Crystallization and structure-functional analysis of mutated motor subunits of type I restriction enzyme EcoR124I

Tatsiana Baikova^1,2, Mikalai Lapkouski¹, Santosh Panjikar³, Ivana Kuta-Smatanova^1,2, Rudiger Ettrich^1,2 and Eva Csefalvay^1,2

¹Institute of Nanobiology and Structural Biology of ISBE, AS CR, 37333 Nove Hrady, Czech Republic

²University of South Bohemia in České Budějovice, 37333 Nove Hrady, Czech Republic

³EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D22603 Hamburg, Germany

Invasion of foreign DNA into microorganisms can be prevented by restriction–modification (R-M) systems. Though Type I R-M enzymes are not site-specific endonucleases they remain an important area of research mainly because of their unusual biochemical properties and their unique mechanisms of DNA translocation and restriction. The crystal structure of the motor subunit, responsible for translocation and restriction, of the type I enzyme EcoR124I , resolved at 2.6 A, shows a square-planar arrangement of globular domains with a prominent cleft that accommodates DNA extending from the two canonical helicase domains to the endonuclease active site [1]. Unexpectedly, ATP bound in the helicase cleft is also engaged by Lys220 of the nuclease domain, potentially coupling the nuclease and translocation activities. To shed light on the coupling mechanism, mutants were designed that should alter the coupling at this position by influencing DNA cleavage but leaving the ATPase activity of EcoR124I unaltered. Three-dimensional structural information is essential to complement experimental data in a way, that lets us draw conclusions about the molecular mechanism. We discuss mutations of Lys220 to Ala, Arg and Glu, and the successfully solved crystals structures of the Arg220 and Glu220 mutants, that we determined to 2.74 A (PDB 2w74) and 2.99A resolution (PDB 2y3t), respectively.

Data collection of Glu220 crystal was performed on beamline X11 at EMBL/DESY (Hamburg). Data from 451 images were integrated and scaled with XDS and XSCALE. The motor subunit Glu220 crystals belong to space group P21 and unit cell dimensions are a=127.11 A, b=123.11 A, c=160.11 A, and β=111.48°. The structure was solved by molecular replacement using Auto-Rickshaw19 and MOLREP programs, and using our previously solved wild type structure (PDB 2w00) as the search model.

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

We gratefully acknowledge support from the Ministry of Education, Youth and Sports of the Czech Republic (MSM6007665808, LC06010), the Academy of Sciences of the Czech Republic (AVOZ60870520), the Grant Agency of the Czech Republic (Nos. P207/10/1934), and joint Czech - US National Science Foundation International Research Cooperation (ME09016 and INT03-09049), Additionally, T.B. was supported by the University of South Bohemia, grant GAJU 170/2010/P.