1. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -1- 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 2 CEITEC – Central European Institute of Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic Study of BsoBI Endonuclease By Molecular Dynamics

2. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -2- BsoBI Nuclease  type II restriction endonuclease from Geobacillus stearothermophilus  optimal cleavage at 65 ˚C  recognizes short palindromatic sequence C|PyCGPuG  cofactor - 2 Mg 2+ atoms 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.  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 catalytic domains monomer A monomer B DNA

3. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -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. fluorescent probes: acceptor and donor FRET – Fluorescence Resonance Energy Transfer efficiency is proportional to acceptor-donor distance single molecule level (diluted solution) Proposed conformational changes

4. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -4- Motivation  find structure of "opened" state(s)  find pathways how DNA enters to the active site  confirm FRET experiments  find structure of pre-reaction complex

5. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -5- Molecular Dynamics - complex  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 protein + DNA

6. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -6- Molecular Dynamics – no DNA only protein DNA removed with DNA (150 ns) no DNA

7. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -7- Molecular Dynamics – no DNA only protein DNA removed d1

8. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -8- Metadynamics history dependent biasing potential converge to the free energy reaction (collective) variables 1 CV – distance between catalytic domains  pmemd v10/PMFLib v4.0  h=0.1 kcal/mol   =0.5 Å  t s =2 ps  CPU only Laio, A.; Parrinello, M. P. Natl. Acad. Sci. USA 2002, 99, 12562–12566.

9. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -9- MTD - Collective Variable Evolutions

10. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -10- MTD - Free Energy Estimates

11. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -11- Principal Component Analysis no DNA MTD Index EigenValue Å2Å2 15390.5 2599.9 3353.7 4177.0 5123.1 682.1 771.3 843.3 929.3 1027.7 Fluctuation matrix Diagonalization Projectionsmode #1mode #2

12. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -12- PCA Projections 0 ns (start) 16 ns (end) no DNA MTD

13. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -13- Opened Enzyme Average distance 16 Å closed with DNA 7Å

14. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -14- 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.

15. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -15- 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

16. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -16- Donor/Acceptor Structures Alexa594 CC 19 Å

17. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -17- Donor/Acceptor Dynamics E153C E290C "closed""open" protein restrained implicit solvent 600 K

18. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -18- Conclusions  Main outcomes

19. MIB VI, 16 th – 19 th September 2013, Mariánské lázně -19- Acknowledgements  Financial support  CEITEC  GACR  Computational resources  MetaCentrum  CERIT-SC