Human farnesyl pyrophosphate synthase is allosterically inhibited by its own product

Abstract

Farnesyl pyrophosphate synthase (FPPS) is an enzyme of the mevalonate pathway and a well-established therapeutic target. Recent research has focused around a newly identified druggable pocket near the enzyme's active site. Pharmacological exploitation of this pocket is deemed promising; however, its natural biological function, if any, is yet unknown. Here we report that the product of FPPS, farnesyl pyrophosphate (FPP), can bind to this pocket and lock the enzyme in an inactive state. The K_d for this binding is 5-6 μM, within a catalytically relevant range. These results indicate that FPPS activity is sensitive to the product concentration. Kinetic analysis shows that the enzyme is inhibited through FPP accumulation. Having a specific physiological effector, FPPS is a bona fide allosteric enzyme. This allostery offers an exquisite mechanism for controlling prenyl pyrophosphate levels in vivo and thus contributes an additional layer of regulation to the mevalonate pathway.

Figure 1. FPP synthesis and mevalonate pathway.

(a) Catalytic steps of FPPS reaction. (b) Overview of mevalonate pathway and downstream metabolites. Enzymes are shown in Italics. Dotted arrows represent multi-enzyme steps. Sites of intervention by current clinical drugs are indicated. Abbreviations: GGPPS, geranylgeranyl pyrophosphate synthase; HMG CoA, hydroxylmethylglutaryl coenzyme A.

Figure 2. Allosteric binding of FPP to FPPS.

(a) Overall structure, discovery map (inset, green mesh, F_o−F_c contoured at 3σ), and phosphorus anomalous signal (inset, magenta, contoured at 3σ). Only one subunit (the crystallographic asymmetric unit) is shown for clarity; the biological assembly is a homodimer. A stereo image of the final 2F_o−F_c map around the bound ligand is shown in Supplementary Fig. 1. (b) Binding interactions by FPP pyrophosphate. (c) Binding interactions by IPP pyrophosphate (PDB ID 4H5E). (d) FPP in space-filling representation. The surface of the binding pocket is also represented. (e) Induced-fit conformational change accompanying FPP binding. The apo-enzyme structure is shown in grey (PDB ID 2F7M). (f–h) Allosteric pocket in unliganded, FPP-bound and fully closed states, respectively.

Figure 3. Ligand binding to FPPS characterized by ITC.

(a) FPP binding in absence of Mg²⁺. The raw thermogram is shown in the upper panel, and the binding isotherm with the fitted curve in the lower panel. (b) DMAPP binding in absence (left panels) and presence (right panels) of Mg²⁺. (c) GPP binding in absence (left panels) and presence (right panels) of Mg²⁺.

Figure 4. Reaction progress kinetic analysis of FPPS.

(a) Same excess experiment. Thermograms are shown in the upper panel, and differential rate data generated from the thermograms in the lower panel. The initial substrate and product concentrations are indicated. (b) Determination of kinetic parameters. The data from the excess IPP experiment (red) were not regression-analysed due to apparent substrate inhibition.

Figure 5. Conformational transition and catalytic cycle of FPPS.

(a) Superimposition of open (FPP bound, cyan) and closed (substrate bound, green) states. DMAPP was modelled in based on the structures of FPPS in complex with substrate analogues (PDB IDs 1RQI and 4H5E). Yellow spheres are Mg²⁺ ions coordinated to the Asp-rich motifs of the enzyme. (b) Schematic representation of FPPS catalytic cycle.