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. 2023 Jun 7;24(12):9849.
doi: 10.3390/ijms24129849.

Incorporation of N7-Platinated Guanines into Thermus Aquaticus (Taq) DNA Polymerase: Atomistic Insights from Molecular Dynamics Simulations

Federica De Castro  1 , Giada Ciardullo  2 , Francesco Paolo Fanizzi  1 , Mario Prejanò  2 , Michele Benedetti  1 , Tiziana Marino  2
Affiliations

Incorporation of N7-Platinated Guanines into Thermus Aquaticus (Taq) DNA Polymerase: Atomistic Insights from Molecular Dynamics Simulations

Federica De Castro et al. Int J Mol Sci. .

Abstract

In this work, we elucidated some key aspects of the mechanism of action of the cisplatin anticancer drug, cis-[Pt(NH3)2Cl2], involving direct interactions with free nucleotides. A comprehensive in silico molecular modeling analysis was conducted to compare the interactions of Thermus aquaticus (Taq) DNA polymerase with three distinct N7-platinated deoxyguanosine triphosphates: [Pt(dien)(N7-dGTP)] (1), cis-[Pt(NH3)2Cl(N7-dGTP)] (2), and cis-[Pt(NH3)2(H2O)(N7-dGTP)] (3) {dien = diethylenetriamine; dGTP = 5'-(2'-deoxy)-guanosine-triphosphate}, using canonical dGTP as a reference, in the presence of DNA. The goal was to elucidate the binding site interactions between Taq DNA polymerase and the tested nucleotide derivatives, providing valuable atomistic insights. Unbiased molecular dynamics simulations (200 ns for each complex) with explicit water molecules were performed on the four ternary complexes, yielding significant findings that contribute to a better understanding of experimental results. The molecular modeling highlighted the crucial role of a specific α-helix (O-helix) within the fingers subdomain, which facilitates the proper geometry for functional contacts between the incoming nucleotide and the DNA template needed for incorporation into the polymerase. The analysis revealed that complex 1 exhibits a much lower affinity for Taq DNA polymerase than complexes 2-3. The affinities of cisplatin metabolites 2-3 for Taq DNA polymerase were found to be quite similar to those of natural dGTP, resulting in a lower incorporation rate for complex 1 compared to complexes 2-3. These findings could have significant implications for the cisplatin mechanism of action, as the high intracellular availability of free nucleobases might promote the competitive incorporation of platinated nucleotides over direct cisplatin attachment to DNA. The study's insights into the incorporation of platinated nucleotides into the Taq DNA polymerase active site suggest that the role of platinated nucleotides in the cisplatin mechanism of action may have been previously underestimated.

Keywords: antimetabolites; antitumor drugs; antiviral drugs; cisplatin; coordination compounds; molecular dynamics; nucleoside analogues; platinum compounds.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Structure of the Thermus Aquaticus DNA Polymerase enzyme in complex with DNA (PDB code 5YTD) [65].
Scheme 1
Scheme 1
Schematic representation of complexes: [Pt(dien)(N7-dGTP)] (1), cis-[Pt(NH3)2Cl(N7-dGTP)] (2), and cis-[Pt(NH3)2(H2O)(N7-dGTP)] (3); dGTP = 5′-(2′-deoxy)-guanosine triphosphate. They are generally indicated in short as [PtL3(N7-dGTP)] on the bottom, together with the relative Pt-containing moieties under investigation, indicated on the top.
Figure 2
Figure 2
The four considered different Taq:DNA:substrate systems: Taq:DNA:dGTP, Taq:DNA:1, Taq:DNA:2 and Taq:DNA:3; {1 = [Pt(dien)(N7-dGTP)]; 2 = cis-[Pt(NH3)2Cl(N7-dGTP)]; 3 = cis-[Pt(NH3)2(H2O)(N7-dGTP)]}.
Figure 3
Figure 3
Root mean square deviation (RMSD) of (a) backbone atoms of the protein and (b) atoms of deoxyribonucleotide triphosphate (dNTP) bases, and (c) root mean square fluctuation (RMSF) calculated for backbone atoms of different Taq:DNA:substrate systems {1 = [Pt(dien)(N7-dGTP)]; 2 = cis-[Pt(NH3)2Cl(N7-dGTP)]; 3 = cis-[Pt(NH3)2(H2O)(N7-dGTP)]}.
Figure 4
Figure 4
Number of hydrogen-bond interactions involving the dNTP species and (a) protein, (b) water molecules, and (c) radial distribution functions calculated for the dNTP-H2O pair {1 = [Pt(dien)(N7-dGTP)]; 2 = cis-[Pt(NH3)2Cl(N7-dGTP)]; 3 = cis-[Pt(NH3)2(H2O)(N7-dGTP)]}.
Figure 5
Figure 5
(a) Superposition of the four investigated systems, with focus on the α-helix dedicated to recognition, and (b) distances between the centers of mass (COM) of the four different dNTP and the α-helix (relative distribution of distances is also shown), {where: 1 = [Pt(dien)(N7-dGTP)]; 2 = cis-[Pt(NH3)2Cl(N7-dGTP)]; 3 = cis-[Pt(NH3)2(H2O)(N7-dGTP)]}.
Figure 6
Figure 6
(a) Superposition of the four investigated systems, focusing on the different dNTP and F667 and Y671 amino acids. (b) Measured N3dNTP-OHY671 and (c) calculated radial distribution function (RDF) for the center of mass of the guanine-dNTP-phenol ring-Y671 pair obtained for the four different systems, {where: 1 = [Pt(dien)(N7-dGTP)]; 2 = cis-[Pt(NH3)2Cl(N7-dGTP)]; 3 = cis-[Pt(NH3)2(H2O)(N7-dGTP)]}.
Figure 7
Figure 7
(a) RDF calculated for guanine-dNTP-cytosin-dC551 pair, and (b) C1′dNTP-C1′dC551 distance observed in the MD simulations of the four investigated systems, {where: 1 = [Pt(dien)(N7-dGTP)]; 2 = cis-[Pt(NH3)2Cl(N7-dGTP)]; 3 = cis-[Pt(NH3)2(H2O)(N7-dGTP)]}.
Figure 8
Figure 8
(a) Distribution of O3′d551−PαdNPT−OαdNPT angle and O3′d551−PαdNPT distance obtained for the four investigated systems {where 1 = [Pt(dien)(N7-dGTP)]; 2 = cis-[Pt(NH3)2Cl(N7-dGTP)]; 3 = cis-[Pt(NH3)2(H2O)(N7-dGTP)]}, and (b) the atoms involved in the aligned/unaligned conformations.
Figure 9
Figure 9
Schematic representation of the DNA-dNTP model adduct adopted in the present study. Residue numbers are reported following the PDB numeration [65]. Star (*) indicates the point of DNA replication process where the insertion of dNTP takes place.
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