HsdR subunit of the type I restriction-modification enzyme EcoR124I: biophysical characterisation and structural modelling.
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.
Obarska-Kosinska
Agnieszka
A
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
Taylor
James E N
JEN
Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, PO1 2DT, UK.
Callow
Philip
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.
Orlowski
Jerzy
J
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
Bujnicki
Janusz M
JM
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland. Electronic address: iamb@genesilico.pl.
Kneale
G Geoff
GG
Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, PO1 2DT, UK. Electronic address: geoff.kneale@port.ac.uk.
D000595:Amino Acid Sequence; D001483:Base Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D018388:Codon, Terminator; D004247:DNA / Q000378:metabolism; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry*; D045565:Holliday Junction Resolvases / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D033363:Neutron Diffraction; D010957:Plasmids; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000201:enzymology; D053838:Scattering, Small Angle; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D048229:Sulfolobus solfataricus / Q000201:enzymology; D013698:Templates, Genetic
D018388:Codon, Terminator; D021122:Protein Subunits; D004247:DNA; D045565:Holliday Junction Resolvases; D015253:Deoxyribonucleases, Type I Site-Specific