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Originally published In Press as doi:10.1074/jbc.M602603200 on May 11, 2006

J. Biol. Chem., Vol. 281, Issue 31, 22004-22012, August 4, 2006
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The Crystal Structure of Human Geranylgeranyl Pyrophosphate Synthase Reveals a Novel Hexameric Arrangement and Inhibitory Product Binding*

Kathryn L. Kavanagh{ddagger}1, James E. Dunford{ddagger}§, Gabor Bunkoczi{ddagger}, R. Graham G. Russell§, and Udo Oppermann{ddagger}2

From the {ddagger}Structural Genomics Consortium, Botnar Research Centre and the §Institute of Musculoskeletal Sciences, Botnar Research Centre, Nuffield Department of Orthopaedic Surgery, University of Oxford, Oxford OX3 7LD, United Kingdom

Received for publication, March 20, 2006 , and in revised form, May 10, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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)3 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 (1, 2). 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 (3, 4). Typical examples of prenylated proteins are small GTPases such as Ras, which is farnesylated, and the Rho family of GTPases, which is geranylgeranylated (5-7). 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 (8, 9).

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 (10). Further targets for drug development presently being explored are the protein prenyltransferases for the treatment of cancer (11) or FPPS from protozoan parasites for the treatment of malaria, Leishmaniasis, and Chagas disease (12, 13).

We recently determined the structure of human FPPS and were able to deduce the molecular mechanism of N-BP inhibition (14). 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.


Figure 1
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  		FIGURE 1.
A, schematic overview of the mevalonate pathway of isoprenoid synthesis. Inhibition by statins and N-BPs is indicated. B, proposed reaction scheme of GGPP synthesis catalyzed by human GGPS. The synthesis of the trans-isoprenoid geranylgeranyl pyrophosphate from farnesyl pyrophosphate and isopentenyl pyrophosphate proceeds through a carbocation intermediate. R indicates a C-5 isoprenoid side chain; OPP indicates pyrophosphate.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
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-beta-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 (15). 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 either P41212 or P43212, and calculation of a Matthews coefficient of 2.5 implied six monomers per asymmetric unit. Attempts at molecular replacement using models with ~20% sequence identity were unsuccessful (after this structure was deposited, the Saccharomyces cerevisiae form of GGPS with 44% identity became available).

Selenomethionine-labeled protein was crystallized by suspending a 3-µl drop containing 26 mg/ml protein, 333 mM NaCl, 0.67 mM MgCl2, 0.67 mM GGPP, 3% glycerol, 15% 2-methyl-2,4-pentanediol, 1.7% PEG 10,000, 6.7 mM HEPES, pH 7.5, over a 1-ml reservoir containing 45% 2-methyl-2,4-pentanediol, 5% PEG 10,000. A single crystal was flashed-cooled in liquid nitrogen. Diffraction data were collected for the selenomethionine derivative at the Swiss Light Source PXII beamline at 0.9791 Å (peak wavelength determined from a fluorescence scan). Data sets were processed using XDS (16), and data statistics are shown in Table 1. Data processing confirmed a P1 unit cell for the derivative crystal, and analysis of solvent content predicted 12 monomers in the unit cell.


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  		TABLE 1
Data processing and refinement statistics Values in parentheses are for data in highest shell. r.m.s.d. indicates root mean square deviation.

 
Structure Solution, Model Building, and Refinement—Before merging the selenomethionine data set, the anomalous signal was assessed by calculating {Delta}F/{sigma} using the program XPREP (Bruker AXS, 2005) indicating significant signal to 4.0 Å. Forty eight selenium sites (corresponding to 12 chains) were easily found using SHELXD (17) with convincing statistics. Noncrystallographic symmetry (NCS) analysis was performed manually by assigning selenium sites to their NCS equivalents, and NCS matrices were calculated using LSQKAB (18). Initial phases were calculated using SHELXE (19), and NCS averaging was performed with dm (20) using an NCS mask calculated from the selenium positions (a 15-Å radius sphere was used around each selenium atom to construct the mask). Visual inspection of the electron density clearly indicated there were two hexamers in the P1 unit cell.

An initial model for a single chain was built into the density-modified P1 maps using the program COOT (21). Subsequently, molecular replacement was performed with PHASER (22) on the native data set testing both enantiomeric space groups in the tetragonal unit cell. A solution was found for six monomers in the P41212 unit cell with similar hexameric arrangement as seen in the P1 cell. Before refinement commenced, 5% of the data were flagged for calculation of Rfree. Iterative rounds of refinement using REFMAC5 (23) and manual fitting in COOT converged to the final model for which statistics are shown in Table 1. Tight main chain and medium side chain NCS restraints were used throughout the refinement process.

Sequence Alignment—Sequences were extracted from NCBI and were aligned using the program ICM (Molsoft, San Diego) with the implemented alignment tool. The four GGPS crystal structures (from human, Thermus thermophilus, Pyrococcus horikoshi, and S. cerevisiae) were superimposed and used as template to compare all primary structures. Obvious misalignments in the resulting output file were manually adjusted to obtain the final alignment.

Determination of Molecular Weight in Solution—The molecular weight of GGPS was measured by gel filtration chromatography using an ÄKTA purifier system. GGPS (3 mg) was applied to a calibrated Superdex 200 10/300 GL (GE Healthcare) column and developed with 100 mM NaCl, 10 mM HEPES, pH 7.5, 1 mM MgCl2, 0.5 mM TCEP at a flow rate of 0.5 ml/min. The molecular mass in solution was estimated by comparing the retention time of GGPS to the standard curve obtained with molecular weight markers (Sigma).

Kinetics of Recombinant Human GGPS—GGPS activity was analyzed by the method of Reed and Rilling (24) with the following modifications. In brief, 80 ng (2 pmol) was assayed in a final volume of 100 µl of buffer containing 50 mM Tris, pH 7.7, 2 mM MgCl2, 1 mM TCEP, 5 µg/ml bovine serum albumin, 0.2% Tween 20. The concentrations of FPP and IPP ([14C]IPP, 400 kBq/µmol) were as indicated and were typically between 0.2 and 20 µM. For inhibition studies, the concentration of GGPP varied from 0.4 to 40 µM. Reactions were initiated by addition of enzyme and were allowed to proceed at 37 °C. Assays were terminated by the addition of 0.2 ml of HCl/methanol (1:4) and incubated for 10 min at 37 °C. The reaction mixtures were extracted with 0.4 ml of ligroin and, after thorough mixing, the amount of radioactivity in the upper phase was determined using a Packard Tricarb 1900CA scintillation counter by adding 0.2 ml of the ligroin to 4 ml of general purpose scintillant. Data were fitted by nonlinear regression to the Michaelis-Menten equation using the Graphpad Prism software package or to a competitive inhibition model using the enzyme kinetics module in Sigmaplot.

Analysis of Reaction Products—Enzyme reactions were performed as described above except that [14C]IPP (2.18 GBq/mmol) was employed at a concentration of 17 µM, and the reactions were carried out in 50-µl volumes. Reactions were initiated by the addition of 1 µg of protein and stopped after 5 min by the addition of 2 µl of 0.5 M EDTA. Enzyme reaction products were analyzed using thin layer chromatography by spotting 5 µl of the reaction mixture onto Silica Gel 60 TLC plates (Merck) that were developed in propan-2-ol/ammonia/water (6:3:1). Standards were visualized by staining with iodine vapor, and radioactivity was visualized using a Storm860 PhosphorImaging system (GE Healthcare).


Figure 2
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  		FIGURE 2.
Biochemical analysis of recombinant human GGPS. A, recombinant GGPS was analyzed on a gel filtration column as described in the text. The inset shows a standard curve of molecular weight markers and the relative position of GGPS indicating a molecular mass of 193 kDa. B, analysis of GGPS reaction products by thin layer chromatography. 1st to 4th lanes show 14C incorporation into products using DMAPP, GPP, FPP, and GGPP as allylic substrates with the addition of 0.2% Tween 20. 5th to 8th lanes show the same reactions in the absence of detergent. C and D, steady-state kinetic analysis of GGPS. Enzyme reactions were performed by varying concentrations of FPP (C) or IPP (D) while the concentration of the other substrate was constant.

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Biochemical Analysis of Recombinant Human GGPS—Size-exclusion chromatography of recombinant GGPS revealed a molecular mass of 193 kDa (Fig. 2A) which, given a monomeric molecular mass of 34.96 kDa, suggests there are 5-6 monomers associated per molecule in solution. This is consistent with previous studies that estimate that GGPS from bovine brain is a 195-kDa homo-oligomer (25) but deviates, however, from a study showing that human GGPS is a 280-kDa octamer (26). Importantly, these data confirm that the crystallographic model containing six monomers (see below) is the biologically relevant unit.

Human GGPS expressed in E. coli as an N-terminal His-tagged protein was used to study steady-state kinetics and to ensure that the enzyme used for crystallization was active. The tagged and TEV-cleaved forms of the enzyme were tested for activity, and it was found that the presence of the tag had no significant effect on enzyme activity (data not shown). GGPS generated 14C-labeled acid-labile products using DMAPP, GPP, or FPP as a substrate. This activity was stimulated by the addition of 0.2% Tween 20 to the reaction (Fig. 2B), which is consistent with the finding that octyl glucoside increases activity for the bovine form of the enzyme (25). Analysis of reaction mixtures by thin layer chromatography showed that the enzyme has a strong preference for FPP as the allylic substrate in agreement with previous observations (26). Significant to the structural results discussed below, the ultimate product of the GGPS-catalyzed reaction is GGPP, and reactions set up using GGPP as allylic substrate showed no further chain elongation.

Steady-state kinetic constants were calculated by varying the concentration of one substrate while holding the concentration of the second substrate constant (Table 2 and Fig. 2, C and D). The enzyme catalyzes the production of GGPP with a kcat = 0.204 s-1 and apparent Km values of 3.0 µM (IPP) and 4.2 µM (FPP). The Michaelis constants are in general agreement with reported values for the bovine brain (Km, IPP = 2 µM and Km, FPP = 0.74 µM) and yeast enzymes (Km, IPP = 0.8 µM and Km, FPP = 3.2 µM) (25, 27). Although the kcat is 2-fold lower than that determined for human FPPS (kcat = 0.42 s-1), it is an order of magnitude higher than the reported value for S. cerevisiae GGPS (kcat = 0.025 s-1) and is therefore within the range expected for this class of enzymes (14, 27). Unlike FPP synthase (28), no substrate inhibition was observed at IPP concentrations up to 100 µM. However, the product GGPP was found to inhibit the enzyme competitively with respect to FPP with a Ki of 25 µM.


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  		TABLE 2
Steady-state kinetic parameters for recombinant human GGPS

 
Overall Structure of Human GGPS—The crystallographic asymmetric unit for the native protein contains six protein chains (A-F), each associated with two Mg2+ ions and one GGPP molecule (Fig. 3A). Except for subunit F in which the GGPP molecule is disordered past C-10, the refined B-factors for the ligand atoms are comparable with the B-factors for the surrounding protein atoms. Similar to FPPS, each chain adopts the all {alpha}-helical trans-prenyltransferase fold, and the monomers associate into dimers. The crystal structures of GGPS from the bacteria T. thermophilus (PDB code 1WMW),4 the Archaea P. horikoshii (PDB code 1WY0),5 and the yeast S. cerevisiae (27) also exhibit this dimeric quaternary structure. In fact, all-trans-prenyltransferases that have previously had their x-ray structures determined are dimeric. However, in both crystal forms that we have characterized three dimers join together to form a hexamer in a 3-bladed propeller arrangement (Fig. 3A). Size-exclusion chromatography (see above) confirms that the protein is hexameric in solution.


Figure 3
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  		FIGURE 3.
Hexameric organization of human GGPS. A, ribbon diagram of the human GGPS hexamer. The dimers are colored blue (chains AB), green (chains CD), and pink (chains EF) with one monomer from each dimer colored darker. Bound magnesiums (cyan) and GGPP (white, carbons; red, oxygens; pink, phosphorus) are rendered in space-filling representation. B, stereoview of the subunit interaction site involved in hexamer formation. Shown are contact areas between chain A (blue), involving segment A in helix {alpha}2 and segment B from the loop between helices {alpha}4 and {alpha}5, (see Fig. 4), and chain E (pink), with contributing residues from helices {alpha}10 and {alpha}11 (segment C). Polar interactions less than 3.5 Å are indicated by dashed yellow lines.

 


Figure 4
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  		FIGURE 4.
Alignment of archaeal, prokaryotic, and eukaryotic trans-prenyltransferases. Sequences of eukaryotic GGPS orthologs from human (GGPS_Homo), murine (GGPS_Mus), bovine (GGPS_Bos), insect (GGPS_Dm, Drosophila melanogaster), plant (GGPP6_AraTh, Arabidopsis thaliana), and yeast (GGPS_Scer, S. cerevisiae) were compared with bacterial (GGPS_Ttherm, T. thermophilus) or archaeal (GGPS_Ph, P. horikoshi) forms as well as to human FPPS (FPPS_Homo). The secondary structure of human GGPS is shown below the alignment, and an additional {alpha}-helix present at the N termini of the other structures is depicted in gray. Conserved sequence motifs found in trans-prenyltransferases are labeled I-V (35) and highlighted by red boxing. The regions involved in hexameric organization of human GGPS are indicated by blue boxing and are labeled A-C. The phenylalanine residues in FPPS proposed to limit the chain elongation pocket are indicated by asterisks above the alignment.

 
As previously seen in other trans-prenyltransferases, the monomers are composed of {alpha}-helices joined together by loop regions. Ten of these helices form a helical bundle that surrounds a central cavity where the active site is located. The N-terminal helix is perpendicular to the core helices and contributes residues that are involved in dimerization as well as hexamerization. Helices {alpha}9-{alpha}11 form a lid over the active site, and in human GGPS this region is also involved in inter-subunit interactions as discussed below.

Each chain associates with its dimer partner and a monomer from each of the other two dimers (Fig. 3B). The average surface area per monomer is 13,573 Å2 with the total surface area buried by hexamer formation equal to 16,750 Å2. The largest contact area occurs at the monomer to dimer level with ~1600 Å2 per monomer buried upon dimer formation. On average, an additional 1200 Å2 per monomer is buried upon hexamerization.


Figure 5
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  		FIGURE 5.
Detail of GGPP binding. A, residues within 4 Å of GGPP (salmon, carbons; red, oxygens; pink, phosphorus) or magnesium (gray spheres) are shown in stick representation and labeled. Residues above the plane of the GGPP have been left out for clarity. Fo - Fc electron density with phases derived from the final model with GGPP and magnesium omitted is contoured at 3{sigma} and superimposed upon the ligands. The environment, which is dominated by aliphatic and aromatic amino acids, is capped with polar and charged residues that ligate the Mg2+ and phosphate groups. B, same as A but rotated 180° around the y axis. C and D, superimposition of the complex of human FPPS with zoledronate (ZOL) and IPP (PDB code 1ZW5, green) onto GGPS (gray). C, overlap showing that helices{alpha}3 and{alpha}7 are closer together in FPPS and would sterically interfere with GGPP binding. Arg-28 is seen in a different conformation than the equivalent residue in FPPS, which binds the IPP through two hydrogen bonds (blue dashed lines). D, GGPP is bound in the center of the helical bundle, which differs from the proposed FPPS chain elongation site. The two phenylalanines that are proposed to delineate the base of the FPPS chain elongation site are shown in red.

 
To investigate whether the regions involved in inter-dimer contacts are conserved in other species, a sequence alignment of trans-prenyltransferases was performed and is shown in Fig. 4. This family of enzymes consists of {alpha}-helical proteins, and the secondary structural elements observed in human GGPS are drawn as red cylinders below the alignment and are labeled {alpha}2-{alpha}13 (Fig. 4). Regions that are involved in inter-dimer contacts in the human GGPS structure are boxed in blue and labeled A-C (Fig. 4). The following discussions concerning equivalent amino acids refer to the residue numbering for human GGPS.

The inter-dimer interface between chains A and E (Fig. 3B) consists of residues from the N-terminal helix {alpha}2 (region A in Fig. 4) and loop 75-83 (region B) on chain A interacting with residues 226-254 from chain E (region C) on the adjacent dimer (Fig. 3B). At the core of this interface is a hydrophobic patch consisting of Tyr-18, Phe-76, Pro-77, Ile-82, and Tyr-83 of chain A contacting Ile-233, Ile-243, and Tyr-246 of chain E. These contacts are mirrored with Ile-233, Ile-243, and Tyr-246 of chain A interacting with Tyr-18, Phe-76, Pro-77, Ile-82, and Tyr-83 on chain D of the third dimer to form a ring-like structure. Similar interactions are observed between chains B, C, and F. These regions are largely conserved in mammalian and Drosophila GGPS but not in bacterial, archaeal, fungal, or plant GGPS or in mammalian FPPS indicating that this hexameric quaternary structure may be limited to a subset of eukaryotic GGPS, including mammalian and insect orthologs.

Analysis of Ligand Pocket—The site where GGPP is bound is a ~25-Å long channel surrounded by mainly aliphatic and aromatic side chains of residues Arg-28, Leu-31, Phe-35, His-57, Leu-122, Leu-155, Phe-156, Ala-159, Val-160, and Phe-184 (Fig. 5, A and B). This pocket is capped by charged and polar residues, including the aspartate-rich motifs on helices {alpha}4 and {alpha}8 (64DDIED68 and 188DDYAN192) that ligate the magnesium ions and the pyrophosphate moieties, and residues Arg-73, Lys-151, Gln-185, Lys-202, and Lys-212 that are also involved in phosphate binding. A similar arrangement of magnesium ions mediating the interaction between acidic side chains and phosphate groups occurs in ligand-bound FPPS structures (14, 29-32).

By analogy with FPPS with which GGPS shares a common fold and conserved motifs known to be involved in catalysis (Fig. 4), the reaction is proposed to proceed by an ionization-condensation-elimination mechanism (33, 34). In this scheme (Fig. 1B), the enzyme-bound allylic substrate undergoes cleavage at the C-1-O bond. The resulting carbocation intermediate is proposed to be stabilized by the accompanying negatively charged pyrophosphate and by residues in the active site, most notably Thr-152 or its equivalent (motif IV, Fig. 4). Condensation of IPP with the first carbocation intermediate results in a second positively charged intermediate, and the final product results from stereospecific elimination of a proton.

In the alignment shown in Fig. 4, the five conserved regions previously identified for trans-prenyltransferases (35) are designated by red boxes and are numbered I-V. We can now propose explanations for each of these consensus sequences. Regions II, III, and V are involved in ligating the magnesiums and the pyrophosphate on the allylic substrate. Regions II and V contain the canonical DDXX(D/N) motifs, whereas region III has a GQXXD motif. Region I (GKXXR) contains two basic residues that are involved in IPP pyrophosphate binding in a ternary FPPS structure, specifically Lys-25 and Arg-28 (14, 29, 31). Region IV is the KT sequence containing Thr-152 proposed to be involved in catalysis by stabilizing a carbocation intermediate. As this residue is an alanine in the P. horikoshii sequence, it may be less important than suggested previously, and the exact contribution of the KT motif to catalysis remains to be experimentally tested.

Structures of human FPPS with inhibitors bound (14, 31) demonstrate that N-BPs directly interact with the lysine carbonyl and the threonine hydroxyl of this KT motif, interactions that are proposed to be important for potent inhibitor binding. In the current GGPS structure the hydroxyl of the equivalent threonine is involved in intramolecular interactions. Although there is a pronounced kink in helix {alpha}7 at this position as in human FPPS (Fig. 5), the lysine carbonyl does not protrude into the active site. However, interactions with the threonine and lysine may become evident only when a polar or charged ligand is present.

As GGPP was not added to the native protein used for crystallization, the GGPP ligand is assumed to be tightly bound and to have co-purified with the protein. This association could be reinforced by the presence of 500 mM NaCl in purification buffers, preventing release of the lipid product. Because of the GGPP C20 aliphatic tail, product release is likely to be energetically unfavorable. An indication that this could be relevant is the fact that inclusion of 0.2% Tween 20 accelerated the reaction rate (see above).

However, the current structure does not represent a product complex where the GGPP pyrophosphate would be bound in the IPP site. Because the GGPP phosphates are actually bound in the allylic site, this instead mimics substrate binding and probably demonstrates an inhibitory conformation. Our data for human GGPS indicates that GGPP is a competitive inhibitor with respect to FPP with Ki ~25 µM. In previous studies, GGPP was found to be a competitive inhibitor with respect to FPP for bovine GGPS with a Ki of ~1.2 µM (25). GGPP has also been shown to specifically inhibit its own synthesis in rabbit reticulocyte lysates (36). The fact that GGPP is likely bound in an inhibitory manner also correlates well with published studies on inhibition of human GGPS by Szabo et al. (37). In this study it was found that bisphosphonate and diphosphate compounds with long aliphatic side chains had increased potency as inhibitors when compared with bisphosphonates in current clinical use, with the most effective inhibitor being the 3-aza analog of GGPP.

It is interesting to note that for FPPS two phenylalanines, which exist in a tunnel leading from the central core to the exterior of the molecule, are implicated in prohibiting synthesis of products longer than C-15 (Figs. 4 and 5). In fact mutation of these residues to Ala and Ser allows formation of longer isoprenoids. Complex structures of avian FPPS with FPP or DMAPP indicate binding in the cavity limited by these phenylalanines (32). More recently, a mutational investigation of S. cerevisiae GGPS demonstrated that residues important in controlling product chain length are located in a channel analogous to the chain elongation site for FPPS. However, in the current GGPS structure, the aliphatic tail of GGPP does not extend toward this region but alternatively inhabits a channel in the central cavity of the protein (Fig. 5). Thus the GGPP binding that we observe apparently indicates an inhibitor-binding site in GGPS distinct from the chain elongation site.

Structural Comparison—We compared the structure of human GGPS with GGPSs from S. cerevisiae (PDB code 2DH4), T. thermophilus (PDB code 1WMW), and P. horikoshii (1WY0) as well as the structure of human FPPS (PDB code 1ZW5). These enzymes share 17-44% identity with human GGPS and have root mean square deviations for {alpha}-carbons in the range of 1.2-2.6 Å. The three other GGPS structures are unliganded and, by analogy to unliganded FPPS structures (29-31, 38), would be expected to have a more open conformation. The structures have similar secondary structural elements even in regions that have no discernible sequence conservation. The most notable differences are the absence of the N-terminal helix for human GGPS and the fact that Arg-28 (on helix {alpha}3) in three of the other structures occupies the same steric volume as part of the GGPP molecule (Fig. 5C). As Arg-28 ligates the IPP phosphates in the FPPS structure, the alternative conformation seen in human GGPS may explain why GGPP is an inhibitor and not a substrate; the conformation of Arg-28 is likely to prevent it from productively binding IPP. A more subtle difference is that in the archaeal and bacterial GGPS and human FPPS structures the base of the internal cavity is narrower because of helices {alpha}3 and {alpha}7 being closer together (Fig. 5C). The fungal GGPS structure also exhibits a wider cavity at the base of the helical bundle similar to the arrangement in human GGPS. Insertion of GGPP in the internal cavity of human GGPS could expand this area or it may be an inherent structural variation.

In summary, we have presented the first crystal structure of a mammalian geranylgeranyl pyrophosphate synthase. It reveals a novel hexameric quaternary structure that is not observed in bacterial, fungal, or archaeal forms of this enzyme. The regions involved in hexamer formation are largely conserved for mammalian and insect GGPS but not for plant, bacterial, fungal, or archaeal GGPS, suggesting insect GGPS may be hexameric as well. Analysis of ligand binding indicates that the GGPP pyrophosphate moiety occupies the allylic substrate site and the aliphatic tail inhabits a channel in the center of the helical bundle. This channel is distinct from the proposed chain elongation site for FPPS and GGPS. As GGPP is an inhibitor and not a substrate for this enzyme, this possibly indicates a method of product regulation whereby sufficient intracellular concentrations of GGPP inhibit further production of this isoprenoid.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2FVI) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence may be addressed. Fax: 44-1865-737231; E-mail: kate.kavanagh{at}sgc.ox.ac.uk. 2 To whom correspondence may be addressed. Fax: 44-1865-737231; E-mail: udo.oppermann{at}sgc.ox.ac.uk.

3 The 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. Back

4 K. Suto, K. Nishio, Y. Nodake, K. Hamada, M. Kawamoto, N. Nakagawa, S. Kuramitu, and K. Miura, unpublished results. Back

5 M. Sugahara and N. Kunishima, unpublished results. Back


   ACKNOWLEDGMENTS
 
Collaboration with the Swiss Light Source synchrotron facility and staff is gratefully acknowledged. The Structural Genomics Consortium is a registered charity (number 1097737) funded by the Wellcome Trust, GlaxoSmithKline, Genome Canada, the Canadian Institutes of Health Research, the Ontario Innovation Trust, the Ontario Research and Development Challenge Fund, the Canadian Foundation for Innovation, Karolinska Institutet, Vinnova, Knut and Alice Wallenberg Foundation, and the Swedish Strategic Research Foundation.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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