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Ternary complex structures of human farnesyl pyrophosphate synthase bound with a novel inhibitor and secondary ligands provide insights into the molecular details of the enzyme's active site closure.

Park J, Lin YS, De Schutter JW, Tsantrizos YS, Berghuis AM - BMC Struct. Biol. (2012)

Bottom Line: Isothermal titration calorimetry experiments demonstrated that PPi binds more tightly to the enzyme-inhibitor complex than IPP, and differential scanning fluorometry experiments confirmed that Pi binding does not induce the tail ordering.In human FPPS, Y349 functions as a safety switch that prevents any futile C-terminal closure and is locked in the "off" position in the absence of bound IPP.The findings of this study can be exploited for structure-guided optimization of existing inhibitors as well as development of new pharmacophores.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Canada.

ABSTRACT

Background: Human farnesyl pyrophosphate synthase (FPPS) controls intracellular levels of farnesyl pyrophosphate, which is essential for various biological processes. Bisphosphonate inhibitors of human FPPS are valuable therapeutics for the treatment of bone-resorption disorders and have also demonstrated efficacy in multiple tumor types. Inhibition of human FPPS by bisphosphonates in vivo is thought to involve closing of the enzyme's C-terminal tail induced by the binding of the second substrate isopentenyl pyrophosphate (IPP). This conformational change, which occurs through a yet unclear mechanism, seals off the enzyme's active site from the solvent environment and is essential for catalysis. The crystal structure of human FPPS in complex with a novel bisphosphonate YS0470 and in the absence of a second substrate showed partial ordering of the tail in the closed conformation.

Results: We have determined crystal structures of human FPPS in ternary complex with YS0470 and the secondary ligands inorganic phosphate (Pi), inorganic pyrophosphate (PPi), and IPP. Binding of PPi or IPP to the enzyme-inhibitor complex, but not that of Pi, resulted in full ordering of the C-terminal tail, which is most notably characterized by the anchoring of the R351 side chain to the main frame of the enzyme. Isothermal titration calorimetry experiments demonstrated that PPi binds more tightly to the enzyme-inhibitor complex than IPP, and differential scanning fluorometry experiments confirmed that Pi binding does not induce the tail ordering. Structure analysis identified a cascade of conformational changes required for the C-terminal tail rigidification involving Y349, F238, and Q242. The residues K57 and N59 upon PPi/IPP binding undergo subtler conformational changes, which may initiate this cascade.

Conclusions: In human FPPS, Y349 functions as a safety switch that prevents any futile C-terminal closure and is locked in the "off" position in the absence of bound IPP. Q242 plays the role of a gatekeeper and directly controls the anchoring of R351 side chain. The interactions between the residues K57 and N59 and those upstream and downstream of Y349 are likely responsible for the switch activation. The findings of this study can be exploited for structure-guided optimization of existing inhibitors as well as development of new pharmacophores.

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Structures of human FPPS ternary complexes at the IPP sub-pocket and the 350KRRK353 tail regions. (A) The cavity of the IPP sub-pocket is filled with ordered water molecules as well as the bound Pi. The water molecules, which are displayed as red spheres, neutralize the surrounding charged residues and also provide structural support via hydrogen bonds (yellow dashes) to this flexible region. The yellow spheres represent the two water molecules present in the FPPS-YS0470 binary complex [PDB: 4DEM] but not in the FPPS-YS0470-Pi ternary complex, and the grey dashes show their interactions with the adjacent entities. (B) The 350KRRK353 tail is in the same conformation as that previously seen in the FPPS-YS0470 binary complex. Compound YS0470 and Pi are bound deeper in the active site and too far from the C-terminal region to have any direct interaction with the tail residues. (C and E) The phosphate groups of the bound PPi and IPP superpose very well. Other bound water molecules and water-mediated hydrogen bonding interactions are omitted for clarity. We have sub-divided the PPi binding site into the alpha- and beta-sites for easier description. (D and F) The 350KRRK353 tail is in the fully closed conformation in the both complexes. The grey meshes represent simulated-annealing omit (Fo-Fc) maps contoured at 3.0 sigma level. Carbon atoms are represented in green, cyan, and magenta, for the FPPS-YS0470-Pi, FPPS-YS0470-PPi, and FPPS-YS0470-IPP ternary complexes, respectively. The color schemes for other atoms (red for oxygen; blue for nitrogen; and orange for phosphorous) are consistent throughout all the figures.
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Figure 2: Structures of human FPPS ternary complexes at the IPP sub-pocket and the 350KRRK353 tail regions. (A) The cavity of the IPP sub-pocket is filled with ordered water molecules as well as the bound Pi. The water molecules, which are displayed as red spheres, neutralize the surrounding charged residues and also provide structural support via hydrogen bonds (yellow dashes) to this flexible region. The yellow spheres represent the two water molecules present in the FPPS-YS0470 binary complex [PDB: 4DEM] but not in the FPPS-YS0470-Pi ternary complex, and the grey dashes show their interactions with the adjacent entities. (B) The 350KRRK353 tail is in the same conformation as that previously seen in the FPPS-YS0470 binary complex. Compound YS0470 and Pi are bound deeper in the active site and too far from the C-terminal region to have any direct interaction with the tail residues. (C and E) The phosphate groups of the bound PPi and IPP superpose very well. Other bound water molecules and water-mediated hydrogen bonding interactions are omitted for clarity. We have sub-divided the PPi binding site into the alpha- and beta-sites for easier description. (D and F) The 350KRRK353 tail is in the fully closed conformation in the both complexes. The grey meshes represent simulated-annealing omit (Fo-Fc) maps contoured at 3.0 sigma level. Carbon atoms are represented in green, cyan, and magenta, for the FPPS-YS0470-Pi, FPPS-YS0470-PPi, and FPPS-YS0470-IPP ternary complexes, respectively. The color schemes for other atoms (red for oxygen; blue for nitrogen; and orange for phosphorous) are consistent throughout all the figures.

Mentions: The overall structure of the human FPPS-YS0470-Pi ternary complex is very similar to that of the binary complex, with the backbone RMSD value of 0.19 Å. The Pi molecule, found at the previously described IPP binding site [9,10], forms direct polar contacts with the main chain of K57 and the side chain of R113 (Figure 2A). In addition, the ligand is further stabilized by an intricate network of water-mediated hydrogen bonds involving a number of residues including N59, R60, E93, and R112 (Figure 2A). The space occupied by Pi in the ternary complex instead accommodates a water molecule with a high B-factor in the binary complex. Another difference between the two structures is the absence of two ordered water molecules (shown as yellow spheres in Figure 2A) near the C-terminal residue K353 in the ternary complex. The reason for this discrepancy is unclear, but these water molecules may contribute to the closing of the 350KRRK353 tail by bridging hydrogen bond interactions between K353 and the residues R112, R113, and T255 (Figure 2A).


Ternary complex structures of human farnesyl pyrophosphate synthase bound with a novel inhibitor and secondary ligands provide insights into the molecular details of the enzyme's active site closure.

Park J, Lin YS, De Schutter JW, Tsantrizos YS, Berghuis AM - BMC Struct. Biol. (2012)

Structures of human FPPS ternary complexes at the IPP sub-pocket and the 350KRRK353 tail regions. (A) The cavity of the IPP sub-pocket is filled with ordered water molecules as well as the bound Pi. The water molecules, which are displayed as red spheres, neutralize the surrounding charged residues and also provide structural support via hydrogen bonds (yellow dashes) to this flexible region. The yellow spheres represent the two water molecules present in the FPPS-YS0470 binary complex [PDB: 4DEM] but not in the FPPS-YS0470-Pi ternary complex, and the grey dashes show their interactions with the adjacent entities. (B) The 350KRRK353 tail is in the same conformation as that previously seen in the FPPS-YS0470 binary complex. Compound YS0470 and Pi are bound deeper in the active site and too far from the C-terminal region to have any direct interaction with the tail residues. (C and E) The phosphate groups of the bound PPi and IPP superpose very well. Other bound water molecules and water-mediated hydrogen bonding interactions are omitted for clarity. We have sub-divided the PPi binding site into the alpha- and beta-sites for easier description. (D and F) The 350KRRK353 tail is in the fully closed conformation in the both complexes. The grey meshes represent simulated-annealing omit (Fo-Fc) maps contoured at 3.0 sigma level. Carbon atoms are represented in green, cyan, and magenta, for the FPPS-YS0470-Pi, FPPS-YS0470-PPi, and FPPS-YS0470-IPP ternary complexes, respectively. The color schemes for other atoms (red for oxygen; blue for nitrogen; and orange for phosphorous) are consistent throughout all the figures.
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Figure 2: Structures of human FPPS ternary complexes at the IPP sub-pocket and the 350KRRK353 tail regions. (A) The cavity of the IPP sub-pocket is filled with ordered water molecules as well as the bound Pi. The water molecules, which are displayed as red spheres, neutralize the surrounding charged residues and also provide structural support via hydrogen bonds (yellow dashes) to this flexible region. The yellow spheres represent the two water molecules present in the FPPS-YS0470 binary complex [PDB: 4DEM] but not in the FPPS-YS0470-Pi ternary complex, and the grey dashes show their interactions with the adjacent entities. (B) The 350KRRK353 tail is in the same conformation as that previously seen in the FPPS-YS0470 binary complex. Compound YS0470 and Pi are bound deeper in the active site and too far from the C-terminal region to have any direct interaction with the tail residues. (C and E) The phosphate groups of the bound PPi and IPP superpose very well. Other bound water molecules and water-mediated hydrogen bonding interactions are omitted for clarity. We have sub-divided the PPi binding site into the alpha- and beta-sites for easier description. (D and F) The 350KRRK353 tail is in the fully closed conformation in the both complexes. The grey meshes represent simulated-annealing omit (Fo-Fc) maps contoured at 3.0 sigma level. Carbon atoms are represented in green, cyan, and magenta, for the FPPS-YS0470-Pi, FPPS-YS0470-PPi, and FPPS-YS0470-IPP ternary complexes, respectively. The color schemes for other atoms (red for oxygen; blue for nitrogen; and orange for phosphorous) are consistent throughout all the figures.
Mentions: The overall structure of the human FPPS-YS0470-Pi ternary complex is very similar to that of the binary complex, with the backbone RMSD value of 0.19 Å. The Pi molecule, found at the previously described IPP binding site [9,10], forms direct polar contacts with the main chain of K57 and the side chain of R113 (Figure 2A). In addition, the ligand is further stabilized by an intricate network of water-mediated hydrogen bonds involving a number of residues including N59, R60, E93, and R112 (Figure 2A). The space occupied by Pi in the ternary complex instead accommodates a water molecule with a high B-factor in the binary complex. Another difference between the two structures is the absence of two ordered water molecules (shown as yellow spheres in Figure 2A) near the C-terminal residue K353 in the ternary complex. The reason for this discrepancy is unclear, but these water molecules may contribute to the closing of the 350KRRK353 tail by bridging hydrogen bond interactions between K353 and the residues R112, R113, and T255 (Figure 2A).

Bottom Line: Isothermal titration calorimetry experiments demonstrated that PPi binds more tightly to the enzyme-inhibitor complex than IPP, and differential scanning fluorometry experiments confirmed that Pi binding does not induce the tail ordering.In human FPPS, Y349 functions as a safety switch that prevents any futile C-terminal closure and is locked in the "off" position in the absence of bound IPP.The findings of this study can be exploited for structure-guided optimization of existing inhibitors as well as development of new pharmacophores.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Canada.

ABSTRACT

Background: Human farnesyl pyrophosphate synthase (FPPS) controls intracellular levels of farnesyl pyrophosphate, which is essential for various biological processes. Bisphosphonate inhibitors of human FPPS are valuable therapeutics for the treatment of bone-resorption disorders and have also demonstrated efficacy in multiple tumor types. Inhibition of human FPPS by bisphosphonates in vivo is thought to involve closing of the enzyme's C-terminal tail induced by the binding of the second substrate isopentenyl pyrophosphate (IPP). This conformational change, which occurs through a yet unclear mechanism, seals off the enzyme's active site from the solvent environment and is essential for catalysis. The crystal structure of human FPPS in complex with a novel bisphosphonate YS0470 and in the absence of a second substrate showed partial ordering of the tail in the closed conformation.

Results: We have determined crystal structures of human FPPS in ternary complex with YS0470 and the secondary ligands inorganic phosphate (Pi), inorganic pyrophosphate (PPi), and IPP. Binding of PPi or IPP to the enzyme-inhibitor complex, but not that of Pi, resulted in full ordering of the C-terminal tail, which is most notably characterized by the anchoring of the R351 side chain to the main frame of the enzyme. Isothermal titration calorimetry experiments demonstrated that PPi binds more tightly to the enzyme-inhibitor complex than IPP, and differential scanning fluorometry experiments confirmed that Pi binding does not induce the tail ordering. Structure analysis identified a cascade of conformational changes required for the C-terminal tail rigidification involving Y349, F238, and Q242. The residues K57 and N59 upon PPi/IPP binding undergo subtler conformational changes, which may initiate this cascade.

Conclusions: In human FPPS, Y349 functions as a safety switch that prevents any futile C-terminal closure and is locked in the "off" position in the absence of bound IPP. Q242 plays the role of a gatekeeper and directly controls the anchoring of R351 side chain. The interactions between the residues K57 and N59 and those upstream and downstream of Y349 are likely responsible for the switch activation. The findings of this study can be exploited for structure-guided optimization of existing inhibitors as well as development of new pharmacophores.

Show MeSH
Related in: MedlinePlus