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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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Ligand binding to human FPPS. (A) The active site structures of the FPPS-YS0470-PPi complex (cyan) and the FPPS-zoledronate-IPP complex (white) [PDB: 1ZW5] are presented superimposed. The IPP molecule in the zoledronate complex is not shown for clear visualization of the bisphosphonates. The hydroxyl ‘hook’ of the bound zoledronate is circled in red. This functional group forms a hydrogen bond with the side chain of D243, which is represented as a grey dashed line in the illustration. The orange dashed lines represent stacking interactions between YS0470 and the residues F99 and Q171. (B) ITC isotherms for the binding of PPi and IPP to the human FPPS-YS0470 complex. Three independent experiments were carried out for each ligand. The binding parameters were determined from each data set by using a one-site model. The reported values are Mean ± SEM from the three experiments. T = 303.15 kelvin. (C) A bar graph showing melting temperatures of human FPPS in various ligand-bound states measured by DSF. In order to ensure complete complex formation, YS0470 and the secondary ligands were added at 10- and 100-fold molar excess of the protein, respectively.
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Figure 3: Ligand binding to human FPPS. (A) The active site structures of the FPPS-YS0470-PPi complex (cyan) and the FPPS-zoledronate-IPP complex (white) [PDB: 1ZW5] are presented superimposed. The IPP molecule in the zoledronate complex is not shown for clear visualization of the bisphosphonates. The hydroxyl ‘hook’ of the bound zoledronate is circled in red. This functional group forms a hydrogen bond with the side chain of D243, which is represented as a grey dashed line in the illustration. The orange dashed lines represent stacking interactions between YS0470 and the residues F99 and Q171. (B) ITC isotherms for the binding of PPi and IPP to the human FPPS-YS0470 complex. Three independent experiments were carried out for each ligand. The binding parameters were determined from each data set by using a one-site model. The reported values are Mean ± SEM from the three experiments. T = 303.15 kelvin. (C) A bar graph showing melting temperatures of human FPPS in various ligand-bound states measured by DSF. In order to ensure complete complex formation, YS0470 and the secondary ligands were added at 10- and 100-fold molar excess of the protein, respectively.

Mentions: A crucial implication of the PPi-induced C-terminal tail closure in human FPPS is that PPi, which is a prevalent cellular metabolite, may have a relevant role in the inhibition of the enzyme in vivo. Based on the structures of our PPi and IPP-bound ternary complexes, which are both in the fully closed state, PPi would at least be equally effective as IPP in stabilizing the protein-inhibitor complex, if not better. The inner surface of the IPP sub-pocket of FPPS is highly hydrophilic, and the hydrophobic isopentenyl tail of IPP is forced to stack against a number of hydrophilic side chain functional groups upon its binding to the enzyme. These interactions may be beneficial for the catalysis and the subsequent release of the product in the isoprenoid elongation reaction, but not for stabilizing the enzyme-bisphosphonate complex in the closed state. With PPi binding, on the other hand, the water molecules bound in place of the isopentenyl tail of IPP may further stabilize the protein-inhibitor complex by linking the ligands and the surrounding residues. These water molecules play an especially important role in the binding of our inhibitor YS0470, which lacks the hydroxyl ‘hook’ present in most of the currently used bisphosphonate drugs, by making up for the missing polar interaction seen in the presence of this functional group (Figure 3A). The absence of this hydroxyl side chain in our series of bisphosphonates is advantageous for the purpose of targeting non-bone tissues, because the presence of this substituent in a bisphosphonate increases its affinity for bone mineral [19,20].


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)

Ligand binding to human FPPS. (A) The active site structures of the FPPS-YS0470-PPi complex (cyan) and the FPPS-zoledronate-IPP complex (white) [PDB: 1ZW5] are presented superimposed. The IPP molecule in the zoledronate complex is not shown for clear visualization of the bisphosphonates. The hydroxyl ‘hook’ of the bound zoledronate is circled in red. This functional group forms a hydrogen bond with the side chain of D243, which is represented as a grey dashed line in the illustration. The orange dashed lines represent stacking interactions between YS0470 and the residues F99 and Q171. (B) ITC isotherms for the binding of PPi and IPP to the human FPPS-YS0470 complex. Three independent experiments were carried out for each ligand. The binding parameters were determined from each data set by using a one-site model. The reported values are Mean ± SEM from the three experiments. T = 303.15 kelvin. (C) A bar graph showing melting temperatures of human FPPS in various ligand-bound states measured by DSF. In order to ensure complete complex formation, YS0470 and the secondary ligands were added at 10- and 100-fold molar excess of the protein, respectively.
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Related In: Results  -  Collection

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Figure 3: Ligand binding to human FPPS. (A) The active site structures of the FPPS-YS0470-PPi complex (cyan) and the FPPS-zoledronate-IPP complex (white) [PDB: 1ZW5] are presented superimposed. The IPP molecule in the zoledronate complex is not shown for clear visualization of the bisphosphonates. The hydroxyl ‘hook’ of the bound zoledronate is circled in red. This functional group forms a hydrogen bond with the side chain of D243, which is represented as a grey dashed line in the illustration. The orange dashed lines represent stacking interactions between YS0470 and the residues F99 and Q171. (B) ITC isotherms for the binding of PPi and IPP to the human FPPS-YS0470 complex. Three independent experiments were carried out for each ligand. The binding parameters were determined from each data set by using a one-site model. The reported values are Mean ± SEM from the three experiments. T = 303.15 kelvin. (C) A bar graph showing melting temperatures of human FPPS in various ligand-bound states measured by DSF. In order to ensure complete complex formation, YS0470 and the secondary ligands were added at 10- and 100-fold molar excess of the protein, respectively.
Mentions: A crucial implication of the PPi-induced C-terminal tail closure in human FPPS is that PPi, which is a prevalent cellular metabolite, may have a relevant role in the inhibition of the enzyme in vivo. Based on the structures of our PPi and IPP-bound ternary complexes, which are both in the fully closed state, PPi would at least be equally effective as IPP in stabilizing the protein-inhibitor complex, if not better. The inner surface of the IPP sub-pocket of FPPS is highly hydrophilic, and the hydrophobic isopentenyl tail of IPP is forced to stack against a number of hydrophilic side chain functional groups upon its binding to the enzyme. These interactions may be beneficial for the catalysis and the subsequent release of the product in the isoprenoid elongation reaction, but not for stabilizing the enzyme-bisphosphonate complex in the closed state. With PPi binding, on the other hand, the water molecules bound in place of the isopentenyl tail of IPP may further stabilize the protein-inhibitor complex by linking the ligands and the surrounding residues. These water molecules play an especially important role in the binding of our inhibitor YS0470, which lacks the hydroxyl ‘hook’ present in most of the currently used bisphosphonate drugs, by making up for the missing polar interaction seen in the presence of this functional group (Figure 3A). The absence of this hydroxyl side chain in our series of bisphosphonates is advantageous for the purpose of targeting non-bone tissues, because the presence of this substituent in a bisphosphonate increases its affinity for bone mineral [19,20].

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