Biochim Biophys Acta. 2015 Feb;1850(2):393-400.

Chemical and thermal influence of the [4Fe-4S]2+ cluster of A/G-specific adenine glycosylase from Corynebacterium pseudotuberculosis.

Eberle RJ1, Coronado MA1, Caruso IP1, Lopes DO2, Miyoshi A3, Azevedo V3, Arni RK4.
  • 1Multiuser Center for Biomolecular Innovation, Departament of Physics, Universidade Estadual Paulista (UNESP), São Jose do Rio Preto, SP 15054-000, Brazil.
  • 2Laboratory of Molecular Biology, Federal University of São João Del-Rei (CCO), Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil.
  • 3Laboratório de Genética Celular e Molecular, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627-Pampulha, CP 486, CEP 31, Belo Horizonte, MG 270-901, Brazil.
  • 4Multiuser Center for Biomolecular Innovation, Departament of Physics, Universidade Estadual Paulista (UNESP), São Jose do Rio Preto, SP 15054-000, Brazil. Electronic address: arni@sjrp.unesp.br.

 

Abstract

The gram-positive bacteria Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis in livestock significantly reduces productivity and often causes death. The adenine/guanine-specific DNA glycosylase (MutY) prevents mutations in the DNA of the pathogen and a unique feature of the MutY protein family is the [4Fe-4S]2+ cluster that interlinks two protein subdomains. MutY from C. pseudotuberculosis was expressed in E. coli and purified, the CD experiments indicate a high content of α-helices and random coiled secondary structure and a typical near-UV CD fingerprint for the [4Fe-4S]2+ cluster. EDTA and copper sulfate possess a strong destabilizing effect on the [4Fe-4S]2+ cluster. UV-vis and fluorescence spectroscopy results demonstrate that between pH3.0 and 4.0 the integrity of the [4Fe-4S]2+ cluster is destroyed. To investigate the thermal stability of the protein differential scanning calorimetry and fluorescence spectroscopy were used and the Tm was determined to be 45°C. The analysis presented provides information concerning the protein stability under different physio-chemical conditions.

KEYWORDS: C. pseudotuberculosis; DNA repair; MutY; Secondary and tertiary structure; Spectroscopic method; [4Fe–4S](2+) cluster

PMID: 25445713

 

Supplement:

The adenine/guanine-specific DNA glycosylase (MutY), a key enzyme in the BER pathway, prevents mutations in DNA resulting from the presence of the oxidatively damaged 8-OxoG. This guanosine derivate can mispair with 2′-deoxycytidine 5′-triphosphate or with 2′-deoxyadenosine triphosphate during DNA replication, forming C*8-oxoG and A*8-oxoG mispairs. If uncorrected, the A*8-oxoG mispairs result in deleterious C:G to A:T transversions. MutY with its DNA glycosylase activity, excises adenine paired with guanine or 8-OxoG (figure 1A).

An unique feature of the MutY protein family is that the enzymes contain a [4Fe-4S]2+ cluster. The [4Fe-4S]2+ cluster connects two subdomains of the MutY proteins (figure 1B).

 

RE fig1

Figure 1. Adenine/guanine-specific DNA glycosylase (MutY) A: Repairing mechanism of DNA damaged by reactive oxygen species (ROS). B: Homology model of C. pseudotuberculosis MutY.

 

The structural investigation of C. pseudotuberculosis MutY is one of primary focus of our research and we combine various techniques of investigation such as protein crystallography, nuclear magnetic resonance (NMR) spectroscopy and other spectroscopic techniques (UV-vis, fluorescence, circular dichroism (CD)) and differential scanning calorimetry (DSC). We investigated the influence of different ions and pH on the secondary and tertiary structures and on the [4Fe-4S]2+ cluster of MutY [1].

The influence of ions on the secondary structure was investigated by CD spectroscopy, the change in the secondary structural elements was evaluated and showed a strong denaturation effect on MutY with all the tested ions except for lithium (figure 2A).

The influence of the pH on the MutY secondary structure was also examined and showed a significant change at pH 4.0 (figure 2B). The experiment showed that the secondary structure of MutY is very stable in the 5.0 to 8.0 pH range.

 

RE fig2

Figure 2. Influence of ions and pH of the MutY secondary structure investigated by CD spectroscopy. A: Influence of ions on MutY secondary structure. B: Influence of pH on MutY secondary structure. The secondary structure elements was evaluated with the program CDpro.

 

As mentioned above MutY interact with DNA, especially with DNA carrying mutations resulting from the presence of the oxidatively damaged 8-OxoG. For the investigation of interaction we need high concentrations of this DNA. As a model system to depict this MutY-DNA interaction we used the purine bases adenine and guanine and their dntps.

A simple and fast method for ligand screening and characterization of protein-ligand interactions is saturation-transfer difference (STD) NMR. The Multiuser Center of Biomolecular Innovation (CMIB) on the Universidade Estadual Paulista (UNESP), São Jose do Rio Preto-SP (Brazil) has a NMR 600 and we establish experiments to determine the interaction of guanine, adenine and their dntps with MutY using this technique.

 

RE fig3

Figure 3. Nuclear magnetic resonance (NMR) experiments with MutY. A: NMR spectroscope 600 in the CMIB. B: Results of the saturation-transfer difference (STD) NMR of MutY with adenine, guanine and their corresponding dntps reveal dATP as the strongest ligand.

 

We found out that dATP is the strongest interaction partner followed by adenine. Guanine and dGTP show more weak interaction. The identified ligands will be used for co-crystallization experiments with MutY. Additionally, we are interested in the influence of pH and ions of the binding mode and the identification of useful inhibitors that block the interaction between MutY and the ligands.

The importance of this study: The results of our study provide basic information about the influence of pH and ions on the MutY stability and ligand binding. Our information is used to improve the quality of the protein solution to investigate the function and structure of MutY under different conditions.

 

Reference:

[1] Eberle, R. J., Coronado, M. A., Caruso, I. P., Lopes, D. O., Miyoshi, A., Azevedo, V., & Arni, R. K. (2015). Chemical and thermal influence of the [4Fe–4S] 2+ cluster of A/G-specific adenine glycosylase from Corynebacterium pseudotuberculosis. Biochimica et Biophysica Acta (BBA)-General Subjects, 1850(2), 393-400.

 

Aknowledgements:

This study was supported by grants from the CNPq (Science without Frontiers), FAPESP, CAPES and FAPEMIG.

 

Contact:

Raphael J. Eberle, Ph.D.

Multiuser Center of Biomolecular Innovation

Department of Physics

Universidade Estadual Paulista (UNESP)

São Jose do Rio Preto-SP

15054-000, Brazil.

Email: eberleraphael@gmail.com

http://www.cmib-unesp.com/

 

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