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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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Dynamic cross correlation map for the topo70 (lower right triangle) and Tyr729Pro mutant (upper left triangle). Correlated values with 0.50 ≤ Cij < 0.65, 0.65 ≤ Cij < 0.80 and 0.80 ≤ Cij < 1 are represented in green, yellow and red, respectively, while the anti-correlation values −0.80 ≤ Cij, <−0.65 and −0.65 ≤ Cij <−0.50 are represented in dark and light blue, respectively. The correlation range 0 ≤ Cij < 0.50 and the anti-correlation range −0.50 < Cij < 0 are represented in a grey scale depending on the absolute value of the correlation. The value 1, along the diagonal (correlation of the residue with itself), is represented in black.
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Figure 6: Dynamic cross correlation map for the topo70 (lower right triangle) and Tyr729Pro mutant (upper left triangle). Correlated values with 0.50 ≤ Cij < 0.65, 0.65 ≤ Cij < 0.80 and 0.80 ≤ Cij < 1 are represented in green, yellow and red, respectively, while the anti-correlation values −0.80 ≤ Cij, <−0.65 and −0.65 ≤ Cij <−0.50 are represented in dark and light blue, respectively. The correlation range 0 ≤ Cij < 0.50 and the anti-correlation range −0.50 < Cij < 0 are represented in a grey scale depending on the absolute value of the correlation. The value 1, along the diagonal (correlation of the residue with itself), is represented in black.

Mentions: The global motions of the proteins have been investigated by means of the DCC map that provides an aggregate picture of the correlated motions occurring between protein residues. Highly positive peaks of the Cij elements of the map (red, yellow and green in Figures 4 and 6), in fact, are indicative of strong correlation in the movement of residues i and j, while negative Cij values (dark and light blue in Figures 4 and 6) indicate that the two residues move along opposite direction (anti-correlated motion). Comparison of the DCC maps for the wild-type and Thr729Lys systems (Figure 4, lower right triangle and upper left triangle, respectively) shows a dramatic change in the protein dynamics. The linker domain in the wild-type system, in fact, moves in a highly anti-correlated way with both the C-terminal domain and the C-terminal region of the core domain (Figure 4, rectangles A1 and C1, respectively). Moreover, these two regions have a strong correlated motion, one with the other (Figure 4, rectangle B1). Mutation of residue 729 in Lys completely abolishes these correlated and anti-correlated motions (Figure 4, rectangles A2, B2 and C2). Therefore, although the fluctuations of the linker domain in the Thr729Lys protein are not significantly different from the wild type (Figure 1), its motion is randomic and it is hindered to participate to the protein domain communications, characterizing the enzymatic functionality of hTop1p (9,33), as also shown by the distributive mode of action described in the accompanying paper (see Figure 6 of the accompanying paper). The perturbation of the local interactions around the mutation site (Figure 2A; and Figure 8 in Supplementary data) suggests that the uncoupling of the correlated motions in the linker domain of the Thr729Lys mutant is triggered by the lack of interactions between helices 21–16 and 21–17, respectively (Figures 2 and 3). The linker domain, in fact, interacts with helix 17 (the only protein region that directly contacts the linker domain also in the X-ray structures) in the three simulations by means of several hydrophobic residues: Val703, Ile714 and Leu716 in helix 19, and Pro613 and Leu617 in helix 17 (Figure 5). Therefore, the helix 17 altered motion in the Thr729Lys mutant, that is a consequence of its different interactions with helix 21, is directly transmitted to the linker domain dynamics.Figure 4.


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)

Dynamic cross correlation map for the topo70 (lower right triangle) and Tyr729Pro mutant (upper left triangle). Correlated values with 0.50 ≤ Cij < 0.65, 0.65 ≤ Cij < 0.80 and 0.80 ≤ Cij < 1 are represented in green, yellow and red, respectively, while the anti-correlation values −0.80 ≤ Cij, <−0.65 and −0.65 ≤ Cij <−0.50 are represented in dark and light blue, respectively. The correlation range 0 ≤ Cij < 0.50 and the anti-correlation range −0.50 < Cij < 0 are represented in a grey scale depending on the absolute value of the correlation. The value 1, along the diagonal (correlation of the residue with itself), is represented in black.
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Related In: Results  -  Collection

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Figure 6: Dynamic cross correlation map for the topo70 (lower right triangle) and Tyr729Pro mutant (upper left triangle). Correlated values with 0.50 ≤ Cij < 0.65, 0.65 ≤ Cij < 0.80 and 0.80 ≤ Cij < 1 are represented in green, yellow and red, respectively, while the anti-correlation values −0.80 ≤ Cij, <−0.65 and −0.65 ≤ Cij <−0.50 are represented in dark and light blue, respectively. The correlation range 0 ≤ Cij < 0.50 and the anti-correlation range −0.50 < Cij < 0 are represented in a grey scale depending on the absolute value of the correlation. The value 1, along the diagonal (correlation of the residue with itself), is represented in black.
Mentions: The global motions of the proteins have been investigated by means of the DCC map that provides an aggregate picture of the correlated motions occurring between protein residues. Highly positive peaks of the Cij elements of the map (red, yellow and green in Figures 4 and 6), in fact, are indicative of strong correlation in the movement of residues i and j, while negative Cij values (dark and light blue in Figures 4 and 6) indicate that the two residues move along opposite direction (anti-correlated motion). Comparison of the DCC maps for the wild-type and Thr729Lys systems (Figure 4, lower right triangle and upper left triangle, respectively) shows a dramatic change in the protein dynamics. The linker domain in the wild-type system, in fact, moves in a highly anti-correlated way with both the C-terminal domain and the C-terminal region of the core domain (Figure 4, rectangles A1 and C1, respectively). Moreover, these two regions have a strong correlated motion, one with the other (Figure 4, rectangle B1). Mutation of residue 729 in Lys completely abolishes these correlated and anti-correlated motions (Figure 4, rectangles A2, B2 and C2). Therefore, although the fluctuations of the linker domain in the Thr729Lys protein are not significantly different from the wild type (Figure 1), its motion is randomic and it is hindered to participate to the protein domain communications, characterizing the enzymatic functionality of hTop1p (9,33), as also shown by the distributive mode of action described in the accompanying paper (see Figure 6 of the accompanying paper). The perturbation of the local interactions around the mutation site (Figure 2A; and Figure 8 in Supplementary data) suggests that the uncoupling of the correlated motions in the linker domain of the Thr729Lys mutant is triggered by the lack of interactions between helices 21–16 and 21–17, respectively (Figures 2 and 3). The linker domain, in fact, interacts with helix 17 (the only protein region that directly contacts the linker domain also in the X-ray structures) in the three simulations by means of several hydrophobic residues: Val703, Ile714 and Leu716 in helix 19, and Pro613 and Leu617 in helix 17 (Figure 5). Therefore, the helix 17 altered motion in the Thr729Lys mutant, that is a consequence of its different interactions with helix 21, is directly transmitted to the linker domain dynamics.Figure 4.

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