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FDPS-zoledronate: Human farnesyl diphosphate synthase in complex with zoledronate, isopentenyl pyrophosphate and magnesium

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PDB Code 1ZW5 Target Class Oxidoreductases

Target FDPSA
Alias FDPS, FPS
Disease Area/Function metabolism, cancer, signalling
Date Deposited 2005-06-03
Authors K.L.KAVANAGH, K.GUO, X.WU, F.VON DELFT, C.ARROWSMITH, M.SUNDSTROM, A.EDWARDS, U.OPPERMANN, STRUCTURAL GENOMICSCONSORTIUM (SGC)
Related Structure 1YV5, 2QIS, 3B7L, 3CP6

Struc Details Tabs

Structure Details
A key branch point enzyme of the mevalonate pathway is farnesyl diphosphate synthase (FDPS, FPPS, EC 2.5.1.10 ), a Mg2+-dependent homodimeric enzyme, localized in peroxisomes. FDPS catalyzes the formation of both geranyl and farnesylpyrophosphate from isopentenyl pyrophosphate and dimethylallyl pyrophosphate. Post-translational modification of C-terminal C AAX sequences by covalent attachment of these isoprenyl chains is crucial for intracellular localization and proper function of small GTPases such as Ras, Rac, Rho , and CDC42.

Nitrogen-containing bisphosphonates (N-BPs) are known inhibitors of farnesyl diphosphate synthase and are currently used to treat osteoporosis, Paget's disease of the bone, and malignant bone tumors. Bisphosphonate therapy, which inhibits bone resorption, reduces the risk of fracture by 50% within one year. The structure of human FDPS in complex with isopentenyl pyrophosphate, magnesium, and the N-BP zoledronate shows the binding mode for this important class of inhibitors. These results will further the understanding of structure-activity relationships among N-BPs and FDPS, and will enable optimization of their pharmacological potential.

Materials & Methods
StructureFDPS
PDB Code1ZW5
Entry clone accession BC010004
Entry clone source MGC (I.M.A.G.E. Consortium CloneID 4132071)
Tag N-terminal: His-tag with integrated TEV protease site: mgsshhhhhhssgrenlyfq*gh(m)
Vector p11
Expression host BL-21(DE3)
Growth method Overnight cultures in LB (10 mL with100 µg/mL ampicillin) were used to inoculate 1 L of LB medium containing 100 µg/mL ampicillin. Cultures were grown at 37°C until they reached an OD600 of 0.6-0.8 and then induced with 1 mM IPTG. The temperature was adjusted to 18°C and expression was allowed to continue overnight. The cells were collected by centrifugation.
Purification procedure Column 1 : Ni-affinity, HisTrap, 1 mL (GE/Amersham)

Buffers: Binding: 50 mM HEPES pH 7.5, 5 mM imidazole, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP; Wash: 50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM imidazole, 5% glycerol, 0.5 mM TCEP ; Elution: 50 mM HEPES pH 7.5, 500 mM NaCl, 250 mM imidazole, 5% glycerol, 0.5 mM TCEP.

The cell extract was loaded on the column at 1 mL/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 column volumes of Lysis buffer, 10 column volumes of wash buffer, and then eluted with elution buffer at 1 mL/min. The eluted peak at A280 was automatically collected.

Column 2 : Hiload 16/60 Superdex 200 prep grade 120 mL

Buffers : 10 mM Hepes pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP.

Crystallization Zoledronate, isopentenyl pyrophosphate, and MgCl2 were prepared as 100 mM aqueous stock solutions and added to the protein to a final concentration of 2 mM each. Crystals were grown at 20°C in 300 ?l sitting drops by mixing 150 ?l of protein solution and 150 ?l of precipitant consisting of 14% PEG 6000, 0.7 M LiCl, and 70 mM citrate pH 4.0.
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