Effects of the Zn²⁺ to Cd²⁺ Exchange on the Structure and Activity of S1 Nuclease

J. Hrubý^1,2, P. Kolenko^1,2, K. Adámková^2,3, B. Husťáková^2,3, M. Malý^1,2, 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

Corresponding authors: hrubyj18@fjfi.cvut.cz (JH); jan.dohnalek@ibt.cas.cz (JD)

S1 nuclease from Aspergillus oryzae is a single-strand specific nuclease widely utilized in biotechnology industry for biochemical analysis of nucleic acids [1,2]. It is a globular protein with its secondary structure predominantly composed of a-helices (Fig. 1). Its activity depends on the presence of three Zn²⁺ ions in the pocket-shaped active site: Two Zn²⁺ ions of the cluster are buried at the bottom of the active site, the third Zn²⁺ ion is situated closer to the surface of the nuclease. The zinc cluster is coordinated by nine amino acid residues.

We studied the possibility of replacing Zn²⁺ with various metals and effects of such exchange on the structure and enzymatic activity. Here we present the results of the Zn²⁺ to Cd²⁺ exchange. S1 nuclease was mixed with chelating agent ethylenediaminetetraacetic acid (EDTA) in molar ratio 1:5, respectively, and dialysed, resulting in EDTA-treated S1. A mixture of EDTA-treated S1 with CdCl₂ in molar ratio 1:10 was successfully crystallized using the vapour diffusion method. The obtained crystals were of sufficient quality for the diffraction experiment on the synchrotron radiation source Bessy II, Helmholtz Zentrum Berlin [3]. Activity of fully EDTA-treated S1 with CdCl₂ towards ssDNA as a substrate was measured using precipitation of undigested nucleic acids and measurement of absorbance at 260 nm.

The diffraction data were collected at three different X-ray energies with the aim of differentiating the presence of Zn²⁺ or Cd²⁺ ions in the active site using anomalous scattering. The structural data showed that the conformation of the surrounding residues of the active site remained conserved. Only one of the inner Zn²⁺ ions remained intact. The other two Zn²⁺ ions were successfully replaced by Cd²⁺ ions. The key data collection statistics are summarized in Table 1. The anomalous difference map is shown in Fig. 2.

The activity studies showed that EDTA-treated S1 is entirely inactive (less than 1% activity of untreated S1). Only minor restoration of the activity was observed after adding CdCl₂, approximately 3% of the activity of untreated S1 activity.

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

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Table 1: Data collection statistics.

Dataset name	Cd-peak	Zn-low	Zn-peak
X-ray energy [keV]	6.600	9.630	9.680
Resolution [Å]	44.71 – 2.30	44.66 – 2.60	44.66 – 2.70
Rpim	0.033	0.074	0.064
CC1/2	0.999	0.996	0.996
Mean I/σ(I)	18.3	10.4	10.8
Avg. anomalous multiplicity	11.9	13.7	13.9
Anomalous completeness [%]	99.2	100.0	100.0

Figure 1. Secondary structure of S1 nuclease (PDB ID 5FB9). Zinc atoms are represented using blue spheres. Graphics created using PyMOL [4].	Figure 2. Anomalous difference map (Cd-peak) at a level of 5σ. Two distinct peaks prove the presence of Cd^2+. The pocket shape of the active site is highlighted by the grey line. Graphics created using CCP4MG [5].

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 (SGS22/114/OHK4/2T/14). We acknowledge CMS-BIOCEV Crystallization and Diffraction, part of Instruct-ERIC, supported by the MEYS CR (LM2018127).