Study of BSOBI Endonuclease by Molecular Dynamics

I. Kabelka², J. Štěpán^1,2, J. Koča^1,2 and P. Kulhánek^1,2

¹CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

²National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic

BsoBI is a type II restriction endonuclease belonging to the EcoRI family. The enzyme was isolated from the bacteria Geobacillus stearothermophilus and crystallized in complex with short DNA duplex [1]. The interesting discovery about this homodimeric enzyme was the formation of long tunnel for DNA binding and recognition. The DNA molecule is completely encircled inside the tunnel. Considering the fact that BsoBI does not break up into monomers while is still capable to cleave circular DNA molecules, some conformational change is needed to enable the DNA binding. The most probable scenario is the separation of two domains, which would cause a formation of a gap large enough to enable the DNA get inside the active site. This scenario is supported by experimental evidence indicating significant conformational changes during the DNA binding [2].

In our project we have been focusing on investigating the process of opening of the BsoBI enzyme using molecular dynamics simulations. Our starting point was the crystallographic structure of the enzyme in complex with short DNA molecule [1]. We have divided the project into two parts: a) the study of the BsoBI-DNA complex and b) the study of the unbound enzyme. Because there is no experimental structure for free enzyme it was prepared artificially by the removal of the DNA molecule from the crystallographic structure. The sampling of opening process was improved by accelarated molecular dynamics employing Metadynamics [3] approach. This method enabled us to model proposed conformational changes and qualitatively evaluate associated free energy differences among various stable protein conformations.

This work was realized in CEITEC - Central European Institute of Technology with research infrastructure supported by the project CZ.1.05/1.1.00/02.0068 financed from European Regional Development Fund. The access to the MetaCentrum computing facilities provided under the program "Projects of Large Infrastructure for Research, Development, and Innovations" LM2010005 funded by the Ministry of Education, Youth, and Sports of the Czech Republic is acknowledged. The access to the CERIT-SC computing facilities provided under the programme Center CERIT Scientific Cloud, part of the Operational Program Research and Development for Innovations, reg. no. CZ. 1.05/3.2.00/08.0144 is appreciated.

[1] M.J. van der Woerd, J.J. Pelletier, S. Xu, A.M. Friedman, Structure, 9, (2001), 133–144.

[2] J. Dikić , http://geb.uni-giessen.de/geb/volltexte/2009/7301, (2009)

[3] A. Laio, M. Parrinello, P. Natl. Acad. Sci. USA , 99, (2002), 12562–12566.