Crystallization of mutated enzymes potentially involved in coupling of endonuclease and translocase functions in EcoR124I

Tatsiana Baikova, Mikalai Lapkouski, Ivana Kuta-Smatanova, Rudiger Ettrich and Eva Csefalvay

Department of Structure and Function of Proteins, Institute of Systems Biology and Ecology ASCR and Institute of Physical Biology USB, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic

Restriction and modification (R-M) systems provide bacteria with protection against infection by DNA-based bacteriophage. R-M classified on three distinct types based on their composition and cofactor requirements, the nature of their target sequence, and the position of the site of DNA cleavage with respect to the target sequence. The type I R-M enzymes are sophisticated and differ significantly from type II enzymes commonly used as molecular biology reagents. RE Type I are hetero-oligomeric enzymes composed from 5 subunits, which are responsible for specificity (HsdS), methylation activity (2*HsdM), ATP-dependent DNA translocation and endonuclease activity (2*HsdR). Structure of HsdR subunit of EcoR124I restriction enzymes (RE) has been described recently by our group. The crystal structure of the motor subunit responsible for DNA translocation and cleavage by the type I enzyme EcoR124I, resolved at 2.6 A, shows a lysine residue on the endonuclease domain to contact N3 on the exposed edge of ATP bound at the helicase domains, potentially coupling endonuclease and translocase functions [1]. To prove this prediction the Lys220 was replaced by Arg, Glu, and Ala. Our preliminary crystallization experiments under the conditions used for WT HsdR yielded crystals of only Lys220Arg and microcrystals of Lys220Ala. The aim of our work is to find suitable crystallization conditions for functionally altered enzymes Lys220Ala and Lys220Glu. Obtained crystals will be measured on synchtron to determine their structure. Structural information will be combined with in vivo and in vitro biochemical testing of the altered enzymes, which will give us complete information about accouplement of endonuclease and translocase functions in EcoR124I and allow drawing conclusions valid for type I R-M complexes in general.

[1] Lapkouski M., Panjikar S., Janscak P., Kuta Smatanova I., Carey J., Ettrich R., Csefalvay E. Nat. Struct. & Mol.Biol, 2009, 16, 94.