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Thr729 in human topoisomerase I modulates anti-cancer drug resistance by altering protein domain communications as suggested by molecular dynamics simulations.

Chillemi G, D'Annessa I, Fiorani P, Losasso C, Benedetti P, Desideri A - Nucleic Acids Res. (2008)

Bottom Line: Both mutants can bind to the DNA substrate and are enzymatically active, but while Thr729Lys is resistant even at high concentration of the camptothecin (CPT) anti-cancer drug, Thr729Pro shows only a mild reduction in drug sensitivity and in DNA binding.MD simulations show that the Thr729Lys mutation provokes a structural perturbation of the CPT-binding pocket.The simulations also show the complete abolishment, in the Thr729Lys mutant, of the protein communications between the C-terminal domain (where the active Tyr723 is located) and the linker domain, that plays an essential role in the control of the DNA rotation, thus explaining the distributive mode of action displayed by this mutant.

View Article: PubMed Central - PubMed

Affiliation: CASPUR Inter-University Consortium for the Application of Super-Computing for Universities and Research, Via dei Tizii 6, Rome 00185, Italy. g.chillemi@caspur.it

ABSTRACT
The role of Thr729 in modulating the enzymatic function of human topoisomerase I has been characterized by molecular dynamics (MD) simulation. In detail, the structural-dynamical behaviour of the Thr729Lys and the Thr729Pro mutants have been characterized because of their in vivo and in vitro functional properties evidenced in the accompanying paper. Both mutants can bind to the DNA substrate and are enzymatically active, but while Thr729Lys is resistant even at high concentration of the camptothecin (CPT) anti-cancer drug, Thr729Pro shows only a mild reduction in drug sensitivity and in DNA binding. MD simulations show that the Thr729Lys mutation provokes a structural perturbation of the CPT-binding pocket. On the other hand, the Thr729Pro mutant maintains the wild-type structural scaffold, only increasing its rigidity. The simulations also show the complete abolishment, in the Thr729Lys mutant, of the protein communications between the C-terminal domain (where the active Tyr723 is located) and the linker domain, that plays an essential role in the control of the DNA rotation, thus explaining the distributive mode of action displayed by this mutant.

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(A) Helix 16, in core domain, and helix 21, in the C-terminal domain, are highlighted in red and light blue colours, respectively. (B–D) Only the helices are shown in representative snapshots of the Tyr729Lys, wild-type and Tyr729Pro simulations, respectively. The side chains of Thr606 and Trp732, in helixes 16 and 17, respectively, are shown in ball and stick. Stable direct hydrogen bonds, when present, are indicated with a white line.
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Figure 3: (A) Helix 16, in core domain, and helix 21, in the C-terminal domain, are highlighted in red and light blue colours, respectively. (B–D) Only the helices are shown in representative snapshots of the Tyr729Lys, wild-type and Tyr729Pro simulations, respectively. The side chains of Thr606 and Trp732, in helixes 16 and 17, respectively, are shown in ball and stick. Stable direct hydrogen bonds, when present, are indicated with a white line.

Mentions: Thr729 is located in the middle of helix 21 (residues 727–735 in the C-terminal domain, Figures 2A and 3A) and its interactions with helices 16 (residues 586–606 in the core domain), 17 (residues 612–629 in core domain) and 20 (713–722 in C-terminal domain) are fundamental for a correct functioning of hTop1p (3). The catalytic residue Tyr723, in fact, is located in the loop between helices 20 (residues 718–722) and 21 and several residues in this region, such as Gly717, Thr718 and Asn722, have been reported to modulate drug sensitivity and enzyme activity, or produce a lethal phenotype in human or yeast topoisomerase I (14,26–28). In the wild-type protein, the only direct helix 21–17 interaction is the hydrogen bond between Thr729 and Tyr619 (Figure 2C), maintained for the whole simulation time. Both the simulations of the two mutated enzymes show the lack of this interaction, but the structural and dynamical effects are different in the two systems. Mutation of residue 729 in Lys, in fact, slightly pulls away helix 17 from the N-terminal portion of helix 21, as observed plotting the distance between the C-alpha atoms of Tyr619 and residue 729 as a function of simulation time (Figure 7 in Supplementary data). However, the most dramatic structural effect caused by this mutation is the occurrence of a new strong interaction between Lys729 and Lys720 that is present for the 80% of the simulation time (Figure 2B). This interaction is never observed in the other two simulations and it induces a conformational perturbation of the drug-binding pocket, as evidenced by the shortening of the Lys729–Asn722 distance when compared to its value in the wild-type and Thr729Pro mutant (Figure 8 in Supplementary data). Note that Asn722, in the crystallographic structure of the ternary topo70–DNA–topotecan complex (8), forms a water-mediated hydrogen bond with the topotecan drug. Moreover, mutation of Ans722 to Ala leads to hTop1p drug resistance (29–31), while mutation to Ser, Asn or His interferes with both enzyme activity and drug sensitivity (27,32).Figure 2.


Thr729 in human topoisomerase I modulates anti-cancer drug resistance by altering protein domain communications as suggested by molecular dynamics simulations.

Chillemi G, D'Annessa I, Fiorani P, Losasso C, Benedetti P, Desideri A - Nucleic Acids Res. (2008)

(A) Helix 16, in core domain, and helix 21, in the C-terminal domain, are highlighted in red and light blue colours, respectively. (B–D) Only the helices are shown in representative snapshots of the Tyr729Lys, wild-type and Tyr729Pro simulations, respectively. The side chains of Thr606 and Trp732, in helixes 16 and 17, respectively, are shown in ball and stick. Stable direct hydrogen bonds, when present, are indicated with a white line.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2553568&req=5

Figure 3: (A) Helix 16, in core domain, and helix 21, in the C-terminal domain, are highlighted in red and light blue colours, respectively. (B–D) Only the helices are shown in representative snapshots of the Tyr729Lys, wild-type and Tyr729Pro simulations, respectively. The side chains of Thr606 and Trp732, in helixes 16 and 17, respectively, are shown in ball and stick. Stable direct hydrogen bonds, when present, are indicated with a white line.
Mentions: Thr729 is located in the middle of helix 21 (residues 727–735 in the C-terminal domain, Figures 2A and 3A) and its interactions with helices 16 (residues 586–606 in the core domain), 17 (residues 612–629 in core domain) and 20 (713–722 in C-terminal domain) are fundamental for a correct functioning of hTop1p (3). The catalytic residue Tyr723, in fact, is located in the loop between helices 20 (residues 718–722) and 21 and several residues in this region, such as Gly717, Thr718 and Asn722, have been reported to modulate drug sensitivity and enzyme activity, or produce a lethal phenotype in human or yeast topoisomerase I (14,26–28). In the wild-type protein, the only direct helix 21–17 interaction is the hydrogen bond between Thr729 and Tyr619 (Figure 2C), maintained for the whole simulation time. Both the simulations of the two mutated enzymes show the lack of this interaction, but the structural and dynamical effects are different in the two systems. Mutation of residue 729 in Lys, in fact, slightly pulls away helix 17 from the N-terminal portion of helix 21, as observed plotting the distance between the C-alpha atoms of Tyr619 and residue 729 as a function of simulation time (Figure 7 in Supplementary data). However, the most dramatic structural effect caused by this mutation is the occurrence of a new strong interaction between Lys729 and Lys720 that is present for the 80% of the simulation time (Figure 2B). This interaction is never observed in the other two simulations and it induces a conformational perturbation of the drug-binding pocket, as evidenced by the shortening of the Lys729–Asn722 distance when compared to its value in the wild-type and Thr729Pro mutant (Figure 8 in Supplementary data). Note that Asn722, in the crystallographic structure of the ternary topo70–DNA–topotecan complex (8), forms a water-mediated hydrogen bond with the topotecan drug. Moreover, mutation of Ans722 to Ala leads to hTop1p drug resistance (29–31), while mutation to Ser, Asn or His interferes with both enzyme activity and drug sensitivity (27,32).Figure 2.

Bottom Line: Both mutants can bind to the DNA substrate and are enzymatically active, but while Thr729Lys is resistant even at high concentration of the camptothecin (CPT) anti-cancer drug, Thr729Pro shows only a mild reduction in drug sensitivity and in DNA binding.MD simulations show that the Thr729Lys mutation provokes a structural perturbation of the CPT-binding pocket.The simulations also show the complete abolishment, in the Thr729Lys mutant, of the protein communications between the C-terminal domain (where the active Tyr723 is located) and the linker domain, that plays an essential role in the control of the DNA rotation, thus explaining the distributive mode of action displayed by this mutant.

View Article: PubMed Central - PubMed

Affiliation: CASPUR Inter-University Consortium for the Application of Super-Computing for Universities and Research, Via dei Tizii 6, Rome 00185, Italy. g.chillemi@caspur.it

ABSTRACT
The role of Thr729 in modulating the enzymatic function of human topoisomerase I has been characterized by molecular dynamics (MD) simulation. In detail, the structural-dynamical behaviour of the Thr729Lys and the Thr729Pro mutants have been characterized because of their in vivo and in vitro functional properties evidenced in the accompanying paper. Both mutants can bind to the DNA substrate and are enzymatically active, but while Thr729Lys is resistant even at high concentration of the camptothecin (CPT) anti-cancer drug, Thr729Pro shows only a mild reduction in drug sensitivity and in DNA binding. MD simulations show that the Thr729Lys mutation provokes a structural perturbation of the CPT-binding pocket. On the other hand, the Thr729Pro mutant maintains the wild-type structural scaffold, only increasing its rigidity. The simulations also show the complete abolishment, in the Thr729Lys mutant, of the protein communications between the C-terminal domain (where the active Tyr723 is located) and the linker domain, that plays an essential role in the control of the DNA rotation, thus explaining the distributive mode of action displayed by this mutant.

Show MeSH
Related in: MedlinePlus