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Insights into the key interactions between human protein phosphatase 5 and cantharidin using molecular dynamics and site-directed mutagenesis bioassays.

Liu JY, Chen XE, Zhang YL - Sci Rep (2015)

Bottom Line: We found that, unlike previous report, Arg 100 contributes less to PP5-inhibitor binding, and the residues His 69, Asn 128, His 129, Arg 225, His 252 and Arg 250 are of importance to PP5-inhibitor binding.The coordination between MN2 and chemical group of inhibitor should be eliminated.This work provides insight into how cantharidin and its analogs bind to PP5c at the atomic level and will facilitate modification of cantharidin-like chemicals to rationally develop more specific and less cytotoxic anti-cancer drugs.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Plant Protection Resources &Pest Management of the Ministry of Education, Northwest A&F University, Yangling 712100, Shaanxi, China.

ABSTRACT
Serine/threonine protein phosphatase 5 (PP5) is a promising novel target for anticancer therapies. This work aims to uncover the key interactions at the atomic level between PP5 and three inhibitors (cantharidin, norcantharidin and endothall). We found that, unlike previous report, Arg 100 contributes less to PP5-inhibitor binding, and the residues His 69, Asn 128, His 129, Arg 225, His 252 and Arg 250 are of importance to PP5-inhibitor binding. The hydrophobic interactions established between the residues Val 254, Phe 271 and Tyr 276, especially Glu 253, are very important to enhance the inhibitive interaction. We suggested that, to increase the inhibitory activity, the interactions of inhibitor with three negatively charged unfavorable interaction residues, Asp 99, Glu 130 and Asp 213, should be avoided. However, the interactions of inhibitor with favorable interaction residue Arg 250 could enhance the inhibitory activity. The Manganese ion 2 (MN2) unfavorably contribute to the total interaction free energies. The coordination between MN2 and chemical group of inhibitor should be eliminated. This work provides insight into how cantharidin and its analogs bind to PP5c at the atomic level and will facilitate modification of cantharidin-like chemicals to rationally develop more specific and less cytotoxic anti-cancer drugs.

No MeSH data available.


Residue-ligand interaction spectrum of (A) the Cantharidin-PP5c complex, (B) the Norcantharidin-PP5c complex and (C) the Endothall-PP5c complex according to the MM-PBSA method.The x-axis denotes the residue number of the PP5c and the y-axis denotes the total interaction free energy contribution for each residue. The key interactions and H bond patterns at the active site observed during MD simulations of cantharidin (D), norcantharidin (E) and endothall (F). The manganese ions are presented as slate spheres. Cantharidin, norcantharidin and endothall are presented with the stick-and-sphere model. Color code: deep cyan, C; red, O; white, H. Key residues are presented with the stick model. Color code: gray, C; red, O; blue, N; white, H; yellow dashed line, coordination bond; red dashed line, H bond.
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f3: Residue-ligand interaction spectrum of (A) the Cantharidin-PP5c complex, (B) the Norcantharidin-PP5c complex and (C) the Endothall-PP5c complex according to the MM-PBSA method.The x-axis denotes the residue number of the PP5c and the y-axis denotes the total interaction free energy contribution for each residue. The key interactions and H bond patterns at the active site observed during MD simulations of cantharidin (D), norcantharidin (E) and endothall (F). The manganese ions are presented as slate spheres. Cantharidin, norcantharidin and endothall are presented with the stick-and-sphere model. Color code: deep cyan, C; red, O; white, H. Key residues are presented with the stick model. Color code: gray, C; red, O; blue, N; white, H; yellow dashed line, coordination bond; red dashed line, H bond.

Mentions: The key residues contributing more than 1 kcal/mol to the total interaction free energies are illustrated in Fig. 3 (left). The positive charged Arg 100 acts as the gatekeeper and the hydrophobic pocket defined by the side chains of Val 254, Phe 271, and Tyr 276 in the active site of PP5c as mentioned in the previous study22, the unfavorable interaction residues and two manganese ions were also involved in the decomposition of interaction free energies on a per-residue level, and the detailed energetic components are listed in Table S4. Figure 3A shows that the residues of the Cantharidin-PP5c complex in the binding surface contribute more than 1 kcal/mol to the total interaction free energies including His 69, Asn 128, His 129, Arg 225, His 252 and Arg 250. According to the Table S4 and Fig. 3D, the sidechain atoms of the residues His 69, Asn 128 and His 252 contribute remarkable electrostatic energies to the total interaction free energies, which is in accordance with our calculation of RESP charge fitting (Figure S4). The prominent contribution to the total interaction free energies of the gatekeeper Arg 100 and Arg 225 are mainly from electrostatic energies, especially the residue Arg 225 that achieved −4.54 kcal/mol. This is a consequence of the hydrogen bond interaction between Arg 225 and cantharidin (Fig. 3D),the H-bond having a distance of 2.82 Å between the NH1 atom (HH11) of the residue Arg 225 sidechain and the carbonyl oxygen atom (O5) derived from the right carboxylate moiety of the cantharidin exhibits a high H-bond occupancy rate (97.09%) (Table 1).The hydrophobic cavity was consistent with the side chains of Val 254, Phe 271, and Tyr 276. The backbone atoms of Glu 253 primarily contribute the Van der Waals energy to the binding (Fig. 3D). The residues Val 254, Phe 271 and Tyr 276 combine as a “claw” in the hydrophobic cavity to make significant favorable contributions to the total interaction free energies. Noticeably, the residue His 129 possesses a negative energy with a high value, −4.79 kcal/mol. It is not surprising since the sidechain atoms of the residue His 129 can form the important hydrogen bond with cantharidin (Fig. 3D), the carbonyl oxygen atom (O4) derived from the left carboxylate moiety of the cantharidin and the NE2 atom (HE2) of the residue His 129 sidechain form an H-bond with a distance of 2.77 Å between atoms revealing a medium H-bond occupancy rate (60.03%) (Table 1).Intriguingly, the residue Arg 250 is far from the active center with the distance of approximately 10 Å (Fig. 3D), however, its contribution is more than 1 kcal/mol. These findings were also observed both in the norcantharidin-PP5c and Endothall-PP5c complexes. On the contrary, the residues Asp 67, Asp 96, Asp99, Glu 130, His 177 and Asp 213 offer unfavorable interaction free energies. This is a consequence of their large unfavorable electrostatic energies derived from sidechain, resulted from the strong negative charge repulsion established between these residues and cantharidin. It is worth noting that Asp 99, Glu 130, and Asp 213 belonging to uncoordinated residues, suggesting that mutation of these three residues might increase the inhibitory activity of PP5c inhibitors. Interestingly, two manganese ions coordinated with cantharidin contribute high electrostatic energies (−50.87 and −54.36 kcal/mol, respectively), however, only MN1 provides favorable interaction free energies benefiting for cantharidin binding. Because the stronger coordination interaction can produce a stronger repulsion of the Van der Waals decreasing the Van der Waals energy contribution, this inference agrees with MN2 contributes higher unfavorable Van der Waals energy.


Insights into the key interactions between human protein phosphatase 5 and cantharidin using molecular dynamics and site-directed mutagenesis bioassays.

Liu JY, Chen XE, Zhang YL - Sci Rep (2015)

Residue-ligand interaction spectrum of (A) the Cantharidin-PP5c complex, (B) the Norcantharidin-PP5c complex and (C) the Endothall-PP5c complex according to the MM-PBSA method.The x-axis denotes the residue number of the PP5c and the y-axis denotes the total interaction free energy contribution for each residue. The key interactions and H bond patterns at the active site observed during MD simulations of cantharidin (D), norcantharidin (E) and endothall (F). The manganese ions are presented as slate spheres. Cantharidin, norcantharidin and endothall are presented with the stick-and-sphere model. Color code: deep cyan, C; red, O; white, H. Key residues are presented with the stick model. Color code: gray, C; red, O; blue, N; white, H; yellow dashed line, coordination bond; red dashed line, H bond.
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Related In: Results  -  Collection

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Show All Figures
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f3: Residue-ligand interaction spectrum of (A) the Cantharidin-PP5c complex, (B) the Norcantharidin-PP5c complex and (C) the Endothall-PP5c complex according to the MM-PBSA method.The x-axis denotes the residue number of the PP5c and the y-axis denotes the total interaction free energy contribution for each residue. The key interactions and H bond patterns at the active site observed during MD simulations of cantharidin (D), norcantharidin (E) and endothall (F). The manganese ions are presented as slate spheres. Cantharidin, norcantharidin and endothall are presented with the stick-and-sphere model. Color code: deep cyan, C; red, O; white, H. Key residues are presented with the stick model. Color code: gray, C; red, O; blue, N; white, H; yellow dashed line, coordination bond; red dashed line, H bond.
Mentions: The key residues contributing more than 1 kcal/mol to the total interaction free energies are illustrated in Fig. 3 (left). The positive charged Arg 100 acts as the gatekeeper and the hydrophobic pocket defined by the side chains of Val 254, Phe 271, and Tyr 276 in the active site of PP5c as mentioned in the previous study22, the unfavorable interaction residues and two manganese ions were also involved in the decomposition of interaction free energies on a per-residue level, and the detailed energetic components are listed in Table S4. Figure 3A shows that the residues of the Cantharidin-PP5c complex in the binding surface contribute more than 1 kcal/mol to the total interaction free energies including His 69, Asn 128, His 129, Arg 225, His 252 and Arg 250. According to the Table S4 and Fig. 3D, the sidechain atoms of the residues His 69, Asn 128 and His 252 contribute remarkable electrostatic energies to the total interaction free energies, which is in accordance with our calculation of RESP charge fitting (Figure S4). The prominent contribution to the total interaction free energies of the gatekeeper Arg 100 and Arg 225 are mainly from electrostatic energies, especially the residue Arg 225 that achieved −4.54 kcal/mol. This is a consequence of the hydrogen bond interaction between Arg 225 and cantharidin (Fig. 3D),the H-bond having a distance of 2.82 Å between the NH1 atom (HH11) of the residue Arg 225 sidechain and the carbonyl oxygen atom (O5) derived from the right carboxylate moiety of the cantharidin exhibits a high H-bond occupancy rate (97.09%) (Table 1).The hydrophobic cavity was consistent with the side chains of Val 254, Phe 271, and Tyr 276. The backbone atoms of Glu 253 primarily contribute the Van der Waals energy to the binding (Fig. 3D). The residues Val 254, Phe 271 and Tyr 276 combine as a “claw” in the hydrophobic cavity to make significant favorable contributions to the total interaction free energies. Noticeably, the residue His 129 possesses a negative energy with a high value, −4.79 kcal/mol. It is not surprising since the sidechain atoms of the residue His 129 can form the important hydrogen bond with cantharidin (Fig. 3D), the carbonyl oxygen atom (O4) derived from the left carboxylate moiety of the cantharidin and the NE2 atom (HE2) of the residue His 129 sidechain form an H-bond with a distance of 2.77 Å between atoms revealing a medium H-bond occupancy rate (60.03%) (Table 1).Intriguingly, the residue Arg 250 is far from the active center with the distance of approximately 10 Å (Fig. 3D), however, its contribution is more than 1 kcal/mol. These findings were also observed both in the norcantharidin-PP5c and Endothall-PP5c complexes. On the contrary, the residues Asp 67, Asp 96, Asp99, Glu 130, His 177 and Asp 213 offer unfavorable interaction free energies. This is a consequence of their large unfavorable electrostatic energies derived from sidechain, resulted from the strong negative charge repulsion established between these residues and cantharidin. It is worth noting that Asp 99, Glu 130, and Asp 213 belonging to uncoordinated residues, suggesting that mutation of these three residues might increase the inhibitory activity of PP5c inhibitors. Interestingly, two manganese ions coordinated with cantharidin contribute high electrostatic energies (−50.87 and −54.36 kcal/mol, respectively), however, only MN1 provides favorable interaction free energies benefiting for cantharidin binding. Because the stronger coordination interaction can produce a stronger repulsion of the Van der Waals decreasing the Van der Waals energy contribution, this inference agrees with MN2 contributes higher unfavorable Van der Waals energy.

Bottom Line: We found that, unlike previous report, Arg 100 contributes less to PP5-inhibitor binding, and the residues His 69, Asn 128, His 129, Arg 225, His 252 and Arg 250 are of importance to PP5-inhibitor binding.The coordination between MN2 and chemical group of inhibitor should be eliminated.This work provides insight into how cantharidin and its analogs bind to PP5c at the atomic level and will facilitate modification of cantharidin-like chemicals to rationally develop more specific and less cytotoxic anti-cancer drugs.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Plant Protection Resources &Pest Management of the Ministry of Education, Northwest A&F University, Yangling 712100, Shaanxi, China.

ABSTRACT
Serine/threonine protein phosphatase 5 (PP5) is a promising novel target for anticancer therapies. This work aims to uncover the key interactions at the atomic level between PP5 and three inhibitors (cantharidin, norcantharidin and endothall). We found that, unlike previous report, Arg 100 contributes less to PP5-inhibitor binding, and the residues His 69, Asn 128, His 129, Arg 225, His 252 and Arg 250 are of importance to PP5-inhibitor binding. The hydrophobic interactions established between the residues Val 254, Phe 271 and Tyr 276, especially Glu 253, are very important to enhance the inhibitive interaction. We suggested that, to increase the inhibitory activity, the interactions of inhibitor with three negatively charged unfavorable interaction residues, Asp 99, Glu 130 and Asp 213, should be avoided. However, the interactions of inhibitor with favorable interaction residue Arg 250 could enhance the inhibitory activity. The Manganese ion 2 (MN2) unfavorably contribute to the total interaction free energies. The coordination between MN2 and chemical group of inhibitor should be eliminated. This work provides insight into how cantharidin and its analogs bind to PP5c at the atomic level and will facilitate modification of cantharidin-like chemicals to rationally develop more specific and less cytotoxic anti-cancer drugs.

No MeSH data available.