Study of HincII endonuclease by molecular dynamics methods

J. Fukal¹, P. Kulhánek¹, E. Fadrná¹, J. Koča¹

¹National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno

enigma@chemi.muni.cz

HincII is one of restriction endonucleases which are present in bacterial cells. They work in defense against alien DNAs (e.g. phages). HincII is homotetramer consisting of two functional dimers which recognize sequence GTPyPuAC. Bound oligonucleotide is double-bent and its specific bending is partially caused by intercalating glutamine 138 on each monomer. HincII enzyme needs ions for its function, Mg²⁺ or Mn²⁺ ions work like natural cofactors and Ca²⁺ inhibits the cleavage reaction.

The goals of this study are as follows: a) what is the influence of intercalated glutamine 138, b) what is the role of ions in the active site, c) describe dynamics of HincII complexes. To address these questions, molecular dynamic simulations were performed on pre-reaction complexes of restriction endonuclease HincII. The crystal structures data from work [1] and [2], deposited in PDB database, were used.
The simulated systems consist of wildtype protein or Q138F mutated protein and of substrate 5’-GTCGAC-3’ or 5’-GTTAAC-3’. From crystals containing Ca²⁺ ions were prepared two variations of each: original with Ca²⁺ ions and changed with Mg²⁺ ions. Force field parm99sb [3] with parmbsc0 [4] was used for all of the simulations.

The analyses show the protein is a rigid part and the DNA is flexible part of the complex. The ions’ analyses demonstrate the ions with 2+ formal charge stay almost in their places but some Na⁺ ions changed their positions. The influence of intercalation of amino acids 138 is studied by anisotropic thermal diffusion method (ATD) [5]. ATD method should reveal how the thermal energy is distributed in the system, in this case, which part of system is affected by the intercalated amino acid. The molecular dynamics simulation of DNA molecule is rather problematic because DNA is sensible for imbalances in parameters. Even though the corrected force field was used, it is observed the DNA is forced to the canonical form but still better than containing artefacts. In spite of existing problems of simulations, usable results were obtained.

Acknowledgements

This work has been supported by Ministry of Education of the Czech Republic (MSM0021622413, LC06030). The access to the METACentrum supercomputing facilities provided under the research intent MSM6383917201 is highly appreciated. The research leading to these results has received funding from the European Community's Seventh Framework Programme under grant agreement № 205872.

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