Structure and function of the C-terminal helical domain of the motor subunit HsdR from the type I restriction-modification system EcoR124I

Pavel Grinkevich^1,2, Amanda Li³, Tatsiana Baikova^1,2, Mikalai Lapkouski¹, Alena Guzanova⁴, Eva Csefalvay^1,2, Marie Weiserova⁴, Rudiger Ettrich^1,2

¹Institute of Nanobiology and Structural Biology of GCRC, Academy of Sciences of the Czech Republic, Nove Hrady, Czech Republic

²Faculty of Sciences, University of South Bohemia, Czech Republic

³Chemistry Department, Princeton University, Princeton, New Jersey, USA

⁴Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

The published structure of the HsdR subunit of EcoR124I [1] suggested the motor subunit to be a planar array of four functionally integrated domains, with the fourth, C-terminal domain being all helical and implied to play a role in complex assembly and/or DNA binding. However, the last 150 amino acids if this domain are unresolved in the crystal structure. A single point mutation lead to a new crystal structure that allowed to trace the backbone of the unresolved C-terminal residues, and homology and energetic modeling was applied to generate an all-atom 3-D model of wild-type HsdR and complemented by in vivo and in vitro studies to establish the function of the helical domain. In vitro DNA cleavage assays, gel mobility shift assays and in vivo restriction tests were performed on WT and mutant HsdR with selectively deleted parts of the helical domain. Our results strongly support the suggested role in subunit interaction and demonstrate the importance of the C-terminus in complex assembly.

1. Lapkouski M., Panjikar S., Janscak P., Kuta Smatanova I., Carey J., Ettrich R., Csefalvay E. (2009) Structure of the motor subunit of type I restriction-modification complex EcoR124I. Nat Struct Mol Biol. Vol 16, pp. 94-95.

We gratefully acknowledge support from the Czech Science Foundation (P207/12/2323). Additionally, Pavel Grinkevich was supported by the University of South Bohemia, GAJU 141/2013/P.