MIB VI, 16 th – 19 th September 2013, Mariánské lázně -1- Ivo Kabelka, 1 Jakub Štěpán, 1,2...

MIB VI, 16th – 19th September 2013, Mariánské lázně -1-

Ivo Kabelka,1 Jakub Štěpán,1,2 Jaroslav Koča1,2, and Petr Kulhánek1,2

[email protected]

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

Study of BsoBI EndonucleaseBy

Molecular Dynamics


BsoBI Nuclease type II restriction endonuclease from

Geobacillus stearothermophilus optimal cleavage at 65 ˚C recognizes short palindromatic sequence

C|PyCGPuG cofactor - 2 Mg2+ 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


Experimental studyDikić, 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


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


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


Molecular Dynamics – no DNA

only proteinDNA removed

with DNA(150 ns)

no DNA


Molecular Dynamics – no DNA

only proteinDNA removed

d1


Metadynamics

)),(()(2

2

MTDMTDi tVVdt

dm rξr

r

r

i

i

),(lim)( MTDMTDt

tVGMTD

ξξ

• history dependent biasing potential • converge to the free energy

reaction (collective) variables

MTD it

iMTDMTD ehtV

0

2

)(2

2

),(

1 CV – distance between catalytic domains pmemd v10/PMFLib v4.0 h=0.1 kcal/mol s=0.5 Å ts=2 ps CPU only

Laio, A.; Parrinello, M. P. Natl. Acad. Sci. USA 2002, 99, 12562–12566.


MTD - Collective Variable Evolutions


MTD - Free Energy Estimates


Principal Component Analysis

no DNAMTDIndex EigenValue l

Å2

1 5390.52 599.93 353.74 177.05 123.16 82.17 71.38 43.39 29.3

10 27.7

Ttt ))()()(( xxxxC

Fluctuation matrix

Diagonalization

iii μCμ

Projections

))(.()( xxμ ttp ii

mode #1 mode #2


PCA Projections

0 ns (start)

16 ns (end)

no DNAMTD


Opened Enzyme

Average distance16 Å

closed with DNA7Å


ContradictionsDikić, 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.


ContradictionsDikić, 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

Alexa594

Cb

19 Å


Donor/Acceptor DynamicsE153C

E290C

"closed" "open"

protein restrainedimplicit solvent

600 K


Conclusions Main outcomes


Acknowledgements Financial support

CEITEC GACR

Computational resources MetaCentrum CERIT-SC

MIB VI, 16 th – 19 th September 2013, Mariánské lázně -1- Ivo Kabelka, 1 Jakub Štěpán, 1,2...

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