, which showed that there was an error in the published sequence. This changed the position of the stop codon and altered the last 17 amino acid residues of the protein sequence. An improved purification procedure was developed to enable HsdR to be purified efficiently for biophysical and structural analysis. Analytical ultracentrifugation shows that HsdR is monomeric in solution, and the frictional ratio of 1.21 indicates that the subunit is globular and fairly compact. Small angle neutron-scattering of the HsdR subunit indicates a radius of gyration of 3.4 nm and a maximum dimension of 10 nm. We constructed a model of the HsdR using protein fold-recognition and homology modelling to model individual domains, and small-angle neutron scattering data as restraints to combine them into a single molecule. The model reveals an ellipsoidal shape of the enzymatic core comprising the N-terminal and central domains, and suggests conformational heterogeneity of the C-terminal region implicated in binding of HsdR to the HsdS-HsdM complex.

Figure 1

(a) SDS-PAGE. Purified HsdR was run on an SDS/12.5 % polyacrylamide gel and stained with Coomassie brilliant blue. The marker lane is labelled accordingly. (b) Dynamic light-scattering measurements of HsdR at 4.5 μM and 10 °C. (c) The published hsdR-gene sequence. (d) The revised hsdR-gene sequence. DNA sequencing revealed an additional nucleotide (red). The resulting frame-shift alters the translated amino acid sequence beyond this point and produces a new stop codon. The published stop codon is shown in yellow, and the new stop codon is shown in cyan. The revised C-terminal amino acid sequence of the R subunit is shown in blue.

Figure 2

Sedimentation velocity of HsdR. (a) Data fitted from a run at 40,000 rpm (in an An50 Ti rotor) at a protein concentration of 4.5 μM, scanning at 280 nm and 10 °C, together with the corresponding residuals. For clarity, scans are shown for every fourth scan measured. (b) and (c) The associated c(S) and c(M) distribution plots.

Figure 3

SANS data for HsdR in 100% H₂O. (a) The points shown correspond to the experimental data; the lines represent the theoretical scattering curves calculated from the p(r) function (continuous blue line) and from the ab initio model. (broken red line). (b) Distance distribution function calculated from the experimental scattering curve.

Figure 4

Low resolution dummy atom model for the HsdR subunit obtained from ab initio modelling of the SANS data. (a), (b) and (c) Three mutually perpendicular views of the structure.

Figure 5

Sequence alignment of the EcoR124I HsdR and its homologs and the templates used for modelling the NTD, RecA-I and RecA-II subdomains. Similar amino acids are coloured according to the physico-chemical properties of their side-chains: negatively charged, red, positively charged, blue, polar, magenta, hydrophobic, green. Sequences are named according to nomenclature from REBASE or PDB codes. Numbers in parentheses indicate how many amino acid residues have been omitted for the sake of clarity. Amino acid residues of the NTD that are predicted to form a catalytic site and residues of the central domain that are predicted to be involved in ATP binding are indicated above the alignment by an asterisk (*). Putative DNA-binding residues are indicated by a hash mark (#). The secondary structure of the EcoR124I domains derived from the model using the DSSP program is shown above the EcoR124I sequence. Secondary structure of the templates: 1hh1 and 2db3 shown below the sequences. β-Strands are shown as arrows and helices are shown as cylinders.

Figure 6

Model of the NTD of EcoR124I HsdR. (a) Model coloured according to MetaMQAP evaluation (well-scored regions are coloured blue, poorly scored regions are coloured red). (b) Residues predicted to be functionally important. The residues predicted to form the active site are shown in blue. The putative DNA-binding residues are shown in green. Two magnesium ions are shown as pink spheres and the DNA molecule is shown as grey lines. The protein chain is coloured according to secondary structure (red, helices; yellow, β-strands; grey, loops).

Figure 7

Model of the central domain of EcoR124I HsdR. (a) Model coloured according to MetaMQAP evaluation (well-scored regions are coloured blue, poorly scored regions are coloured red). (b) Active site of the central domain: ATP moiety (cyan) and magnesium ion (pink) coordinates copied from 2db3 structure. The residues predicted to be involved in ATP-binding (shown in blue) are located in a cleft between two NTPase domains. The model is coloured according to secondary structure (red, helices; yellow, β-strands; grey, loops). (c) Electrostatic potential mapped onto the molecular surface of the central domain (positively and negatively charged regions are coloured in blue and red, respectively). (d) Putative DNA-binding residues (coloured green).

Figure 8

Determination of the structure of the entire HsdR subunit against SANS data. NTD (blue), central domain (green) and coiled-coil regions (red) are shown as cartoons, the linker between NTD and central domain (cyan) and CTD (magenta) are shown in a low-resolution dummy atom model. The ATP molecule is shown in yellow. (a) Three representative reconstructions made using homology models of NTD and central domains with (a1) χ = 1.51 (a2) χ = 1.54 (a3) χ = 1.59. (b) Three representative reconstructions made using homology models of NTD, central domain and coiled-coil regions of CTD (b1) χ = 1.41 (b2) χ = 1.46 (b3) χ = 1.49. (c) Domain architecture of the EcoR124I HsdR subunit.

Supplementary

Supplementary

Supplementary