Fig 1.
Mevalonate pathway and FPP synthesis.
(A) Overview of mevalonate pathway and downstream metabolites. Enzyme names are in Italics. Dotted arrows represent multi-enzyme processes. (B) Reactions carried out by hFPPS.
Fig 2.
Bisphosphonate inhibitors of hFPPS.
Carbon hydrogens in the R2 side chains are omitted for simplicity. IC50 values were reported previously [9, 10]. SASA (total solvent accessible surface area) and FOSA (hydrophobic component of SASA) were calculated with QikProp 3.2 by using a virtual probe of 1.4 Å radius.
Fig 3.
Binding of bisphosphonates risedronate (A) and YS04070 (B) to hFPPS.
(A) The enzyme undergoes an open-to-closed conformational change upon DMAPP/GPP or bisphosphonate binding. This enzyme closure fully shapes the second substrate binding site, the IPP subpocket. The inset shows details of the Mg2+ (yellow spheres)-mediated binding interactions between the bisphosphonate moiety of risedronate and the DDXXD motifs. Mg2+-coordinated water molecules (red spheres) and the active site hydrophobic cavity (surface) are also represented. Only one subunit of the homodimer is shown for clarity. (B) The hydrophobic cavity accommodates the bulky side chain of YS04070. The Mg2+-mediated interactions are identical to those seen with risedronate.
Table 1.
Crystallization conditions.
Table 2.
Data collection and structure refinement statistics.
Fig 4.
Binding of PNP-BPs and risedronate to hFPPS.
(A) Co-crystal structure with YS04070. (B) Docking output structure with JDS05120 [10]. (C), (D), and (E) Co-crystal structures with the new inhibitors JDS05119, YS05035, and JDS05120, respectively. See S1 Fig for the ligand discovery maps. The two additional structures with JDS05120 (PDB entries 4NFJ and 4NFK, Table 2) do not show significant differences and are thus not shown. (F) Co-crystal structure with risedronate. (G) The JDS05120-bound structure is superimposed onto the YS04070-bound structure (protein residues in magenta; YS04070 in green). The conformational differences in the key residues and inhibitors are indicated. The cartoon representation of the protein is omitted for unobstructed view of the bound bisphosphonates. Select H-bond interactions are shown as yellow dashed lines, while relevant non-H-bond distances are indicated by grey dashed lines.
Fig 5.
ITC characterization of hFPPS and PNP-BP binding.
(A) YS04070, (B) YS05035, (C) JDS05119, and (D) JDS05120. The upper panels present raw thermograms; for clarity, only a single representative run is shown for each compound. The lower panels present the binding isotherms fitted to the means of three independent experiments. SE values are shown as bars.
Table 3.
Thermodynamic parameters of hFPPS and bisphosphonate binding determined by ITC.