The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants

Abstract

Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding.

Keywords: Active site mutant; Bisphosphonate; Drug binding; Farnesyl pyrophosphate synthase; Inhibition mechanism; Substrate binding.

Fig. 1

Optimal catalytic activity of wtFPPS and Tyr204 FPPS mutants is observed in different pH environment. The catalytic profile of wtFPPS is a bell shape curve with optimal catalysis observed at pH values between 6 to 8. Optimal catalysis in Y204F and Y204A FPPS mutants is observed in an acidic environment. Graph represents means obtained from four individual experiments.

Fig. 2

Interactions in the IPP binding site. Hydrogen-bonding interactions for Gln96 and Arg60 are shown for the WT + ZOL + IPP structure (grey carbons, 1ZW5) as dashed yellow dotted lines. A) Gln96 and Arg60 electrostatic interactions with IPP, Tyr204 and Ser205 as seen in WT + ZOL + IPP. Distances for H-bonds are given in Å. B) Overlay of apo wtFPPS (yellow, 2F7M), WT + RIS (pink, 1YV5) and WT + ZOL + IPP FPPS crystal structures showing conserved orientation of Gln96 and Arg60. C) In FPPS tyrosine mutants Y204A + ZOL (bluegreen, 4KQ5) and Y204A + PAM (light green, 4KPJ) Gln96 adopts an extended conformation that pushes Arg60 away from its optimal IPP binding position. D) In Thr201 FPPS mutants, T201A + ZOL (purple, 4KFA) and T201S + RIS (light blue, 2QIS), Gln96 and Arg60 adopt similar conformations as described for Tyr204 mutants.

Fig. 3

Structures of Thr201 mutants with N-BPs. A) Overlay of WT + RIS (grey, 1YV5), T201S + RIS (light blue, 2QIS) and T201A + RIS (plum, 4Q23). H-bonds are shown as yellow dashed lines and distances (Å) are given for RIS in the T201S structure. B) T201A + ZOL (purple, 4KFA) superimposed onto WT + ZOL + IPP (grey, 1ZW5). H-bond distances (Å) for the ZOL nitrogen are shown in yellow for wtFPPS and grey for T201A.

Fig. 4

Structures of Tyr204 mutants with bound inhibitor. A) Overlay of WT + RIS (grey, 1YV5), Y204F + RIS + IPP (darkgreen, 4KPD) and Y204A + RIS + IPP (lightgreen, 4KQS). H-bond distances (Å) for the RIS nitrogen are given for wtFPPS and RIS position is unaffected by the Y204 mutation. B) Y204A + ZOL (bluegreen, 4KQ5) superimposed onto WT + ZOL + IPP (grey, 1ZW5). H-bond distances (Å) for the ZOL nitrogen are given for Y204A. C) Superimposition of WT + ZOL + IPP (grey, 1ZW5), Y204F + RIS + IPP (darkgreen, 4KPD), Y204A + RIS + IPP (lightgreen, 4KQS) and Y204A + ALN + IPP (yellow, 4KQU). In wtFPPS the IPP is sterically constrained between Asp243 and the Tyr204 hydroxyl (distances (Å) shown as dashed yellow lines). In Y204A and Y204F, lack of the hydroxyl allows the IPP C4 to shift ~ 0.9 Å into a more hydrophobic pocket. Distance from the IPP C4 in Y204F + RIS + IPP to the position where the Tyr hydroxyl would be is shown in grey.