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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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Novel bisphosphonate inhibitors of human FPPS. (A) General structure of 2-aminopyridine-based bisphosphonate inhibitors (see ref. [12] for the complete list of functional groups at the substitution site). (B) Compound YS0470.
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Figure 1: Novel bisphosphonate inhibitors of human FPPS. (A) General structure of 2-aminopyridine-based bisphosphonate inhibitors (see ref. [12] for the complete list of functional groups at the substitution site). (B) Compound YS0470.

Mentions: The bisphosphonates in current clinical use, such as risedronate and zoledronate, are highly hydrophilic. The resultant low membrane permeability, as well as their extreme affinity for bone tissue, compromises the full antineoplastic potential of these drugs. In an effort to develop FPPS inhibitors with superior physicochemical properties, we have recently identified potent and selective 2-aminopyridine-based bisphosphonates (Figure 1A) [12]. The crystal structure [PDB: 4DEM] of human FPPS in complex with one of our new inhibitors, YS0470 (Figure 1B), revealed a series of novel interactions and conformations in and around the active site cavity [12]. Most interestingly, the 350KRRK353 tail of the enzyme was partially ordered, with its main chain covering over the IPP sub-pocket but its side chains fully flexible [12]. This finding provided the first report of such a conformational state in human FPPS and raised a question whether the IPP sub-pocket in the enzyme-YS0470 complex is still accessible to a ligand. In order to address this question, 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. Prior to the present communication there have been only two IPP-bound human FPPS structures available, determined independently but both co-bound with zoledronate [9,10], and none with bound PPi. Crystal structure analysis and complementary isothermal titration calorimetry (ITC) and differential scanning fluorometry (DSF) experiments reveal new insights into the details of the ligand-induced conformational changes in human FPPS and suggest a mechanism for the enzyme’s C-terminal tail closure. A better understanding of the human FPPS tail closure may help develop new classes of anticancer drugs that function by targeting this crucial conformational change.


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)

Novel bisphosphonate inhibitors of human FPPS. (A) General structure of 2-aminopyridine-based bisphosphonate inhibitors (see ref. [12] for the complete list of functional groups at the substitution site). (B) Compound YS0470.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539973&req=5

Figure 1: Novel bisphosphonate inhibitors of human FPPS. (A) General structure of 2-aminopyridine-based bisphosphonate inhibitors (see ref. [12] for the complete list of functional groups at the substitution site). (B) Compound YS0470.
Mentions: The bisphosphonates in current clinical use, such as risedronate and zoledronate, are highly hydrophilic. The resultant low membrane permeability, as well as their extreme affinity for bone tissue, compromises the full antineoplastic potential of these drugs. In an effort to develop FPPS inhibitors with superior physicochemical properties, we have recently identified potent and selective 2-aminopyridine-based bisphosphonates (Figure 1A) [12]. The crystal structure [PDB: 4DEM] of human FPPS in complex with one of our new inhibitors, YS0470 (Figure 1B), revealed a series of novel interactions and conformations in and around the active site cavity [12]. Most interestingly, the 350KRRK353 tail of the enzyme was partially ordered, with its main chain covering over the IPP sub-pocket but its side chains fully flexible [12]. This finding provided the first report of such a conformational state in human FPPS and raised a question whether the IPP sub-pocket in the enzyme-YS0470 complex is still accessible to a ligand. In order to address this question, 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. Prior to the present communication there have been only two IPP-bound human FPPS structures available, determined independently but both co-bound with zoledronate [9,10], and none with bound PPi. Crystal structure analysis and complementary isothermal titration calorimetry (ITC) and differential scanning fluorometry (DSF) experiments reveal new insights into the details of the ligand-induced conformational changes in human FPPS and suggest a mechanism for the enzyme’s C-terminal tail closure. A better understanding of the human FPPS tail closure may help develop new classes of anticancer drugs that function by targeting this crucial conformational change.

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