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.

Prediction of bacterial proteins carrying a nuclear localization signal and nuclear targeting of HsdM from Klebsiella pneumoniae

“…It has been known that a complex of HsdS and HsdM recognizes and methylates 5'-GA m6 A(6N)TGCC-3' or 5'-GGC m6 A(6N)TTC-3' (Lee et al, 1997;Obarska-Kosinska et al, 2008). However, our results showed that rHsdM alone had the ability to transfer a methyl group in eukaryotic DNA.…”

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

“…In contrast to the situation with type II restriction enzymes, it is only very recently that partial atomic structures for type I RM subunits have emerged (Calisto et al 2005;Kim et al 2005;Obarska et al 2006;Obarska-Kosinska et al 2008;Kennaway et al 2009;Lapkouski et al 2009;Uyen et al 2009;Taylor et al 2010;Gao et al 2011), and their mechanisms, which most dramatically involve the translocation of thousands of base pairs of DNA with the formation of supercoiled loops (Yuan et al1980;Endlich and Linn 1985;Studier and Bandyopadhyay 1988;García and Molineux 1999), still present many questions. The type I RM enzymes are complex structures with two HsdR restriction (R) subunits, two HsdM modification (M) subunits, and one HsdS sequence specificity (S) subunit (Murray 2000;Loenen 2003), each with a number of domains (Supplemental Fig.…”

Crystal structure of a novel domain of the motor subunit of the Type I restriction enzyme EcoR124 involved in complex assembly and DNA binding

“…As demonstrated for a related Type I R-M enzyme EcoKI in partial proteolysis experiments, cleaving off 155 terminal residues of HsdR produced a fragment stable in the presence of ATP but unable to bind to MTase (24). De novo modelling of HsdR from EcoR124 supported the notion of the C-terminus actively participating in complex formation, albeit revealing little about its structural organization except for a relatively short coil-coiled fragment and an overall disordered nature (25). Another known crystal structure of HsdR from Vibrio vulnificus also lacks ~220 C-terminal residues and folds into three domains (endonuclease and two RecA-like helicases), providing no further insight into the 3D structure of the C-terminus.…”

“…Within the Type IC family, full sequences are only available for EcoR124 and NgoAV from Neisseria gonorrhoeae (27) that display ~75% sequence identity. An alignment between members of different families was nevertheless achieved in endonuclease and helicase I and II domains due to the presence of clearly defined motifs (25,21). The helical and C-terminal domains exhibit an even lower sequence identity than the rest of the protein, thereby prohibiting a direct identification of conserved regions through multiple pairwise alignment.…”