The Crystal Structure of Human Geranylgeranyl Pyrophosphate Synthase Reveals a Novel Hexameric Arrangement and Inhibitory Product Binding

Modification of GTPases with isoprenoid molecules derived from geranylgeranyl pyrophosphate or farnesyl pyrophosphate is an essential requisite for cellular signaling pathways. The synthesis of these isoprenoids proceeds in mammals through the mevalonate pathway, and the final steps in the synthesis are catalyzed by the related enzymes farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase. Both enzymes play crucial roles in cell survival, and inhibition of farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates is an established concept in the treatment of bone disorders such as osteoporosis or certain forms of cancer in bone. Here we report the crystal structure of human geranylgeranyl pyrophosphate synthase, the first mammalian ortholog to have its x-ray structure determined. It reveals that three dimers join together to form a propeller-bladed hexameric molecule with a mass of ∼200 kDa. Structure-based sequence alignments predict this quaternary structure to be restricted to mammalian and insect orthologs, whereas fungal, bacterial, archaeal, and plant forms exhibit the dimeric organization also observed in farnesyl pyrophosphate synthase. Geranylgeranyl pyrophosphate derived from heterologous bacterial expression is tightly bound in a cavity distinct from the chain elongation site described for farnesyl pyrophosphate synthase. The structure most likely represents an inhibitory complex, which is further corroborated by steady-state kinetics, suggesting a possible feedback mechanism for regulating enzyme activity. Structural comparisons between members of this enzyme class give deeper insights into conserved features important for catalysis. Modification of GTPases with isoprenoid molecules derived from geranylgeranyl pyrophosphate or farnesyl pyrophosphate is an essential requisite for cellular signaling pathways. The synthesis of these isoprenoids proceeds in mammals through the mevalonate pathway, and the final steps in the synthesis are catalyzed by the related enzymes farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase. Both enzymes play crucial roles in cell survival, and inhibition of farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates is an established concept in the treatment of bone disorders such as osteoporosis or certain forms of cancer in bone. Here we report the crystal structure of human geranylgeranyl pyrophosphate synthase, the first mammalian ortholog to have its x-ray structure determined. It reveals that three dimers join together to form a propeller-bladed hexameric molecule with a mass of ∼200 kDa. Structure-based sequence alignments predict this quaternary structure to be restricted to mammalian and insect orthologs, whereas fungal, bacterial, archaeal, and plant forms exhibit the dimeric organization also observed in farnesyl pyrophosphate synthase. Geranylgeranyl pyrophosphate derived from heterologous bacterial expression is tightly bound in a cavity distinct from the chain elongation site described for farnesyl pyrophosphate synthase. The structure most likely represents an inhibitory complex, which is further corroborated by steady-state kinetics, suggesting a possible feedback mechanism for regulating enzyme activity. Structural comparisons between members of this enzyme class give deeper insights into conserved features important for catalysis. Synthesis of isoprenoids is intrinsic to all organisms and leads to a vast array of metabolites with diverse functions. In humans and other mammals, the products of this pathway include essential molecules such as cholesterol, heme A, ubiquinone, dolichol, and farnesoids (Fig. 1A). The latter products include farnesyl pyrophosphate (FPP) 3The abbreviations used are: FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; FPPS, farnesyl pyrophosphate synthase; GGPP, geranylgeranyl pyrophosphate; GGPS, geranylgeranyl pyrophosphate synthase; GPP, geranyl pyrophosphate; IPP, isopentenyl pyrophosphate; N-BP, nitrogen-containing bisphosphonate; NCS, noncrystallographic symmetry; TCEP, tris(2-carboxyethyl)phosphine; TEV, tobacco etch virus; PDB, Protein Data Bank; PEG, polyethylene glycol. and geranylgeranyl pyrophosphate (GGPP), which are precursors for protein prenylation and might serve as nuclear receptor ligands for the receptors farnesoid X receptor or liver X receptor (1Murthy S. Tong H. Hohl R.J. J. Biol. Chem. 2005; 280: 41793-41804Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 2Niesor E.J. Flach J. Lopes-Antoni I. Perez A. Bentzen C.L. Curr. Pharm. Des. 2001; 7: 231-259Crossref PubMed Scopus (51) Google Scholar). The post-transcriptional modification of proteins with isoprenoids consists of farnesylation and geranylgeranylation of proteins with a C-terminal CaaX motif (where a is any aliphatic residue) by protein prenyltransferases (3Sinensky M. Biochim. Biophys. Acta. 2000; 1529: 203-209Crossref PubMed Scopus (62) Google Scholar, 4Sinensky M. Biochim. Biophys. Acta. 2000; 1484: 93-106Crossref PubMed Scopus (208) Google Scholar). Typical examples of prenylated proteins are small GTPases such as Ras, which is farnesylated, and the Rho family of GTPases, which is geranylgeranylated (5Coxon F.P. Ebetino F.H. Mules E.H. Seabra M.C. McKenna C.E. Rogers M.J. Bone. 2005; 37: 349-358Crossref PubMed Scopus (92) Google Scholar, 6Coxon F.P. Helfrich M.H. Van't Hof R. Sebti S. Ralston S.H. Hamilton A. Rogers M.J. J. Bone Miner. Res. 2000; 15: 1467-1476Crossref PubMed Scopus (352) Google Scholar, 7Coxon F.P. Rogers M.J. Calcif. Tissue Int. 2003; 72: 80-84Crossref PubMed Scopus (84) Google Scholar). Prenylation has been shown to be crucial to the targeting and activity of GTPases that are involved in cell growth and survival, motility, cytoskeletal regulation, intracellular transport, and secretion (8Coxon F.P. Helfrich M.H. Larijani B. Muzylak M. Dunford J.E. Marshall D. McKinnon A.D. Nesbitt S.A. Horton M.A. Seabra M.C. Ebetino F.H. Rogers M.J. J. Biol. Chem. 2001; 276: 48213-48222Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 9Molnar G. Dagher M.C. Geiszt M. Settleman J. Ligeti E. Biochemistry. 2001; 40: 10542-10549Crossref PubMed Scopus (64) Google Scholar). In mammals, as in most eukaryotes, isoprenoid synthesis proceeds through the mevalonate pathway starting from acetyl-CoA with the intermediates hydroxymethylglutaryl-CoA, mevalonate, isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), and FPP (Fig. 1A). Farnesyl pyrophosphate synthase (FPPS) resides at a key branch point of the pathway, because it produces precursors for all isoprenoids. Several enzymes in the pathway constitute important and well established drug targets, for example statins are used to lower cholesterol levels by inhibiting the rate-limiting enzyme in the pathway, hydroxymethylglutaryl-CoA reductase. Another class of drugs in clinical use are the nitrogen-containing bisphosphonates (N-BPs) that inhibit FPPS, used to treat disorders characterized by bone resorption such as osteoporosis, Paget disease, or multiple myeloma (10Russell R.G. Rogers M.J. Bone. 1999; 25: 97-106Crossref PubMed Scopus (768) Google Scholar). Further targets for drug development presently being explored are the protein prenyltransferases for the treatment of cancer (11Basso A.D. Kirschmeier P. Bishop W.R. J. Lipid Res. 2006; 47: 15-31Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) or FPPS from protozoan parasites for the treatment of malaria, Leishmaniasis, and Chagas disease (12Cheng F. Oldfield E. J. Med. Chem. 2004; 47: 5149-5158Crossref PubMed Scopus (51) Google Scholar, 13Garzoni L.R. Waghabi M.C. Baptista M.M. de Castro S.L. de Meirelles Mde N. Britto C.C. Docampo R. Oldfield E. Urbina J.A. Int. J. Antimicrob. Agents. 2004; 23: 286-290Crossref PubMed Scopus (96) Google Scholar). We recently determined the structure of human FPPS and were able to deduce the molecular mechanism of N-BP inhibition (14Kavanagh K.L. Guo K. Dunford J.E. Wu X. Knapp S. Ebetino F.H. Rogers M.J. Russell R.G.G. Oppermann U. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7829-7834Crossref PubMed Scopus (451) Google Scholar). In this study we describe the structure of human geranylgeranyl pyrophosphate synthase (GGPS), the enzyme producing the isoprenoid molecule essential for geranylgeranylation of proteins. The enzyme is a potential drug target for oncology and bone disorders. GGPS predominantly catalyzes the condensation of IPP with FPP to obtain the C20 product GGPP, although it can utilize DMAPP or GPP as alternate allylic substrates (Fig. 1B). The enzyme is 17% identical to human FPPS, sharing key consensus regions and possibly a common catalytic mechanism. Currently, structural information is only available for one archaeal (PDB code 1WY0), one fungal (PDB code 2DH4), and one bacterial ortholog (PDB code 1WMW) sharing ∼20-44% sequence identity and displaying different quaternary structures. Cloning, Expression, and Purification of Human GGPS—A clone encoding human GGPS encompassing residues 1-300 (derived from clone accession number gi 4758430) as an N-terminally His6-tagged fusion protein with a TEV protease cleavage site was expressed in Escherichia coli BL21(DE3). In brief, 10 ml of overnight culture were used to inoculate 1 liter of Terrific Broth containing 100 μg/ml kanamycin. Cells were grown at 37 °C to an A600 of 1 and were then cooled to 18 °C before being induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside and cultured overnight. Cells were harvested by centrifugation, and the pellet was resuspended in 20 ml of binding buffer (500 mm NaCl, 5% glycerol, 50 mm HEPES, pH 7.5, 5 mm imidazole, 0.5 mm TCEP) with protease inhibitors (Complete, Roche Applied Science), followed by lysis using a high pressure cell disrupter. The lysate was cleared by centrifugation before applying to a pre-equilibrated nickel-nitrilotriacetic acid (Qiagen) column with a 3-ml bed volume. The column was washed with 20 column volumes of binding buffer, 10 column volumes of wash buffer (500 mm NaCl, 5% glycerol, 50 mm HEPES, pH 7.5, 30 mm imidazole, 0.5 mm TCEP), and eluted in 12 ml of the same buffer containing 250 mm imidazole. The hexahistidine tag was removed by incubation with His-tagged TEV protease (50 μg/mg of recombinant GGPS) for 48 h at 4 °C, followed by removal of His-tagged protein by passing the digest over nickel-nitrilotriacetic acid resin and collecting the unbound fraction. The TEV-cleaved protein was further purified by gel filtration chromatography using a Superdex 200 column on an ÄKTA purifier system (GE Healthcare). Purity and integrity of GGPS were confirmed by SDS-PAGE and liquid chromatography/mass spectrometry (Agilent). Selenomethionine Labeling—Selenomethionine-substituted protein was produced using cells grown in SelenoMet medium (Molecular Dimensions) in the presence of 75 mg/liter selenomethionine together with amino acids suppressing de novo synthesis of methionine (15Doublie S. Methods Enzymol. 1997; 276: 523-530Crossref PubMed Scopus (796) Google Scholar). Labeled GGPS was purified as described for the native protein, and the incorporation of selenomethionine was confirmed by liquid chromatography/mass spectrometry. Crystallization and Data Collection—Crystals of native protein were grown at 20 °C in sitting drops by mixing 200 nl of 90 mg/ml protein in 10 mm HEPES, pH 7.5, 500 mm NaCl, 5% glycerol with 100 nl of precipitant solution consisting of 25% PEG 3350, 200 mm magnesium formate, pH 5.5, and equilibrating against 100 μl of the precipitant solution. A single crystal was transferred to a cryo-protectant prepared with 20% glycerol, 80% well solution and flash-cooled in liquid nitrogen. A native data set was collected at a wavelength of 1.008 Å at the Swiss Light Source PXII beamline. Data processing indicated the space group was or and of a of at molecular using with sequence identity were this structure was the form of GGPS with identity protein was by a containing mg/ml protein, mm NaCl, mm mm GGPP, glycerol, PEG mm HEPES, pH 7.5, over a containing 5% PEG A single crystal was in liquid nitrogen. data were collected for the selenomethionine at the Swiss Light Source PXII at Å wavelength determined from a Data were using E. for and data are shown in Data processing confirmed a cell for the and of 12 in the processing and in are for data in cell and of of in a and the selenomethionine data the was by using the to to 12 were using M. I. E. for F. Scholar) with was by to and were using Scopus Google Scholar). were using Scopus Google and was with K. and on Protein Scholar) using an from the was used to the of the indicated were in the for a single chain was into the using the P. K. Biol. 2004; PubMed Scopus Google Scholar). molecular was with R.J. Biol. 2005; PubMed Scopus Google Scholar) on the native data set space in the A solution was for in the cell with hexameric as in the 5% of the data were for of of using Biol. 1997; PubMed Scopus Google Scholar) and in to the final for which are shown in chain and medium chain were used the were from and were using the with the The GGPS crystal and S. were and used as to all structures. in the were to obtain the final of in molecular of GGPS was by gel filtration chromatography using an ÄKTA purifier GGPS was to a Superdex 200 (GE column and with 100 mm NaCl, 10 mm HEPES, pH 7.5, 1 mm 0.5 mm at a of 0.5 The molecular mass in solution was by the of GGPS to the with molecular of Human activity was by the of and Biochemistry. 15: PubMed Scopus Google Scholar) with the In brief, was in a final of 100 μl of buffer containing 50 mm pH mm 1 mm TCEP, 5 μg/ml The of FPP and IPP were as indicated and were between and 20 inhibition the of from to were by of enzyme and were to at 37 were by the of ml of and for 10 at 37 The were with ml of the of in the was determined using a by ml of the to 4 ml of Data were by to the using the or to a inhibition using the enzyme in of were as described that was at a of and the were in were by the of 1 of protein and 5 by the of μl of 0.5 products were using chromatography by 5 μl of the that were in were by with and was using a system (GE Healthcare). of Human chromatography of recombinant GGPS a molecular mass of (Fig. a molecular mass of are molecule in solution. is with that that GGPS from is a H. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar) from a study that human GGPS is a I. H. K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). these data that the containing is the Human GGPS expressed in E. coli as an His-tagged protein was used to study steady-state and to that the enzyme used for was The and TEV-cleaved forms of the enzyme were for and it was that the presence of the tag on enzyme activity GGPS products using DMAPP, GPP, or FPP as a activity was by the of 20 to the (Fig. which is with the that activity for the form of the enzyme H. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of by chromatography that the enzyme has a for FPP as the allylic in with I. H. K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). to the structural the product of the is GGPP, and set using as allylic further chain were by the of one the of the and and The enzyme catalyzes the of with a and of and The are in with for the IPP and FPP and enzymes IPP and FPP H. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Guo J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). the is lower that determined for human FPPS it is an of the for S. GGPS and is the for this class of enzymes (14Kavanagh K.L. Guo K. Dunford J.E. Wu X. Knapp S. Ebetino F.H. Rogers M.J. Russell R.G.G. Oppermann U. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7829-7834Crossref PubMed Scopus (451) Google Scholar, Guo J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). FPP synthase G. Biochim. Biophys. Acta. PubMed Scopus Google inhibition was observed at IPP to 100 the product was to inhibit the enzyme with to FPP with a of for recombinant human as in a of Human for the native protein protein with and one molecule (Fig. for in which the molecule is the for the are with the for the protein to FPPS, chain the all and the into The crystal of GGPS from the (PDB code K. K. M. N. S. and K. the P. (PDB code 1WY0), and N. and the S. Guo J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) also exhibit this dimeric quaternary In that have x-ray determined are in crystal forms that we have characterized three dimers join together to form a in a (Fig. chromatography that the protein is hexameric in solution. in other the are of together by of these form a that a cavity the site is The is to the and residues that are involved in as well as form a over the and in human GGPS this is also involved in as chain with its and a from of the other dimers (Fig. The is with the by to The at the to with an is the regions involved in are conserved in other a sequence of was and is shown in family of enzymes consists of and the structural observed in human GGPS are as the and are (Fig. that are involved in in the human GGPS structure are in and (Fig. The amino acids to the for human The between A and (Fig. consists of residues from the A in and on chain A with residues from chain on the (Fig. the of this is a consisting of and of chain A and of chain E. are with and of chain A with and on chain of the to form a are observed between and F. regions are conserved in mammalian and GGPS in bacterial, archaeal, fungal, or plant GGPS or in mammalian FPPS that this hexameric quaternary structure be to a of GGPS, mammalian and insect of site is bound is a by aliphatic and of residues and (Fig. A and is by and the on and and that the magnesium and the pyrophosphate and residues and that are also involved in A of magnesium the between and in FPPS (14Kavanagh K.L. Guo K. Dunford J.E. Wu X. Knapp S. Ebetino F.H. Rogers M.J. Russell R.G.G. Oppermann U. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7829-7834Crossref PubMed Scopus (451) Google Scholar, A. J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Oldfield E. Docampo R. 2006; PubMed Scopus Google Scholar, F. E. M. R. M. S. P. S. A. Chem. Med. Chem. 2006; Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). with FPPS with which GGPS a common and conserved to be involved in (Fig. the is to by an mechanism J. Biol. Chem. Full Text PDF PubMed Google Scholar, Biochemistry. PubMed Scopus Google Scholar). In this (Fig. the allylic cleavage at the The is to be by the pyrophosphate and by residues in the most or its of IPP with the first in a and the final product from of a In the shown in the conserved regions for A. Protein Sci. PubMed Scopus Google Scholar) are by and are We can for of these consensus and are involved in the and the pyrophosphate on the allylic and the whereas has a residues that are involved in IPP pyrophosphate binding in a FPPS and (14Kavanagh K.L. Guo K. Dunford J.E. Wu X. Knapp S. Ebetino F.H. Rogers M.J. Russell R.G.G. Oppermann U. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7829-7834Crossref PubMed Scopus (451) Google Scholar, A. J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, F. E. M. R. M. S. P. S. A. Chem. Med. Chem. 2006; Scopus Google Scholar). is the sequence containing to be involved in by a this is an in the P. it be important and the of the motif to to be of human FPPS with inhibitors bound (14Kavanagh K.L. Guo K. Dunford J.E. Wu X. Knapp S. Ebetino F.H. Rogers M.J. Russell R.G.G. Oppermann U. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7829-7834Crossref PubMed Scopus (451) Google Scholar, F. E. M. R. M. S. P. S. A. Chem. Med. Chem. 2006; Scopus Google Scholar) that with the and the of this that are to be important for In the GGPS structure the of the is involved in is a in at this as in human FPPS (Fig. the into the with the and only a or is was to the native protein used for the is to be tightly bound and to have with the be by the presence of 500 mm in of the of the C20 aliphatic product is likely to be that this be is the that of 20 the the structure a product the pyrophosphate be bound in the IPP the are bound in the allylic this binding and an inhibitory data for human GGPS that is a with to FPP with In was to be a with to FPP for GGPS with a of H. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). has also been shown to inhibit its synthesis in R.J. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The that is likely bound in an inhibitory also well with on inhibition of human GGPS by S. S. J.A. A. H. Oldfield E. J. Med. Chem. PubMed Scopus Google Scholar). In this study it was that and with aliphatic as inhibitors with bisphosphonates in clinical with the most being the of It is to that for FPPS which in a from the to the of the are in synthesis of products 4 and In of these residues to and of isoprenoids. of FPPS with FPP or DMAPP binding in the cavity by these Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). a of S. GGPS that residues important in product chain are in a to the chain elongation site for in the GGPS the aliphatic of this a in the cavity of the protein (Fig. the binding that we an site in GGPS distinct from the chain elongation Structural the structure of human GGPS with from S. (PDB code 2DH4), (PDB code and P. as well as the structure of human FPPS (PDB code enzymes identity with human GGPS and have for in the of The three other GGPS are by to FPPS A. J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Oldfield E. Docampo R. 2006; PubMed Scopus Google Scholar, F. E. M. R. M. S. P. S. A. Chem. Med. Chem. 2006; Scopus Google Scholar, M. Biochemistry. PubMed Scopus Google be to have a The have structural in regions that have sequence The most are the of the for human GGPS and the that in three of the other the same as of the molecule (Fig. the IPP in the FPPS the in human GGPS is an and a the of is likely to it from binding A is that in the archaeal and bacterial GGPS and human FPPS the of the cavity is because of and being together (Fig. The fungal GGPS structure also a cavity at the of the to the in human of in the cavity of human GGPS this or it be an structural In we have the first crystal structure of a mammalian geranylgeranyl pyrophosphate synthase. It reveals a hexameric quaternary structure that is observed in bacterial, fungal, or archaeal forms of this The regions involved in are conserved for mammalian and insect GGPS for bacterial, fungal, or archaeal GGPS, suggesting insect GGPS be hexameric as of binding that the pyrophosphate the allylic site and the aliphatic a in the of the is distinct from the chain elongation site for FPPS and is an and a for this this possibly a of product intracellular of inhibit further of this with the Swiss Light Source and is The Structural is a by the the of the the and the for and and the

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