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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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Residues involved in the human FPPS C-terminal tail closure. (A) The structures of the FPPS-YS0470-Pi (green) and FPPS-YS0470-PPi (cyan) complexes are superimposed. The conformational changes that occur prior to the rigidification of the R351 side chain are indicated with black arrows. The residues Y349, F238, and Q242 are in the anchor-blocking conformation in the Pi-bound complex and in the anchor-accepting conformation in the PPi-bound complex. (B) A schematic representation of the Y349 switch activation: the K57 side chain rigidifies and attracts the C-terminal tail; N59 interacts with K347 via a water molecule; and the Y349 side chain rotates out due to the torsion created by these two forces.
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Figure 4: Residues involved in the human FPPS C-terminal tail closure. (A) The structures of the FPPS-YS0470-Pi (green) and FPPS-YS0470-PPi (cyan) complexes are superimposed. The conformational changes that occur prior to the rigidification of the R351 side chain are indicated with black arrows. The residues Y349, F238, and Q242 are in the anchor-blocking conformation in the Pi-bound complex and in the anchor-accepting conformation in the PPi-bound complex. (B) A schematic representation of the Y349 switch activation: the K57 side chain rigidifies and attracts the C-terminal tail; N59 interacts with K347 via a water molecule; and the Y349 side chain rotates out due to the torsion created by these two forces.

Mentions: Analysis of our FPPS structures suggests that proper positioning and ordering of the R351 side chain also requires a series of preceding conformational changes in the residues Q242, F238, and Y349. In the absence of bound PPi/IPP, Q242 forms a hydrogen bond to a nearby water molecule and together with it blocks the anchoring of the R351 side chain to the 221G-E247 helix (Figure 4A). The conformational change in Q242, in turn, requires a ~20° rotational translocation of the F238 side chain, which is prohibited due to steric hindrance by the Y349 side chain in the absence of PPi/IPP (Figure 4A). In this anchor-blocking conformation, the Y349 side chain is stacked tightly in position between the side chains of F238 and Y322, and is further stabilized via a polar interaction with the residue S321 (Figure 4A). In the anchor-accepting conformation, on the other hand, the side chain of Y349, as well as those of the adjacent aromatic residues F238 and Y322, has significantly greater freedom of movement, as evident from the electron density maps and the refined B-factors (Additional file 2: Figure S1). The above findings suggest that Y349, lying upstream in the cascade of these conformational changes, functions as a safety switch, which is normally locked in the “off” mode to prevent any futile C-terminal tail closure. Q242, on the other hand, plays the role of a gatekeeper in the enzyme, which directly controls the anchoring of R351. The greater structural freedom of the three aromatic residues (i.e. F238, Y322, and Y349) in the fully closed form of the enzyme may contribute to compensate for the reduction in conformational entropy caused by the ordering of the tail.


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)

Residues involved in the human FPPS C-terminal tail closure. (A) The structures of the FPPS-YS0470-Pi (green) and FPPS-YS0470-PPi (cyan) complexes are superimposed. The conformational changes that occur prior to the rigidification of the R351 side chain are indicated with black arrows. The residues Y349, F238, and Q242 are in the anchor-blocking conformation in the Pi-bound complex and in the anchor-accepting conformation in the PPi-bound complex. (B) A schematic representation of the Y349 switch activation: the K57 side chain rigidifies and attracts the C-terminal tail; N59 interacts with K347 via a water molecule; and the Y349 side chain rotates out due to the torsion created by these two forces.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Residues involved in the human FPPS C-terminal tail closure. (A) The structures of the FPPS-YS0470-Pi (green) and FPPS-YS0470-PPi (cyan) complexes are superimposed. The conformational changes that occur prior to the rigidification of the R351 side chain are indicated with black arrows. The residues Y349, F238, and Q242 are in the anchor-blocking conformation in the Pi-bound complex and in the anchor-accepting conformation in the PPi-bound complex. (B) A schematic representation of the Y349 switch activation: the K57 side chain rigidifies and attracts the C-terminal tail; N59 interacts with K347 via a water molecule; and the Y349 side chain rotates out due to the torsion created by these two forces.
Mentions: Analysis of our FPPS structures suggests that proper positioning and ordering of the R351 side chain also requires a series of preceding conformational changes in the residues Q242, F238, and Y349. In the absence of bound PPi/IPP, Q242 forms a hydrogen bond to a nearby water molecule and together with it blocks the anchoring of the R351 side chain to the 221G-E247 helix (Figure 4A). The conformational change in Q242, in turn, requires a ~20° rotational translocation of the F238 side chain, which is prohibited due to steric hindrance by the Y349 side chain in the absence of PPi/IPP (Figure 4A). In this anchor-blocking conformation, the Y349 side chain is stacked tightly in position between the side chains of F238 and Y322, and is further stabilized via a polar interaction with the residue S321 (Figure 4A). In the anchor-accepting conformation, on the other hand, the side chain of Y349, as well as those of the adjacent aromatic residues F238 and Y322, has significantly greater freedom of movement, as evident from the electron density maps and the refined B-factors (Additional file 2: Figure S1). The above findings suggest that Y349, lying upstream in the cascade of these conformational changes, functions as a safety switch, which is normally locked in the “off” mode to prevent any futile C-terminal tail closure. Q242, on the other hand, plays the role of a gatekeeper in the enzyme, which directly controls the anchoring of R351. The greater structural freedom of the three aromatic residues (i.e. F238, Y322, and Y349) in the fully closed form of the enzyme may contribute to compensate for the reduction in conformational entropy caused by the ordering of the tail.

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