Although EcoR124 is one of the better-studied Type I restriction-modification enzymes, it still presents many challenges to detailed analyses because of its structural and functional complexity and missing structural information. In all available structures of its motor subunit HsdR, responsible for DNA translocation and cleavage, a large part of the HsdR C terminus remains unresolved. The crystal structure of the C terminus of HsdR, obtained with a crystallization chaperone in the form of pHluorin fusion and refined to 2.45 Å, revealed that this part of the protein forms an independent domain with its own hydrophobic core and displays a unique α-helical fold. The full-length HsdR model, based on the WT structure and the C-terminal domain determined here, disclosed a proposed DNA-binding groove lined by positively charged residues. In vivo and in vitro assays with a C-terminal deletion mutant of HsdR supported the idea that this domain is involved in complex assembly and DNA binding. Conserved residues identified through sequence analysis of the C-terminal domain may play a key role in protein-protein and protein-DNA interactions. We conclude that the motor subunit of EcoR124 comprises five structural and functional domains, with the fifth, the C-terminal domain, revealing a unique fold characterized by four conserved motifs in the IC subfamily of Type I restriction-modification systems. In summary, the structural and biochemical results reported here support a model in which the C-terminal domain of the motor subunit HsdR of the endonuclease EcoR124 is involved in complex assembly and DNA binding.

• Keywords
• C-terminal domain, DNA binding protein, DNA endonuclease, EcoR124, Escherichia coli (E. coli), GFP fusion, HsdR, X-ray crystallography, crystal structure, restriction-modification,
• MeSH
• Biophysical Phenomena MeSH
• DNA-Binding Proteins chemistry   genetics   MeSH
• Escherichia coli chemistry   genetics   MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Multiprotein Complexes chemistry   genetics   MeSH
• Protein Subunits chemistry   genetics   MeSH
• Protein Domains genetics   MeSH
• Escherichia coli Proteins chemistry   genetics   MeSH
• Deoxyribonucleases, Type I Site-Specific chemistry   genetics   MeSH
• Amino Acid Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• endodeoxyribonuclease EcoR124I MeSH Browser
• HsdR protein, E coli MeSH Browser
• Multiprotein Complexes MeSH
• Protein Subunits MeSH
• Escherichia coli Proteins MeSH
• Deoxyribonucleases, Type I Site-Specific MeSH