Ni-Replacement in Zn-Dependent S1 Nuclease

J. Hrubý1,2, P. Kolenko1,2, K. Adámková2,3, B. Husťáková2,3, M. Malý1,2, L.H. Østergaard4, T. Kovaľ2, J. Dohnálek2

1Czech Technical University in Prague, Břehová 7, 115 19 Prague, Czech Republic

2Institute of Biotechnology of the Czech Academy of Sciences, Biocev, Průmyslová 595, Vestec,

3University of Chemical and Technology Prague, Technická 5, Prague, Czech Republic

4Dept. of Agile Protein Screening, Novozymes A/S, Krogshoejvej 36, Bagsvaerd, Denmark

hrubyj18@fjfi.cvut.cz

S1 nuclease from Aspergillus oryzae is a single-strand-specific endonuclease widely used for biochemical analysis of nucleic acids [1,2]. Its activity depends on presence of three Zn2+ ions in the active site composed of nine residues coordinating the zinc cluster. The cluster and its adjacent residues are conserved across the whole S1-P1 family of nucleases. Two of the Zn2+ ions of the cluster are buried on the bottom of the active site. The third Zn2+ ion is closer to the nuclease’s surface.

A possibility of replacement of Zn2+ by Ni2+ was studied using biophysical assays and mainly the X-ray anomalous dispersion. Various molar ratios of S1 nuclease, chelating agent ethylenediaminetetraacetic acid (EDTA) and NiCl2 were analysed. The mixture of S1:EDTA:NiCl2 in molar ratio 1:5:10 was crystallized using a vapor diffusion method. The obtained crystals were of a good quality for the diffraction experiment on synchrotron radiation source Bessy II, Helmholtz Zentrum Berlin [3].

The diffraction data were collected at three different wavelengths in close proximity of the Ni- and Zn-absorption edges, respectively. The anomalous difference maps obtained from the collected data confirmed exchange of one Zn2+ ion by Ni2+, whilst the remaining two Zn2+ ions remained unaffected. No structural changes of the surrounding residues were observed.

 

1.       T. Koval’, L. H. Oestergaard, J. Lehmbeck, Allan Nørgaard, Petra Lipovová, Jarmila Dušková, Tereza Skálová, Mária Trundová, Petr Kolenko, Karla Fejfarová, Jan Stránský, Leona Švecová, Jindřich Hašek, Jan Dohnálek, PLoS ONE, 11, 2016, e0168832.

2.       T. Koval’, J. Dohnálek, Biotechnology Advances, 36, 2018, pp. 603-612.

3.       U. Mueller, R. Foerster, M. Hellmig, F. U. Huschmann, A. Kastner, P. Malecki, S. Puehringer, M. Roewer,  K. Sparta, M. Steffien, M. Uehlein, P. Wilk, M. S. Weiss. The European Physics Journal Plus, 130, 2015, pp. 141/1-10.

 

This work was supported by the MEYS CR (projects CAAS – CZ.02.1.01/0.0/0.0/16_019/0000778 and ELIBIO – CZ.02.1.01/0.0/0.0/15_003/0000447) from the ERDF fund, by the Czech Academy of Sciences (grant No. 86652036), and by the GA CTU in Prague (SGS19/189/OHK4/3T/14).