and low bone tissue mass are currently estimated to be a major public health risk affecting >50% of the female population over the age of 50. complex occurs with inhibitor binding. Isothermal titration calorimetry indicates that binding of N-BPs to the apoenzyme is usually entropy-driven presumably through desolvation entropy effects. These experiments reveal the molecular binding characteristics of an important pharmacological target and provide a route for further optimization of YIL 781 these important drugs. and their antiresorptive potency and other eukaryotic microorganisms such as and parasites YIL 781 (13-15). The exact mechanism by which N-BPs inhibit FPPS remains unclear however. Recent structural studies of prokaryotic FPPS exhibited that N-BPs can bind to the GPP/DMAPP substrate pocket (16). Studies of RIS in complex with FPPS revealed that the side chain is positioned in the hydrophobic cleft that normally accommodates an isoprenoid lipid and the phosphonate groups are bound to YIL 781 a cluster of three Mg2+ ions chelated by two aspartate-rich motifs that are conserved in FPPS sequences (17). Previous analyses based on molecular modeling and binding studies examined the potential mode of inhibition of vertebrate FPPS (13 18 Several 38231 studies suggested that N-BPs bind to the GPP substrate-binding site because N-BPs might mimic the structure of the enzyme’s natural substrates GPP/DMAPP and act as carbocation transition state analogs (18). However kinetic studies with recombinant human FPPS indicated that both the GPP and IPP substrate-binding sites might be occupied by N-BPs (19). A two-site binding model was further considered in studies because docking analysis of N-BPs into the GPP pocket of a homology model of human FPPS based on the avian structure did not offer a full qualitative explanation for the binding differences of compounds with dramatic differences in potency (19 20 To clarify YIL 781 the mode of N-BP drug binding to its human target we decided high-resolution structures of human FPPS in complexes with the clinically used N-BPs ZOL and RIS and its substrate IPP and we studied in detail the mode of inhibition and binding by using isothermal titration calorimetry (ITC) and kinetic analysis. Results Structure Determination of Human FPPS. Human FPPS was crystallized and its structure was decided in complex with Mg2+ and RIS and in complex with Mg2+ ZOL and IPP at resolutions of 2.0 and 2.3 ? respectively (Table 3 which is published as supporting information on the PNAS web site). Human FPPS exhibits the all α-helical prenyltransferase YIL 781 fold described earlier for the avian form of FPPS (17). FPPS is a dimer with 13 α-helices and connecting loops per subunit (Fig. 1structure (Protein Data Bank ID code 1RTR) the ligand-bound human structures have a more closed conformation (16 17 This observation suggests that N-BP binding causes a structural rearrangement accompanied by a decrease in the size of the internal cavity (Fig. 1of Thr-201 (Fig. 2proton is likely to be a pyrophosphate oxygen bound through interactions with conserved Arg-112 and Lys-257 side chains located on the flexible loops shielding the active site from bulk solvent during catalysis (16). The N-BPs bind in a nearly identical manner and root-mean-square deviations for α-carbon positions between the RIS and ZOL structures are 0.44 ?. The largest conformational change involves the basic residues at the C terminus 350 which become ordered at IPP binding. Although they were predicted to bind the IPP phosphates a direct conversation between these residues and the IPP is not observed. Alternatively the IPP phosphates are coordinated by six water molecules Gln-96 Arg-113 Arg-60 and Lys-57. Additionally there is a network..