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Ivo Kabelka, 1 Jakub Štěpán, 1,2 Jaroslav Koča 1,2 , and Petr Kulhánek 1,2

Study of BsoBI Endonuclease By Molecular Dynamics. Ivo Kabelka, 1 Jakub Štěpán, 1,2 Jaroslav Koča 1,2 , and Petr Kulhánek 1,2 kulhanek@chemi.muni.cz 1 National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic

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Ivo Kabelka, 1 Jakub Štěpán, 1,2 Jaroslav Koča 1,2 , and Petr Kulhánek 1,2

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  1. Study of BsoBI Endonuclease By Molecular Dynamics Ivo Kabelka,1 Jakub Štěpán,1,2 Jaroslav Koča1,2, andPetr Kulhánek1,2 kulhanek@chemi.muni.cz 1National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic 2CEITEC – Central European Institute of Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic

  2. BsoBINuclease • type II restriction endonuclease from Geobacillusstearothermophilus • optimal cleavage at 65 ˚C • recognizes short palindromatic sequence C|PyCGPuG • cofactor - 2 Mg2+ atoms • homodimeric enzyme - both in free form and in complex with DNA (2x323 AA) • capable of cleaving both linear and circular DNA molecules • 20 Å long tunnel for DNA recognition • complete encirclement of DNA helical domains monomer A DNA monomer B catalytic domains Ruan, H.; Lunnen, K. D.; Scott, M. E.; Moran, L. S.; Slatko, B. E.; Pelletier, J. J.; Hess, E. J.; Benner, J., 2nd; Wilson, G. G.; Xu, S. Y. Cloning and sequence comparison of AvaI and BsoBI restriction-modification systems. Mol. Gen. Genet. 1996, 252, 695–699.

  3. Experimental study Dikić, J. The conformational dynamics of BsoBI, analyzed by fluorescence spectroscopy down to the single molecule level. Thesis, University of Justus Liebig, Giessen, 2009. • FRET – Fluorescence Resonance Energy Transfer • efficiency is proportional to acceptor-donor distance • single molecule level (diluted solution) Proposed conformational changes fluorescent probes: acceptor and donor

  4. Motivation • find structure of "opened" state(s) • confirm FRET experiments • find structure of pre-reaction complex • find pathways how DNA enters to the active site

  5. Molecular Dynamics - complex protein + DNA • input structure PDB ID: 1DC1 • pmemd from AMBER v12 • parm99SBbsc0 + TIP3P water • c(NaCl) ~ 0.15 M • 298 K and 1 bar • apo-enzyme (no ion cofactors) • CPU/GPU

  6. Molecular Dynamics – no DNA only protein DNA removed with DNA (150 ns) no DNA

  7. Molecular Dynamics – no DNA only protein DNA removed d1

  8. Metadynamics • history dependent biasing potential • converge to the free energy reaction (collective) variables Laio, A.; Parrinello, M. P. Natl. Acad. Sci. USA2002, 99, 12562–12566. 1 CV – distance between domains • pmemd v10/PMFLib v4.0 • h=0.1 kcal/mol • s=0.5 Å • ts=2 ps • CPU only

  9. Helical Domain Opening

  10. MTD - Collective Variable Evolutions

  11. MTD - Free Energy Estimates

  12. Principal Component Analysis Fluctuation matrix Diagonalization no DNA MTD Projections mode #1 mode #2

  13. PCA Projections no DNA MTD 0 ns (start) 16 ns (end)

  14. Opened Enzyme closed with DNA 7Å Average distance 16 Å

  15. Electrostatic potential – "opened" negative positive

  16. Electrostatic potential – "closed" negative positive

  17. Contradictions Dikić, J. The conformational dynamics of BsoBI, analyzed by fluorescence spectroscopy down to the single molecule level. Thesis, University of Justus Liebig, Giessen, 2009. In the apo-enzyme (no cofactor present) both distances are shorter than in the complex.

  18. Contradictions Dikić, J. The conformational dynamics of BsoBI, analyzed by fluorescence spectroscopy down to the single molecule level. Thesis, Giessen, 2009. In the apo-enzyme (no cofactor present) both distances are shorter than in the complex. MD simulations

  19. Donor/Acceptor Structures Cb 19 Å Alexa594

  20. Donor/Acceptor Dynamics E153C "closed" "open" protein restrained implicit solvent 600 K E290C

  21. Conclusions • opening of catalytic domains confirmed • preliminary structure of "opened" state • mechanical origin of opening (bending of helices connecting catalytic and helical domains) • partial contradictions with FRET experiments

  22. Acknowledgements • Financial support • CEITEC • GACR • Computational resources • MetaCentrum • CERIT-SC

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