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 et al.; licensee BioMed Central Ltd. 2012
Received: 5 October 2012
Accepted: 7 December 2012
Published: 12 December 2012
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.
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.
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.
KeywordsMevalonate pathway Farnesyl pyrophosphate synthase Isopentenyl pyrophosphate Bisphosphonates C-terminal tail closure Cancer chemotherapeutics
The mevalonate pathway produces essential lipid molecules, such as steroids and isoprenoids, in mammalian cells. Occupying the first branching point of the mevalonate pathway, farnesyl pyrophosphate synthase (FPPS) catalyzes the sequential elongation of dimethylallyl pyrophosphate (DMAPP) to geranyl pyrophosphate (GPP) and then to farnesyl pyrophosphate (FPP) via successive condensation of two isopentenyl pyrophosphate (IPP) molecules. The subsequent condensation of FPP and another IPP unit in the pathway produces geranylgeranyl pyrophosphate (GGPP). Covalent attachment of FPP and GGPP (i.e. prenylation) is critical for the proper subcellular localization and function of many proteins, including small GTPases that regulate a wide variety of cellular processes . FPPS, therefore, is an attractive point of pharmacological intervention. Bisphosphonate inhibitors of FPPS, for instance, are widely used to treat a number of bone disorders, such as Paget’s disease, hypercalcemia, metastatic osteolysis, and osteoporosis currently . Given the crucial roles of small GTPases in cancer development [3–5], the ability of bisphosphonates to inhibit FPPS and consequently the prenylation of these proteins qualifies them also as potential cancer chemotherapeutics. In addition, FPPS inhibition produces a secondary anticancer effect via the accumulation of IPP, which activates human γδ T immune cells [6, 7]. Recent clinical studies have demonstrated that bisphosphonate drugs enhance the antitumor effects of various existing therapeutics synergistically and improve survival in patients with prostate cancer, breast cancer, and multiple myeloma .
The mechanism by which bisphosphonates inhibit human FPPS has been examined through characterization of X-ray crystal structures. These compounds bind at the DMAPP/GPP sub-pocket of the enzyme’s active site, mimicking and competing against these substrates [9, 10]. The enzyme inhibition also involves a sequence of conformational changes [9, 10]. Bisphosphonate binding to the “open” form of the enzyme drives a rigid body movement that closes the entrance to the DMAPP/GPP sub-pocket. This conformational change fully shapes the second substrate binding site, the IPP sub-pocket. Subsequent IPP binding to the now “partially closed” enzyme induces the closing of the 350KRRK353 C-terminal tail, which is disordered in the absence of bound IPP, over the IPP entry site. These C-terminal basic residues are essential for catalysis , and upon closing secure the ligands into position and sequester the catalytic cavity from water. With the enzyme in this “fully closed” state, replacement of the deeply buried bisphosphonate inhibitor by a competing substrate is very difficult, and hence the binding of the bisphosphonate is deemed nearly irreversible. The exceptional in vivo efficacy of bisphosphonate drugs, therefore, is thought to arise in part from the stabilization of the enzyme-inhibitor complex by the binding of the accumulating substrate IPP . Despite its importance, however, the molecular details responsible for the tail closure in human FPPS are largely uncharacterized. That this conformational change is induced by IPP is especially intriguing, as the ligand does not make any direct contact with the 350KRRK353 tail when bound to the enzyme.
Crystal-soaking and data collection
Expression and purification of recombinant human FPPS, as well as crystallization of the enzyme-YS0470 binary complex, were carried out as previously described . Aqueous solutions of Pi and PPi were prepared at 5 mM concentration. IPP was prepared at 4.5 mM concentration in 1 M Tris buffer. For each ligand, 0.4 μL of the stock solution was added to a 2 μL hanging drop containing two to three single crystals. The coverslips holding the ligand-added drops were resealed over the same reservoir wells in the original crystallization tray, which was then incubated overnight to ensure maximal diffusion of the ligand into the crystals. Diffraction data were collected from a single crystal for each ligand under a nitrogen cryo-stream as 0.5° oscillation images by using a Rigaku RUH3R X-ray generator with a rotating Copper anode and a Rigaku R-AXIS IV++ area detector.
Data processing and structure refinement
Data collection and refinement statistics
Unit cell dimension (Å)
a = b = 110.98, c = 66.79
a = b = 111.53, c = 66.24
a = b = 111.24, c = 65.65
Resolution range (Å)
No. protein atoms
No. ligand atoms
No. ion atoms
No. solvent atoms
Bond length (Å)
Bond angle (°)
Human FPPS structure models analyzed in this study
Isothermal titration calorimetry
ITC experiments were performed with a VP-ITC titration calorimeter from GE Healthcare. The human FPPS sample was prepared at 50 μM concentration in the presence of 150 μM YS0470 and 200 μM MgCl2 in a buffer containing 10 mM HEPES (pH 7.5), 500 mM NaCl, 2 mM β-mercaptoethanol, and 5% glycerol. Ligand solutions were prepared in the same buffer at concentrations ranging from 0.1 to 1 mM. Titrations were carried out at 30 °C, and the data were analyzed with the Origin 7 software provided by the manufacturer.
Differential scanning fluorometry
The samples were prepared to the final volume of 40 μL in the following concentrations: 4 μM protein, 10 mM HEPES (pH 7.5), 10 mM NaCl, 5 mM MgCl2, and 5× SYPRO Orange dye (Invitrogen, commercial stock solution is 5000×). When added, the final concentration of YS0470 was 40 μM, and those of the secondary ligands (Pi, PPi, and IPP) were up to 400 μM. All samples were prepared in triplicate. Fluorescence was measured by using an iCycler RT-PCR instrument with an iQ5 detector (Bio-Rad) while heating the samples in a gradient from 30 to 90°C in 0.5°C steps every 10 s. The midpoint temperature of the unfolding transition (T m ) was calculated by using the software package Bio-Rad iQ5.
Results and discussion
Structures of the human FPPS ternary complexes
We were able to obtain crystals of human FPPS in ternary complex with different ligands. X-ray diffraction analysis of these crystals has clearly identified the secondary ligands (i.e. Pi, PPi, and IPP) bound in the IPP sub-pocket, as well as the inhibitor YS0470 in the DMAPP/GPP sub-pocket. Co-binding of the secondary ligands did not affect the pre-bound inhibitor, as the binding location and orientation of this inhibitor, as well as its interaction with the enzyme, remain unchanged in the three ternary complexes from those previously observed in the binary complex [PDB: 4DEM] .
The 350KRRK353 tail of the Pi-bound ternary complex is similar to that of the binary complex, with flexible and thus undefined side chains (Figure 2B). Although the backbone atoms of the tail could be modeled in and refined without much difficulty during the structure refinement process, the electron density around this area was weak (Figure 2B), suggesting partial ordering. A direct hydrogen bond between the side chain of E318 and the main chain nitrogen of K350, and a water-mediated hydrogen bond between the main chain nitrogen of D251 and the main chain oxygen of R352, may be responsible for the partial ordering of the tail at this location (Figure 2B). The water molecule mediating the latter hydrogen bonding interaction is well ordered, as also seen in the binary complex previously . The side chain nitrogen of Q242 is within the hydrogen bonding range to the main chain oxygen of R351 (Figure 2B), but such an interaction is not likely, as the angle between the hydrogen donor and acceptor atoms is suboptimal in the current conformational state.
The overall structures of the PPi- and IPP-bound ternary complexes are more similar to each other (RMSD: 0.16 Å) than to those of the binary complex and the Pi-bound ternary complex (RMSD: 0.25-0.31 Å). The PPi and IPP-binding sites fully overlap the Pi-binding site, which we refer to as the beta phosphate site (Figure 2A, C, and E). The space that accommodates the alpha phosphate moiety of IPP, or the alpha phosphate site (Figure 2E), is instead occupied by a single water molecule in both the Pi-bound ternary complex (Figure 2A) and the binary complex [PDB: 4DEM]. The phosphate moiety at the alpha site provides additional charge interactions with the residues R60 and Q112 in both the PPi- and IPP-bound complexes (Figure 2C and E). The isopentenyl tail of the bound IPP ligand extends toward the bisphosphonate inhibitor YS0470 (Figure 2E), displacing water molecules otherwise present in the area (Figure 2A and C). These water molecules in the PPi-bound complex form a tight set of hydrogen bonds connecting the PPi and bisphosphonate molecules and the residues Y204 and Q240 (Figure 2C). Other water molecules and water-mediated interactions around the secondary ligands in the PPi- and IPP-bound complexes are analogous to those in the Pi-bound ternary complex and the binary complex.
The 350KRRK353 tail in both the PPi- and IPP-bound ternary complexes is closed and fully ordered, as evidenced by the clearly defined electron density (Figure 2D and F), as well as the average B-factors of the four C-terminal residues (Additional file 1: Table S1). This conformational state is most notably characterized by the rigidification of R351 (Figure 2D and F), as also observed in the human FPPS-zoledronate-IPP ternary complex [PDB: 1ZW5 and 2F8Z] previously [9, 10]. All three side chain nitrogen atoms of R351 are involved in either direct or water-mediated hydrogen bonding to the neighbouring residues F239, Q242, D243, and K353, thereby holding the tail tightly in position (Figure 2D and F). The side chain of K57 in the PPi- and IPP-bound complexes is also more ordered compared to that of the Pi-bound complex, forming hydrogen bonds to the secondary ligand PPi or IPP and the terminal oxygen atom of K353 (Figure 2B, D, and F). The structures of our PPi- and IPP-bound enzyme complexes thus represent the “fully closed” state, in contrast to the structure of our “partially closed” Pi-bound complex (summarized in Table 2).
Binding of Pi, PPi, and IPP to the human FPPS-YS0470 binary complex
In this work, we have shown for the first time that the binding of PPi induces the full closure of the 350KRRK353 tail in human FPPS. This finding is interesting because PPi, unlike IPP, is not a substrate for the enzyme. The PPi molecule in our ternary complex does not represent a product state either, although the FPPS reaction produces PPi, as it is in the IPP sub-pocket. The transfer of the allylic chain in this reaction, which is from DMAPP or GPP to IPP [17, 18], should leave the product PPi in the DMAPP/GPP sub-pocket. The PPi-induced C-terminal tail closure also demonstrates that the presence of the isopentenyl moiety on the secondary ligand is not a necessary condition for this conformational change in the human FPPS ternary complex. Rather, it is the phosphate moiety occupying the alpha phosphate site that is decisively responsible for the tail closure of the protein. The structure of our Pi-bound ternary complex, as well as those previously determined by others (Table 2), indicates that the occupancy of the beta phosphate site by a single phosphate group alone cannot induce this conformational change.
In order to confirm that PPi indeed forms a tighter complex with human FPPS and YS0470 than IPP, we have carried out ITC experiments in which the secondary ligands were titrated into a protein sample pre-saturated with the inhibitor and magnesium ions. The dissociation constant (Kd) was determined to be lower for PPi (Figure 3B), indicating that PPi-binding is indeed stronger than IPP-binding. The higher binding affinity of PPi is due to a more favorable (more negative) enthalpy change (ΔH, Figure 3B), which corroborates our prediction that the strength of interactions between the ligand and the protein-inhibitor complex is greater for PPi. The entropic component of binding (T ΔS), on the other hand, was shown to be more favorable (less negative) for IPP (Figure 3B), although not sufficient to fully counter the enthalpic effect. The difference in the entropy change is likely due to desolvation effects: more water molecules would be released from the binding site and the ligand itself upon IPP-binding, resulting in greater degrees of freedom in the system. In contrast to the solvation entropy change, the conformational entropy change should be slightly less favorable for IPP binding, as IPP with more rotatable bonds within the molecule than PPi would lose greater degrees of conformational freedom upon binding. Changes in protein conformational entropy resulting from ligand binding should be similar between PPi and IPP, based on our structural data.
We have also examined the binding of Pi to the human FPPS-YS0470 complex by ITC, but could not reliably determine its energetic profile due to a low heat signal. The weak thermodynamic signature likely reflects low affinity binding, as well as the absence of a major conformational change (e.g., the C-terminal tail closure) induced by the binding. Complimentary DSF experiments indeed confirmed the lack of any significant conformational change induced by Pi binding. As seen in Figure 3C, the T m of human FPPS increases ~10°C in the presence of YS0470, indicating that the enzyme is more thermally stable in its partially closed state than in the open state. Addition of the secondary ligands PPi and IPP further stabilizes the enzyme, likely via the full closure of the enzyme, whereas Pi does not provide any additional thermal protection, indicating the lack thereof. It is interesting here that the human FPPS complex shows a higher T m in the presence of IPP (80°C) than with PPi (75°C). These values are seemingly at odds with the results of the ITC experiments, suggesting that IPP forms a tighter complex with human FPPS and YS0470 than PPi. However, as described earlier, PPi binding results in a more favorable enthalpy change (ΔH) than IPP binding but a less favorable entropy change (ΔS). The entropic effects of course become increasingly pronounced for ligand binding as the temperature rises. Based on the ΔH and ΔS values determined from the ITC experiments (Figure 3B), the binding of IPP to the human FPPS-YS0470 complex becomes more favorable than that of PPi only at temperatures above ~70°C.
Mechanistic details of the C-terminal tail closure in human FPPS
As mentioned previously, the molecular details responsible for the tail closing action in human FPPS are largely unknown, despite its functional importance. What is clear, however, is that the role of the R351 side chain is absolutely critical in the full closing of the 350KRRK353 tail. This side chain not only anchors the residue itself to the 221G-E247 helix, one of the longest central helices of human FPPS, but also helps hold the last residue K353 in position by providing a salt bridge (as seen in Figure 2D and F). The electron density observed for our Pi-bound complex has demonstrated that the side chain of R351 can still be entirely flexible, while the main chain of the C-terminal tail is partially ordered and structured (as seen in Figure 2B). This finding suggests that the recruitment of the tail to the approximate region occurs first, where the tail is held loosely by other interactions perhaps involving those described earlier (Figure 2A and B), prior to the rigidification of the R351 side chain.
Despite the many currently available FPPS structures, it is still unclear how PPi/IPP binding turns on the Y349 switch in the human enzyme. This process is particularly intriguing, as the binding site for the secondary ligands is quite far (> 10 Å) from the tyrosine residue, whose conformational change is yet very drastic (i.e. ~80° rotation of the side chain). Comparison of the new ternary structures has allowed us to propose the following putative mechanism. Simultaneous occupancy of the alpha and beta phosphate sites by a pyrophosphate group in the IPP sub-pocket rigidifies the otherwise flexible K57 side chain (Figure 4B). The side chain of K57 in turn attracts the 350KRRK353 tail, which is partially present and/or structured in the vicinity, by forming a salt bridge with the terminal carboxyl group (Figure 4B). This ~0.7 Å shift of the tail is not likely a result of a modeling artefact, as it is accompanied by a similar movement in the adjacent 250G-T255 helix-turn segment (Figure 4B), of which such a shift induced by IPP binding has been also observed in the zoledronate-bound enzyme complexes [PDB: 2F8C, 2F8Z, and 1ZW5]. In addition to the rigidification of the K57 side chain, PPi/IPP binding results in a ~10° angular shift of the N59 side chain, which is translated into a similar movement of the residues K347 and I348 via a water molecule (Figure 4B). Interestingly, no water molecule has ever been observed at this position in human FPPS, except in the presence of bound PPi or IPP. These PPi/IPP-induced interactions between K57 and the residues downstream of Y349, and between N59 and those immediately upstream, may create a torque strong enough to rotate the Y349 side chain out of its “off” conformation (Figure 4B).
Implications of the C-terminal tail closure on drug development
The activity of human FPPS involves a dynamic behavior, in which the enzyme alternates between the substrate-binding (open/partially closed) and catalytic (fully closed) states. As biasing this equilibrium either way results in enzyme inhibition, compounds that can inhibit the enzyme’s C-terminal tail closure can also inhibit its function. The non-bisphosphonate inhibitors of human FPPS identified in a recent fragment-based screening study  are of much relevance. These inhibitors, which act by binding to an allosteric site adjacent to the IPP sub-pocket, have completely different chemical scaffolds from the existing bisphosphonate drugs and thus a great potential as chemotherapeutic agents to target non-bone tissues. The mechanism of inhibition by this series of compounds is thought to involve a carboxylic functional group that interferes with the binding of IPP by electrostatic repulsion . In addition, some of these compounds that are bulkier (e.g., FBS_04 and NOV_980, in the PDB structures 3N45 and 3N46, respectively) directly inhibit the closing of the C-terminal tail by steric hindrance . Intriguingly, the location of the newly identified allosteric pocket is such that N59 and K347, which mediate the communication between the IPP sub-pocket and the tyrosine switch according to our proposed mechanism (as seen in Figure 4B), form opposite walls of this pocket. We predict that, therefore, compounds which occupy the allosteric pocket of human FPPS would inhibit its tail closure even in the presence of bound IPP, by disrupting the interaction between N59 and K347. Based on this prediction, then, elimination of the carboxylic group from the aforementioned allosteric inhibitors should not affect their ability to inhibit the enzyme, although their binding affinity would likely be changed. Removal of this highly charged moiety or its replacement with a preferable functional substituent may improve the pharmacokinetic properties of these compounds, proving the usefulness of the novel insights regarding the human FPPS tail closure provided in the present communication. Alternatively, by taking advantage of the new information, it may be possible to develop inhibitors that can trigger the C-terminal tail closure without IPP and lock the enzyme in the fully closed state, as the concept for such inhibitors was proposed recently .
We have previously shown that the 350KRRK353 tail in the human FPPS-YS0470 binary complex is only partially structured. Here we have demonstrated that the binding of PPi or IPP to the IPP sub-pocket of the binary complex, but not that of Pi, fully structures the enzyme’s tail, which seals off the active site from the solvent environment and thereby stabilizes the protein-inhibitor complex. By examining the human FPPS structures presented in this report and those previously determined by others, we have also identified key residues and interactions responsible for this C-terminal rigidification. The tail closure is controlled by a safety mechanism involving the residue Y349, which is trapped in the “off” conformation in the absence of bound PPi/IPP. Turning on the tyrosine switch allows the gatekeeper Q242 to take on the anchor-accepting conformation, which in turn results in the clamping of R351 to the enzyme’s main frame. The rigidification of the anchor arginine is likely the final step in the sequence of events required for the complete closure of the 350KRRK353 tail. The process by which PPi/IPP-binding activates the tyrosine switch is not entirely clear, but we speculate that it involves subtle rotations and shifts initiated in the residues around the IPP sub-pocket and transmitted to those directly preceding and succeeding the tyrosine switch. The structures of the new ternary complexes of human FPPS will prove useful for optimizing clinically relevant inhibitors. Further, the novel insights with respect to the enzyme’s tail closing mechanism may help develop entirely new pharmacophores.
Farnesyl pyrophosphate synthase
Isothermal titration calorimetry
Differential scanning fluorometry
The authors thank members of the YST lab and the AMB lab for helpful discussions and technical advice, especially Mr. Dmitry Rodionov for the diffraction data collection and Mr. Brahm Yachnin for the data processing. The authors also thank Ms. Nozhat Safaee from the Kalle Gehring lab at McGill Univ. for her help with the ITC experiments. Financial support for this research was provided by a grant from NSERC awarded to YST and a grant from CIHR awarded to AMB. We also thank other funding agencies: JP is a recipient of the FRSQ (Fonds de la Recherche en Santé du Québec) Postdoctoral Training Award; Y-SL and JWDS are recipients of awards from the CIHR DDTP (McGill University); and AMB holds a Canada Research Chair in Structural Biology.
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