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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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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
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.
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
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
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
Molecular Dynamics – no DNA only protein DNA removed with DNA (150 ns) no DNA
Molecular Dynamics – no DNA only protein DNA removed d1
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
Principal Component Analysis Fluctuation matrix Diagonalization no DNA MTD Projections mode #1 mode #2
PCA Projections no DNA MTD 0 ns (start) 16 ns (end)
Opened Enzyme closed with DNA 7Å Average distance 16 Å
Electrostatic potential – "opened" negative positive
Electrostatic potential – "closed" negative positive
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.
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
Donor/Acceptor Structures Cb 19 Å Alexa594
Donor/Acceptor Dynamics E153C "closed" "open" protein restrained implicit solvent 600 K E290C
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
Acknowledgements • Financial support • CEITEC • GACR • Computational resources • MetaCentrum • CERIT-SC