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.

• Klíčová slova
• C-terminal domain, DNA binding protein, DNA endonuclease, EcoR124, Escherichia coli (E. coli), GFP fusion, HsdR, X-ray crystallography, crystal structure, restriction-modification,
• MeSH
• biofyzikální jevy MeSH
• DNA vazebné proteiny chemie   genetika   MeSH
• Escherichia coli chemie   genetika   MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• multiproteinové komplexy chemie   genetika   MeSH
• podjednotky proteinů chemie   genetika   MeSH
• proteinové domény genetika   MeSH
• proteiny z Escherichia coli chemie   genetika   MeSH
• restrikční endonukleasy typu I chemie   genetika   MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• endodeoxyribonuclease EcoR124I MeSH Prohlížeč
• HsdR protein, E coli MeSH Prohlížeč
• multiproteinové komplexy MeSH
• podjednotky proteinů MeSH
• proteiny z Escherichia coli MeSH
• restrikční endonukleasy typu I MeSH