High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase ß-subunit C-terminal mutants

Zhen Wu1, Mark Demma, Corey L. Strickland, Rosalinda Syto, Hung V. Le, William T. Windsor and Patricia C. Weber

Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA


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 Abstract
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 Materials and methods
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 Discussion
 References
 
Protein farnesyltransferase (FPT) is a 97 000 Da heterodimeric enzyme that catalyzes post-translational farnesylation of many cellular regulatory proteins including p21 Ras. To facilitate the construction of site-directed mutants, a novel translationally coupled, two-cistron Escherichia coli expression system for rat FPT has been developed. This expression system enabled yields of >5 mg of purified protein per liter of E.coli culture to be obtained. The E.coli-derived FPT demonstrated an activity comparable to that of protein isolated from other sources. The reported expression system was used to construct three ß-subunit C-terminal truncation mutants, {Delta}5, {Delta}10 and {Delta}14, which were designed to eliminate a lattice interaction between the ß-subunit C-terminus of one molecule and the active site of a symmetry-related molecule. Steady-state kinetic analyses of these mutants showed that deletion up to 14 residues at the C-terminus did not reduce the value of kcat; however, Km values for both peptide and FPP increased 2–3-fold. A new crystalline form of FPT was obtained for the {Delta}10 C-terminal mutant grown in the presence of the substrate analogs acetyl-Cys-Val-Ile-Met-COOH peptide and {alpha}-hydroxyfarnesylphosphonic acid. The crystals diffract to beyond 2.0 Å resolution. The refined structure clearly shows that both substrate analogs adopt extended conformations within the FPT active site cavity.

Keywords: ß-subunit C-terminal mutants/crystallization/high-level expression/protein farnesyltransferase/SPA assay


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 Abstract
 Introduction
 Materials and methods
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Protein prenyltransferases are a class of {alpha}/ß heterodimeric zinc metalloenzymes (Zhang and Casey, 1996Go). They catalyze the transfer of the alkyl groups from allylic prenyl diphosphates to the cysteine residues of many eukaryotic proteins containing C-terminal recognition motifs of CaaX, CC or CXC, where C is cysteine, `a' is an aliphatic amino acid and X is the residue that determines protein substrate specificity (Clarke, 1992Go). Three types of protein prenyltransferases have been identified and characterized. Protein farnesyltransferase (FPT) catalyzes the transfer of the C15 farnesyl group from farnesyl diphosphate (FPP) to proteins where the X residue in the C-terminal CaaX motif is Ser, Met, Ala, Cys or Gln (Clarke, 1992Go). This enzyme was originally purified to homogeneity from rat and bovine brain using peptide affinity chromatography (Reiss et al., 1990aGo,bGo; Moores et al., 1991Go). The gene encoding the enzyme was subsequently cloned from rat (Chen et al., 1991aGo,bGo), human (Omer et al., 1993Go) and yeast (He et al., 1991Go). The second prenyltransferase, protein geranylgeranyltransferase-I (GGPT-I), utilizes geranylgeranyl diphosphate as a substrate and catalyzes the C20 geranylgeranylation of CaaX-containing proteins where the X residue is Leu or Phe (Moores et al., 1991Go; Yokoyama et al., 1991Go, 1995Go; Zhang et al., 1994Go). FPT and GGPT-I share an identical {alpha}-subunit, but have distinct ß-subunits that exhibit 30% sequence identity. A third enzyme, protein geranylgeranyltransferase-II (GGPT-II), recognizes the C-terminal CC or CXC motif of Rab proteins and catalyzes the C20 alkylation of both cysteine residues (Kinsella and Maltese, 1991Go; Moores et al., 1991Go; Armstrong et al., 1993Go). This enzyme, unlike the other two prenyltransferases, requires an auxiliary protein REP to present the protein substrate for catalysis and for the release of product (Seabra et al., 1992Go).

A wide variety of eukaryotic proteins have been identified as the endogenous substrates of protein prenyltransferases (Clarke, 1992Go; Zhang and Casey, 1996Go). They include the Ras superfamily and Ras-related proteins (Rap, Rab, Rac and Ral), large G proteins, nuclear lamins, rhodopsin kinase and the retinal cGMP phosphodiesterases. Mutation(s) in some of these proteins could cause uncontrolled cellular proliferation resulting in a neoplastic phenotype. One of the most common oncogenic proteins is the Ras protein, a family of 21 kDa guanine nucleotide-binding proteins that are encoded by N-, K- and H-ras genes. Ras is a critical regulatory protein in the signal transduction pathway that is responsible for numerous physiological functions including cell growth and differentiation (Lowy and Willumsen, 1993Go). It has been estimated that about 20–30% of all human cancers contain mutations in ras genes, with the most lethal pancreatic cancer having a ras mutation rate as high as 90% (Bos, 1989Go). Functional maturation of Ras requires post-translational farnesylation at its C-terminus for localization in plasma membrane. The farnesylated Ras links upstream membrane-bound receptors to downstream cytoplasmic effector proteins in the signal transduction pathway. Therefore, inhibition of Ras farnesylation should constitute an effective approach to anti-cancer chemotherapy (Gibbs et al., 1994Go). Based on this concept, many classes of FPT inhibitors have been developed to mimic either the Ras CaaX motif or farnesyl diphosphate moiety (Buss and Marsters, 1995Go; Leonard, 1997Go). FPT inhibitors have been shown to reverse the oncogenic phenotype of cells containing mutated ras genes (James et al., 1993Go; Kohl et al., 1993Go; Bishop et al., 1995Go). Administration of an FPT inhibitor to mice bearing tumors induced by an oncogenic H-ras transgene resulted in almost complete tumor regression (Kohl et al., 1995Go).

The crystal structure of rat FPT at 2.25 Å resolution revealed many unique features of the heterodimeric enzyme (Park et al., 1997Go). In this crystal form, the enzyme active site is partially occupied by C-terminal residues of the ß-subunit from an adjacent molecule in the crystal lattice (Park et al., 1997Go). The residues occupying the active site were displaced by soaking the crystals in solutions containing FPP, but the resolution decreased from 2.3 to 3.5 Å (Long et al., 1998Go). A 2.4 Å resolution structure of the ternary complex of FPT:{alpha}-hydroxyphosphonic acid ({alpha}HFP):Ac-Cys-Val-Ile-Met(Se)-COOH was determined from crystals grown by co-crystallization (Strickland et al., 1998Go). The FPT used in the above crystal structures was purified from Sfq cells, an expression system with relatively low FPT levels.

To facilitate the purification of large quantities of protein for crystallographic and thermodynamic studies of FPT:inhibitor interactions and provide a system for the rapid production of site-directed mutants, an Escherichia coli expression system for FPT was developed. Here, we describe a novel translationally coupled two-cistron expression system for rat FPT in E.coli. Using this efficient expression system, several FPT ß-subunit C-terminal truncations were made, purified and kinetically characterized. A unique crystal form has been obtained using a C-terminal {Delta}10 truncation mutant. This crystal diffracts to high resolution and contains two substrate analogs bound in the active site.


    Materials and methods
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 Abstract
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 Materials and methods
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Materials

[1-3H]FPP (21.5 Ci/mmol) was purchased from New England Nuclear Life Science Products (Boston, MA). Streptavidin-coated scintillation beads were obtained from Amersham (Arlington Heights, IL). Biotinylated KKSKTKCVIM was supplied by Analytical Biotechnology Services (Boston, MA). Peptides acetyl-EDAVTSDPATD and NH2-KTKCVFM were synthesized with a standard Fmoc method. Human FPT from Sf9 cells was expressed and purified as described (Bishop et al., 1995Go). Plasmids pUC18-rat{alpha} and pUC18-ratß contain genes encoding rat FPT {alpha}- and ß-subunits, respectively. Expression vectors pET15 and pET28 and E.coli strain BL21(DE3) were purchased from Novagen (Madison, WI). Human {alpha}-thrombin was purchased from Enzyme Research Laboratories (South Bend, IN). All restriction enzymes and Vent polymerase were obtained from New England Biolab (Beverly, MA). Oligonucleotides were purchased from Genosys (Woodlands, TX). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) was performed with precast Tris–glycine gel purchased from Bio-Rad (Hercules, CA). Ni–NTA (nickel–nitrilotriacetic acid) resin was supplied by Qiagen (Chatsworth, CA). Q-Sepharose Fast Flow resin was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Centriprep concentrators were purchased from Amicon (Beverly, MA). Protease inhibitors, aprotinin, Pefabloc SC, leupeptin, E64 and pepstatin were purchased from Boehringer Mannheim (Indianapolis, IN). Unless specified otherwise, all other materials were purchased from Sigma (St Louis, MO).

Preparation of E.coli FPT expression constructs

Two plasmids suitable for expression of the His-tagged FPT [FPT(+His)] were constructed in T7-promoter based pET vectors with genes encoding two FPT subunits in the order ß–{alpha} (pZWF01) or {alpha}–ß (pZWF02) (Table IGo). All subcloning was performed in E.coli DH5{alpha} and production of protein was carried out in E.coli BL21(DE3). Construction of plasmid pZWF01 was started with PCR reactions to obtain DNA fragments that encode the {alpha}- and ß-subunit, respectively. A pair of primers was designed to isolate the coding region of the ß-subunit from pUC18-ratß. The starter F-1 had a unique NdeI site (underlined) incorporated 5' to the initiation codon (bold face) (5'-GATTATTCCATATGGCTTCTTCGAGTTCCTTCACCTATTAT-3') and the end primer F-2 (antiparallel sequence) included a unique EcoRI site (underlined) right after the stop codon (bold face) (5'-CGGGATCCGAATTCAGTCAGTGGCAGGATCTGAGGTCAC-3') for DNA subcloning. Another set of primers was designed to amplify the coding region of the {alpha}-subunit from plasmid pUC18-rat{alpha}. The start primer F3 contained a unique EcoRI site (underlined), the bacteriophage T7 gene 10 ribosome binding site (rbs) and translational spacer element (italics) and the beginning codons of the {alpha}-subunit open reading frame (ORF) (5'-CGGAATTCAAGAAGGAGATATACCATGGCGGCCACTGAGGGTGTCGGTGAATCTGCG-3'). The end primer F-4 (antiparallel sequence) added a unique BamHI site (underlined) after the stop codon (bold-faced) (5'-CGGGATCCAAGCTTATACACTCGCCGGTATGTCACT-3'). The resulting PCR product from primers F1/F2 was digested with NdeI/EcoRI and the PCR product from primers F3/F4 was digested with EcoRI/BamHI. These two DNA fragments were then three-way ligated into a NdeI/BamHI-digested pET15b vector. The new plasmid, pZWF01, was transformed into the production strain E.coli BL21(DE3). Plasmid pZWF02 was constructed in a similar way to pZWF01. A pair of primers were designed to prepare the {alpha}-subunit coding region (F5: 5'-GATTATTCCATATGGCGGCCACTGAGGGTGTCGGTGAATCTG-3'; F6: 5'-CGGGATCCGAATTCATACACTCGCCGGTATGTCACT-3'). Another pair of primers was used to amplify the ß-subunit ORF (F7: 5'-CGGAATTCAAGAAGGAGATATACCATGGCTTCTTCGAGTTCCTTCACCTATTAT-3'; F8: 5'-CGGGATCCAAGCTTAGTCAGTGGCAGGATCTGAGGTCAC-3'). The resulting PCR products were digested with appropriate restriction enzymes and were ligated into a NdeI/HindIII-digested pET28b vector. The new plasmid, pZWF02, was transformed into E.coli BL21(DE3) for production of FPT. The DNA inserts in pZWF01 and pZWF02 were sequenced to ensure that no mutations occurred during the PCR and cloning procedures.


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Table I. Configurations of theE.coli two-cistron expression constructs for FPTa

 
Construction of FPT ß-subunit C-terminal truncation mutants

The ß-subunit C-terminal truncation mutants were prepared in pZWF02 by replacing the full-length ß-subunit ORF with a shorter DNA fragment encoding a truncated ß-subunit ORF. A truncated ORF was synthesized by PCR reaction with start primer F7 and an end primer corresponding to a truncated C-terminal end. The antiparallel sequences of end primers are 5'-CGGGATCCAAGCTTATGAGGTCACCGCATCTTCGCA-TTC-3' for the {Delta}5 mutant, 5'-CGGGATCCAAGCTTATTCGCATTCCTCAAAGCCTGGGAC-3' for the {Delta}10 mutant and 5'-CGGGATCCAAGCTTAAAAGCCTGGGACCGGCTTCTGCAG-3' for the {Delta}14 mutant. The resulting PCR product was double digested with EcoRI/HindIII. Plasmid pZWF02 was double digested with EcoRI/HindIII to remove the ß-subunit ORF and then ligated with the digested PCR product. The resulting plasmid, containing a shorter ß-subunit ORF, was transformed into the production strain E.coli BL21(DE3). All three mutants were expressed in E.coli and purified in the same way as the full-length FPT as described below. The inserted ß-subunit DNA fragments were sequenced to confirm the C-terminal truncation.

Purification of His-tagged FPT from E.coli

Protein purification was conducted at 4°C. At each stage of the purification, the eluted proteins were detected by measuring the absorbance at 280 nm and the enzyme fractions were assayed as described below. Twelve liters of E.coli BL21(DE3)/pZWF02 were grown at 37°C to an absorbance of 3 at 595 nm in Terrific Broth containing kanamycin (25 µg/ml). IPTG was added to a final concentration of 0.8 mM to induce FPT expression. Cells were grown for an additional 5 h post-induction and were harvested by centrifugation at 10 000 g for 10 min. The cell pellet was resuspended in 300 ml of homogenization buffer containing 50 mM Tris, pH 7.5, 1 µg/ml E-64, 2 µg/ml aprotinin, 0.7 µg/ml pepstatin, 0.1 mM leupeptin, 1 mM Pefabloc SC and 2 mM ß-mercaptoethanol (BME). The resuspended cells were disrupted by two passages through a French press at 16 000 psi. Cell debris was removed by ultracentrifugation at 100 000 g for 1 h. Supernatant was then loaded on to a Fast Flow Q-Sepharose column (5x10 cm). The column was washed with 1.2 l of 20 mM Tris, pH 7.5, 100 mM NaCl and 5 mM BME, followed by a salt gradient from 100 to 600 mM NaCl. FPT activity eluted at about 300 mM NaCl. The FPT fractions were pooled, adjusted to 25 mM imidazole and loaded on to a Ni–NTA chelating column (3x10 cm). The column was washed with 500 ml of 20 mM Tris, pH 7.5, 200 mM NaCl, 25 mM imidazole and 10 mM BME, followed by a gradient from 25 to 250 mM imidazole. FPT eluted at 100 mM imidazole. The active fractions were pooled and dialyzed three times against buffer containing 20 mM Tris, pH 7.7, 20 mM KCl, 10 µM ZnCl2 and 1 mM DTT to remove the imidazole from the protein solution. After dialysis, the purified protein was concentrated using a Centriprep concentrator with a 30 kDa cut-off and exchanged to a storage buffer containing 50 mM Tris, pH 7.7, 5 mM MgCl2, 5 µM ZnCl2, 0.01% Triton X-100, 2 mM DTT and 15% glycerol. The resulting protein solution was stored at –80°C until used.

SDS–PAGE analysis was performed as described (Laemmli, 1970Go). Western blotting during protein purification was carried out according to a standard protocol using a BCIP/NBT kit (Kirkegaard and Perry Laboratories, Gaithersburg, MD) with an anti-{alpha}-subunit polyclonal antibody raised against two peptide sequences of FPT {alpha}-subunit and an anti-ß-subunit polyclonal antibody obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The molecular weight of FPT was determined by MALDI mass spectrometry with sinapinic acid as the matrix on a Voyager DE TOF mass spectrometer (PerSeptive Biosystem, Framingham, MA). FPT concentrations were determined either by the method of Bradford (1976) using a Bio-Rad assay kit with bovine serum albumin (BSA) as a standard or using UV absorption at 280 nm with a molar extinction coefficient of 1.5x105 M–1 cm–1. Circular dichroism (CD) measurement of the purified FPT was performed on a JASCO J-500C spectropolarimeter with a protein concentration of 0.07 mg/ml in 10 mM phosphate, pH 7.4, and 1 mM DTT.

Thrombin cleavage of the N-terminal His-tag

The N-terminal His-tag on the {alpha}-subunit of FPT was removed by incubation of the purified FPT with human {alpha}-thrombin. Proteolysis was carried out at 4°C in 20 mM Tris, pH 7.4, 200 mM NaCl and 10 mM BME with 0.16 mg of purified FPT per unit of thrombin. The cleaved FPT, denoted FPT(–His), was separated from the His-tagged protein by passing through a Ni–NTA chelating column. Thrombin was removed from the FPT sample by size-exclusion chromatography using a Pharmacia Sephacryl S-300 column.

FPT activity assay

FPT fractions were assayed using the Amersham Scintillation Proximity Assay (SPA) kit. Assays were performed at 23°C in 200 µl with biotinylated human K-Ras-4B C-terminal peptide KKSKTKCVIM (100 nM) and [1-3H]FPP (90 nM) as substrates in a buffer of 50 mM Tris, pH 7.7, 5 mM MgCl2, 5 µM ZnCl2, 0.01% Triton X-100, 0.2 mg/ml BSA and 2 mM DTT. Reactions were initiated by adding FPT fractions (25–250 ng) to the substrate mixture. The reaction mixture was incubated for 30 min before it was terminated by three reaction volumes of a quench solution containing 5 mg/ml scintillation beads, 500 mM EDTA, pH 8.0, and 0.5% BSA. After rocking the quenched solution at room temperature for 30 min, the radioactivity incorporated into the product was directly counted in a 96-well plate format with a Wallac 1204 Betaplate BS liquid scintillation counter.

Steady-state kinetic analysis of FPT

Steady-state kinetic analysis of FPT was performed by SPA assay in a 96-well plate format as described above. The assay was initiated by adding FPT (0.5–5 nM) to a mixture of biotinylated-KKSKTKCVIM (25–500 nM) and [1-3H]FPP (5–120 nM) in a volume of 200 µl containing 50 mM Tris, pH 7.7, 5 mM MgCl2, 5 µM ZnCl2, 0.01% Triton X-100, 0.2 mg/ml BSA and 2 mM DTT. An aliquot of 50 µl was withdrawn from the reaction at 1.5 and 3 min and the reaction was immediately stopped by 150 µl of quench solution. After incubation of the quenched mixture for 30 min, the radioactivity in the product was determined for calculation of the initial velocity, Vi. The initial velocities at various concentrations of both substrates were globally fitted into Equation 1 for simultaneous calculation of kcat, KmP and KmF.


In Equation 1, kcat is the enzyme turnover number and [E]0 is the total enzyme concentration. The parameters KmP and KmF are the Michaelis constants (Km) for peptide and FPP at an infinite second substrate concentration, respectively. The non-linear least-squares fit of data was performed with Sigma Plot (Jandel Scientific, Corte Madera, CA).

The SPA assay was originally developed for high-throughput screening (Hart and Greenwald, 1978Go). Using a streptavidin-coated bead suspension, radioactivity incorporated into products can be measured directly through a biotin–streptavidin interaction without the need to separate products from reactants. Scintillant is built inside the streptavidin-coated beads and only a small volume of reaction is needed for the determination of radioactivity in a 96-well format. Although these useful features dramatically reduce the workload of kinetic analysis, they also cause a lower counting efficiency. The counting efficiency using the Wallac 1204 Betaplate BS liquid scintillation counter in an SPA format was compared with that from a Beckman Model LS3801 liquid scintillation counter. The lower counting efficiency of the Betaplate scintillation counter was compensated by a factor of 1.6 for calculation of the enzyme turnover number. In the calculation of kcat, the c.p.m. (counts per minute) of radioactivity directly read from the scintillation counter was converted to d.p.m. (disintegrations per minute) using a factor of 2.9.

Substrate inhibition of FPT

Substrate inhibition studies of FPT with biotinylated KKSKTKCVIM were performed under conditions similar to the steady-state kinetic studies, except that the [3H]FPP concentration was fixed at 100 nM and the peptide concentration varied from 25 to 2000 nM. The initial velocities at various peptide concentrations were determined and fitted to Equation 2 for the calculation of the substrate inhibition constant, Ki.


In Equation 2, Kmapp is the apparent Michaelis constant for the peptide substrate and Vmax is the velocity at an infinite peptide substrate concentration. The non-linear least-squares fit of the data was performed using KaleidaGraph (Synergy Software, Reading, PA)

Inhibition studies of FPT

Inhibition studies of FPT by a peptide corresponding to the last 11 amino acid residues of the ß-subunit C-terminus, acetyl-EDAVTSDPATD, or a known FPT inhibitor, NH2-KTKCVFM, were performed at 23°C with fixed concentrations of biotinylated-KKSKTKCVIM (150 nM) and FPP (100 nM). Initial velocities at various concentrations of inhibitor were determined under conditions similar to those for the steady-state kinetic analyses described above. The apparent inhibition constant was determined by plotting 1/Vi vs inhibitor concentrations using the Dixon equation (Dixon, 1953Go).

Crystallization and data collection

Crystals of FPT complexed with {alpha}-hydroxyphosphonic acid, an inactive FPP analog and the Ac-Cys-Val-Ile-Met-COOH peptide were grown using the {Delta}10 C-terminal truncation mutant. The ternary complex of enzyme, FPP analog and CaaX tetrapeptide was prepared by incubating 108 µM FPT (10 mg/ml) with 150 µM {alpha}HFP (Calbiochem-Novabiochem) for 15 min prior to adding 150 µM Ac-Cys-Val-Ile-Met-COOH (AnaSpec). {alpha}HFP was added in a 1.3 µl aliquot of a 23.6 mM ethanol solution and the peptide in 0.6 µl of a 50 mM DMSO solution. The mixture was incubated at 4°C for an additional 4.5 h. Crystals suitable for structure determination were grown by the hanging drop vapor diffusion method when the droplet contained 4 µl of the {alpha}HFP:peptide:FPT complex and 4 µl of the reservoir solution (0.1 M KCl, 0.1 M sodium acetate, pH 5.0). Crystallization trays were incubated at 4°C and after 2–3 weeks, hexagonal rods (0.1x0.3 mm) appeared. Although grown under different conditions, the crystals belong to the same space group (P61) as those used to solve the FPT:{alpha}HFP:Ac-Cys-Val-Ile-SelenoMet-COOH structure using full-length, Sf9 derived FPT (Strickland et al., 1998Go).

Crystals were flash frozen at 95 K using the crystallization reservoir supplemented with 40% glycerol as a cryoprotectant. X-ray intensity data were collected using a Raxis-IIc image plate area detector mounted on a Rigaku-R200 rotating anode X-ray generator operating at 50 kV and 100 mA. The X-ray beam intensity was increased by focusing with `bent mirrors' (J.Johnson and Z.Otwinowski, Yale University; Molecular Structure Corp.). With the detector set at 2{theta} = 0° and a crystal-to-detector distance of 135 mm, data were collected in 180 contiguous 0.30° oscillation images each exposed for 12 min. Diffraction intensities were reduced to structure factors using HKL (Otwinowski and Minor, 1997Go). Although the crystal diffracted beyond 2.0 Å, only a 2.4 Å data set was collected.

Structure determination and refinement

The structure of FPT bound to {alpha}HFP and Ac-Cys-Val-Ile-SelenoMet-COOH, solved using crystals grown from full length, Sf9-derived FPT, was used as a starting model (Strickland et al., 1998Go). After rigid body refinement, using an FPT model with the ligands and solvent molecules located near the active site removed, clear density for the {alpha}HFP and CVIM was seen. Refinement by iterative cycles of model building (CHAIN; Sack, 1988Go) and refinement (XPLOR; Brunger et al., 1987Go), resulted in a final model with an R-factor of 16.8% (8.0–2.4 Å). Positions of discrete water molecules were taken from positive 3{sigma} (Fo – Fc), {alpha}calc difference density peaks if a hydrogen bonding pattern to protein, peptide or solvent atoms could be established.


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Expression and purification of FPT

Heterodimeric rat FPT was expressed using two different translationally coupled two-cistron E.coli expression systems. The first construct, pZWF01, had the ß-subunit encoded by the first cistron followed by the {alpha}-subunit ORF. The second, pZWF02, had a reverse order of {alpha}–ß (Table IGo). Both constructs produced soluble and active FPT in E.coli. The first cistron of both constructs was expressed at a high level, but pZWF02 had a better expression of the second cistron (ß-subunit), resulting in a 5-fold higher yield of the purified FPT than pZWF01. For this reason, construct pZWF02 was chosen for all of the subsequent expression and purification experiments. His-tagged FPT, denoted FPT(+His), from pZWF02 was purified through a two-step chromatography procedure, which yielded 5 mg of purified protein per liter of E.coli culture (Table IIGo). Based on SDS–PAGE analysis, the protein was judged to be ~95% pure and contained two equal intensity bands with apparent molecular weights of 50 and 44 kDa (Figure 1Go, lane 2). N-Terminal sequencing and Western blotting with subunit-specific antibodies identified the 50 kDa band as the {alpha}-subunit and the 44 kDa band as the ß-subunit. Molecular weights of both subunits were determined by MALDI mass spectrometry and the {alpha}- and ß-subunits had masses of 45.9 and 48.4 kDa, respectively. These masses are consistent with the predicted molecular weight of the {alpha}-subunit, 46.2 kDa (44046.6 Da plus the N-terminal His-tag of 2106.2 Da) and the ß-subunit, 48.7 kDa. The higher apparent molecular weight of the {alpha}-subunit on SDS–PAGE may be due to an N-terminal proline-rich region (Omer et al., 1993Go).


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Table II. Purification of the recombinant rat FPT(+His) from E.colia
 


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Fig. 1. SDS–PAGE analysis of the purified FPT and the ß-subunit C-terminal truncation mutants from E.coli. The positions of both the {alpha}- and ß-subunits are marked. Lane 1, molecular mass standards (in kDa); lane 2, FPT(+His); lane 3, FPT(–His), after thrombin cleavage of the N-terminal His-tag; lanes 4–7, the {Delta}5, {Delta}10, {Delta}14 and {Delta}14(+13aa) mutants, respectively.

 
The heterodimeric FPT expressed from construct pZWF02 has a His-tag containing an extra 20 amino acid residues including six histidine residues at the N-terminus of the {alpha}-subunit. The first 17 residues of this tag were proteolytically removed by thrombin. Based on SDS–PAGE analysis, more than 95% of the 17-residue tag was cleaved after a 4 h incubation. Following thrombin cleavage, the resulting protein, denoted FPT(–His), contained three additional residues, Gly-Ser-His, at the N-terminus of the {alpha}-subunit. FPT(–His) was purified to homogeneity by a two-step procedure including a second Ni-chelating chromatographic step and size-exclusion chromatography. The purified FPT(–His) was judged to be more than 95% pure on SDS–PAGE (Figure 1Go, lane 3). The far-UV CD spectrum of the purified enzyme was measured and compared with the His-tagged protein. There was no significant difference in the secondary structure following the N-terminal proteolysis.

Design and preparation of ß-subunit C-terminal truncation mutants

Analysis of the FPT crystal structure (Park et al., 1997Go) revealed that the ß-subunit C-terminal tail, primarily the last five amino acid residues, interacts with the active site through a combination of hydrogen bonds and hydrophobic interactions (Figure 2Go). The active site residues involved in the binding were mainly from the ß-subunit, including residues W102ß, H248ß, K264ß, R291ß, Y300ß, C345ß, S357ß and two {alpha}-subunit residues Y166{alpha} and D196{alpha}. Based on this analysis, three ß-subunit C-terminal truncation mutants, {Delta}5, {Delta}10 and {Delta}14, were designed to eliminate the C-terminal tail from the active site. The {Delta}5 truncation would remove the majority of the interaction, while the {Delta}10 and {Delta}14 would further eliminate the binding residues of the C-terminal tail. These three mutants were constructed in E.coli. One of the {Delta}14 clones sequenced was found to have its stop codon for the ß-subunit ORF mutated, resulting in an extra 13 residues of KLAAALEHHHHHH at the C-terminus of the ß-subunit of the {Delta}14 mutant. This mutant, denoted {Delta}14(+13aa), was also expressed in E.coli. The three deletion mutants and {Delta}14(+13aa) were purified in the same way as the full-length protein described above. There was no obvious change of behavior in chromatography during purification. The purified proteins were judged by SDS–PAGE to be more than 90% pure (Figure 1Go, lanes 4–7). The molecular weight of each mutant was determined by MALDI mass spectrometry and was consistent with the predicted mass. Far-UV CD analysis of each purified mutant was also performed, with no obvious spectral changes being observed in the C-terminal truncation mutants.



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Fig. 2. Hydrogen bonding and hydrophobic interactions between the ß-subunit C-terminal tail and the FPT active site residues mapped from the reported crystal structure (Park et al., 1997Go). The structure of the last 14 amino acid residues of the ß-subunit C-terminus and the interacting residues in the active site are shown. The bonding distances were determined using Insight II (Biosym Technologies, San Diego, CA). The positions of the three C-terminal truncations, {Delta}5, {Delta}10 and {Delta}14, are indicated by arrows.

 
Steady-state kinetic analysis

Steady-state kinetic analysis of FPT(+His) was performed using the SPA assay method with [1-3H]FPP and biotinylated human K-Ras-4B C-terminal peptide (KKTKSKCVIM) as substrates. Results from initial velocity experiments showed that Vi increased with increasing concentration of peptide up to 600 nM (Figure 3Go). Above 600 nM peptide, the Vi value decreased, suggesting that high concentrations of peptide lead to substrate inhibition (Dolence et al., 1995Go). The substrate inhibition constant, Ki, calculated from Equation 2, was 1.3 ± 0.1 µM and the apparent Michaelis constant for the peptide, Kmapp, was 150 ± 16 nM. The Ki value was essentially the same as that reported for yeast FPT using dansyl-GCVIA as the peptide substrate (Dolence et al., 1995Go). Because of the complication of substrate inhibition, the peptide concentration used in the steady-state kinetic analysis was set from 25 to 500 nM.



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Fig. 3. Substrate inhibition studies of FPT with biotinylated KKSKTKCVIM as peptide substrate. The initial velocity was determined by the SPA assay containing [3H]FPP (100 nM), biotinylated KKSKTKCVIM (25–2000 nM) and 3.6 nM FPT(+His) at 23°C. The curve was fitted using Equation 2.

 
The steady-state kinetic parameters of the purified FPT(+His) were determined from a non-linear least-squares fit of the data to Equation 1 (data not shown); kcat was 0.54 min–1 and KmF and KmP were 14 and 63 nM, respectively. These values yielded a secondary rate constant, kcat/(KmPKmF), of 1.0x1013 M–2 s–1 (Table IIIGo), which reflected the overall catalytic efficiency of FPT(+His). The kinetic parameters of human FPT from Sf9 cells were also determined; the results are given in Table IIIGo and are compared with the values of the E.coli-derived FPT(+His). The kcat value of the human FPT was 1.4 min–1, which was 2.6-fold higher than that of the FPT(+His). The KmF values were almost identical (~14 nM) and the KmP of the human FPT was 130 nM, which was 2-fold higher than that of the E.coli-derived enzyme. The resulting catalytic efficiency for the human FPT, 1.2x 1013 M–2 s–1, was similar to that of the E.coli-derived rat FPT.


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Table III. Steady-state kinetic parameters of FPT and the ß-subunit C-terminal truncation mutantsa
 
The steady-state kinetics of FPT(–His) were investigated to assess the effect of the {alpha}-subunit N-terminal His-tag on the enzyme activity. These results are summarized in Table IIIGo. After cleavage of the first 17 amino acid residues from the His-tag, kcat increased by almost 2-fold to 0.95 min–1 and was close to the turnover number of the human FPT from SF9 cells. The Km values for both substrates decreased slightly, resulting in a catalytic efficiency of 3.0x1013 M–2 s–1 (Table IIIGo).

The kinetic profiles of the C-terminal truncation mutants, {Delta}5, {Delta}10, {Delta}14 and {Delta}14(+13aa), were determined by SPA assay (Figure 4Go) and are summarized in Table IIIGo. Compared with the full-length FPT(+His), the {Delta}5 truncation yielded an increased kcat of 1.6 min–1 with almost no change in Km for both substrates. The secondary rate constant of 2.5x1013 M–2 s–1 reflected a 2.5-fold increase in the enzyme activity. Deletions at {Delta}10 or {Delta}14 reduced the kcat value to 0.68 min–1, which is slightly higher than that of the full-length enzyme. These additional deletions, however, caused an elevation of Km for both FPP and peptide. In comparison with the full-length enzyme, KmF of the {Delta}14 mutant increased 2-fold to 28 nM, while KmP increased 3.3-fold. Mutant {Delta}14(+13aa) has 13 different amino acid residues in place of the last 14 C-terminal residues of the ß-subunit. Its KmF and KmP values were similar to those of the control species FPT(+His), but the kcat value, 0.38 min–1, was lower, resulting in a catalytic efficiency of 1.4x1013 M–2 s–1.



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Fig. 4. Steady-state kinetic analysis of the FPT ß-subunit C-terminal truncation mutants. Only one set of data for the {Delta}5 mutant is shown here. The assay was performed with [3H]FPP (5–100 nM), biotinylated KKSKTKCVIM (25–500 nM) and the purified {Delta}5 mutant (1.5 nM) at 23°C.

 
Inhibition studies of FPT

Both His-tagged FPT and FPT(–His) were used for inhibition studies with a peptide corresponding to the last 11 amino acid residues of the ß-subunit C-terminus, acetyl-EDAVTSDPATD. There was no inhibitory effect on the FPT activity up to 2 mM peptide. As a positive control, a known FPT inhibitor, NH2-KTKCVFM, was also tested under similar conditions. An apparent inhibition constant, Ki, of 120 nM for FPT(–His) was determined from a Dixon plot. This value is in the same range of IC50 reported in the literature (Reiss et al., 1991Go).

Structure of FPT

The 2.4 Å resolution structure of the {Delta}10 FPT:{alpha}HFP:Ac-Cys-Val-Ile-Met-COOH complex has a 0.4 and 0.8 Å r.m.s. deviation for a C{alpha} and an all-atom comparison, respectively, versus the reported 2.4 Å resolution FPT:{alpha}HFP:Ac-Cys-Val-Ile-SelenoMet-COOH structure (Strickland et al., 1998Go). The atomic positions for the peptide, {alpha}HFP and zinc are more highly conserved with an all-atom r.m.s. deviation of 0.3, 0.3 and 0.1 Å, respectively (Figure 5Go). Hence neither the new crystallization conditions nor the presence of a SelenoMet result in significant differences in the binding of the ligands.



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Fig. 5. Comparison of the bound conformation of (a) Cys-Val-Ile-Met-COOH and (b) {alpha}HFP from the E.coli-derived {Delta}10 FPT:{alpha}HFP:Ac-Cys-Val-Ile-Met-COOH (thin line) and the Sf9-derived FPT:{alpha}HFP:Ac-Cys-Val-Ile-SelenoMet-COOH (thick line) structures (Strickland et al., 1998Go). The non-carbon atoms are shown as small spheres. The sulfur in the peptide and the phosphate in the {alpha}HFP are shown as larger spheres.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recombinant rat FPT obtained from the insect cell (Sf9)/baculovirus expression system (Chen et al., 1993Go; Bishop et al., 1995Go) was successfully utilized to solve the FPT crystal structure (Park et al., 1997Go). However, the preparation of a baculovirus expression system is tedious and is not a preferred method for structure–function studies using site-directed mutagenesis. Given the ability of E.coli to grow rapidly and to manipulate DNA easily, an E.coli expression system should be the choice for the mutant construction. Four E.coli expression systems have been reported for FPT, one for the rat enzyme (Fu et al., 1996Go), one for yeast FPT (Mayer et al., 1993Go) and two for the human enzyme (Omer et al., 1993Go; Tsao and Waugh, 1997Go). The expression system for rat FPT used a dual plasmid system with the two subunits expressed from two compatible vectors (Fu et al., 1996Go). Because the in vivo copy numbers of two vectors differ by 10-fold, it was difficult to achieve a balanced expression for both subunits. One of the human FPT E.coli expression systems also employed a dual plasmid construct (Tsao and Waugh, 1997Go). By deletion of a negative regulator for control of the plasmid copy number and a mutation in the origin of replication, comparable copy numbers of both plasmids were achieved, resulting in an FPT expression level of 15–25% of the total intracellular proteins. In this system, however, there is no description of the protein solubility, enzyme activity and purification of the expressed protein available. The other two E.coli expression systems for human and yeast FPT utilized a single plasmid for expression of both subunits as two tightly coupled cistrons (Schoner et al., 1990Go). The ribosome binding site for the second cistron was constructed within the 3' end of the first cistron to enhance translational efficiency for the second cistron. In these constructs, an extra four residues were fused to the C-terminus of the first cistron to introduce a ribosome binding site sequence and also acted as an epitope to facilitate protein purification using affinity chromatography.

In this work, our E.coli expression constructs also employed the same tight cistron coupling approach but they contain an improved configuration (Table IGo). The ribosome binding site for the second cistron was inserted downstream from the first cistron to avoid the generation of a fusion protein. To obtain equal expression of both subunits, a T7 gene 10 rbs was designed for the second cistron (Shi et al., 1994Go). The spacer between the two cistrons was minimized in order to achieve tight coupling for translation. In addition, two rare codons for glycine, GGG, located at the beginning of the {alpha}-subunit ORF were changed to the more frequent E.coli codon GGT to decrease the high GC content and to increase protein translational efficiency. Vectors pET15b and pET28b were chosen for a strong T7 promoter, a T7 gene 10 rbs and a cleavable N-terminal His-tag to facilitate protein purification.

Construct pZWF01 had a two-cistron order of ß–{alpha}, while pZWF02 had a reverse order of {alpha}–ß. The higher expression level of pZWF02 over pZWF01 was consistent with the result from the yeast FPT E.coli constructs where the two-cistron order of {alpha}–ß generated a higher amount of active enzyme than the ß–{alpha} construct (Mayer et al., 1993Go). The construct pZWF02 provided a yield of more than 5 mg of purified FPT(+His) per liter of culture, which was about 5–10-fold higher than the reported yield for other E.coli expression systems (Mayer et al., 1993Go; Omer et al., 1993Go; Fu et al., 1996Go). The higher expression of our constructs is probably due to a strong T7 promoter, tightly coupled translation and a balanced expression of the two subunits in the {alpha}–ß cistrons. Steady-state kinetic studies of the recombinant FPT(+His) showed that it had a very similar overall catalytic efficiency to the Sf9-derived human FPT (Table IIIGo). Its kinetic profile was also similar to that of the reported wild-type human FPT with Ras-CVIM as substrate (Omer et al., 1993Go).

Steady-state kinetic analysis of the FPT was performed using an SPA assay. The kinetic parameters determined by the SPA assay were quantitative and reproducible, with the kinetic parameters being similar to those determined by fluorescence or by a filter assay (Pompliano et al., 1992Go; Dolence et al., 1995Go). FPT catalyzes an ordered sequential reaction with FPP binding first to form a binary complex, followed by the binding of CaaX-containing protein/peptide substrate (Pompliano et al., 1993Go; Mathis and Poulter, 1997Go). The ternary complex undergoes chemical reaction and subsequently releases products. The slowest step in the reaction is product release (Furfine et al., 1995Go). Therefore, the kcat from the steady-state kinetic analysis is a measure of the rate of product release and is not likely to reflect the rate of the chemical step.

In the reported crystal structure of rat FPT, the enzyme active site was partially occupied by the last nine residues of the ß-subunit C-terminus, AVTSDPATD. Inhibition studies of FPT with a peptide corresponding to the last 11 amino acid residues of the ß-subunit C-terminus indicated that this sequence had little intrinsic affinity for FPT. The observed occupancy of the FPT active site by the ß-subunit C-terminus is probably due to a fortuitous crystal packing interaction. Although the occupation of the active site by the ß-subunit C-terminus seems to be physiologically irrelevant, this steric obstruction could hinder enzyme ligands from binding to the active site, cause local conformational changes and substantially reduce crystal diffraction resolution (Long et al., 1998Go). To solve this problem, a series of ß-subunit C-terminal truncation mutants were sought to screen for a crystal form that contained an open active site.

Based on the analysis of the interaction between the ß-subunit C-terminal tail and the active site, three C-terminal truncation mutants, {Delta}5, {Delta}10 and {Delta}14, were constructed using the E.coli expression system. Since the N-terminal His-tag on the {alpha}-subunit of the full length FPT had only a modest effect on the enzyme activity, all the mutants were expressed with the N-terminal His-tag. Steady-state kinetic analysis of the C-terminal truncation mutants indicated that a C-terminal deletion up to 14 residues did not decrease the kcat value. In fact, the {Delta}5 mutant had a kcat almost 3-fold higher than the full-length protein. However, the C-terminal truncation appeared to alter the substrate affinity of the enzyme. KmF stayed essentially the same and KmP values increased ~3-fold for the {Delta}10 mutant, while they increased 2- and 3.3-fold, respectively, for the {Delta}14 mutant. The overall catalytic efficiency of FPT decreased gradually with increasing C-terminal deletion (Table IIIGo). Although 14 residues were deleted from the ß-subunit C-terminus, the {Delta}14 mutant retained 20% of the catalytic efficiency of the full-length enzyme. Since the ß-subunit C-terminus is far from the protein core, it is likely that the observed alteration in the kinetic parameters for the C-terminal truncation mutants is due to a long-range conformational effect on substrate binding. Based on these results, it appears that deletion of up to 14 amino acid residues from the ß-subunit C-terminus marginally alters the enzyme activity. The kinetic studies of the mutant {Delta}14(+13aa) reinforced this proposition. Addition of a patch of hydrophobic residues and six histidine residues to the truncated ß-subunit C-terminus of the {Delta}14 mutant recovered the lost affinity (Km) for both substrates. The {Delta}14(+13aa) mutant has almost an identical kinetic profile to the full-length enzyme. The fact that the crystal structure of the E.coli-derived {Delta}10 C-terminal mutant is identical with the Sf9-derived full-length protein structure demonstrates the usefulness of this engineered material. This result suggests that the 3-fold change in KmP for the {Delta}10 mutant does not affect the peptide binding to the active site.

In summary, rat FPT can be expressed to a high level in E.coli from a translationally coupled two-cistron expression system. This efficient expression system should greatly facilitate the mutant construction for studying structure–function relationships. Based on this expression system, a series of ß-subunit C-terminal truncation mutants were prepared. Steady-state kinetic analysis showed that these mutants were active with truncation effects reflecting on the affinity to the substrates. The structure of the {Delta}10 FPT complexed with {alpha}HFP and Ac-Cys-Val-Ile-Met-COOH has been solved and will allow further structural analysis of the FPT active site, helping to elucidate the enzymatic reaction mechanism.


    Acknowledgments
 
We thank Dr Richard Bond and Ms Lynn Wang for supplying plasmids encoding the {alpha}- and ß-subunits and Dr Yan-Hui Liu for performing the MALDI mass spectral analysis. We also thank Dr Rumin Zhang and Mr James Durkin for supporting peptide synthesis and Dr Charles McNemar for performing N-terminal sequencing.


    Notes
 
1 To whom correspondence should be addressed. E-mail: zhen.wu{at}spcorp.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Armstrong,S.A., Seabra,M.C., Sudhof,T.C., Goldstein,J.L. and Brown,M.S. (1993) J. Biol. Chem., 268, 12221–12229.[Abstract/Free Full Text]

Bishop,W.R. et al. (1995) J. Biol. Chem., 270, 30611–30618.[Abstract/Free Full Text]

Bos,J.L. (1989) Cancer Res., 49, 4682–4689.[Abstract]

Bradford,M.M. (1976) Anal. Biochem., 72, 248–254.[ISI][Medline]

Brunger,A., Kuriyan,J. and Karplus,M. (1987) Science, 235, 458–460.[ISI]

Buss,J.E. and Marsters,J.C. (1995) Chem. Biol., 2, 787–791.[ISI][Medline]

Chen,W.-J. Andres,D.A., Goldstein,J.L. and Brown,M.S. (1991a) Proc. Natl Acad. Sci. USA, 88, 11368–11372.[Abstract]

Chen,W.-J. Andres,D.A., Goldstein,J.L., Russell,D.W. and Brown,M.S. (1991b) Cell, 66, 327–334.[ISI][Medline]

Chen,W.-J., Moomaw,J.F., Overton,L., Kost,T.A. and Casey,P.J. (1993) J. Biol. Chem., 268, 9675–9680.[Abstract/Free Full Text]

Clarke,S. (1992) Annu. Rev. Biochem., 61, 355–386.[ISI][Medline]

Dixon,M. (1953) Biochem. J., 55, 170–171.[ISI]

Dolence,J.M., Cassidy,P.B., Mathis,J.R. and Poulter,C.D. (1995) Biochemistry, 34, 16687–16694.[ISI][Medline]

Fu,H.-W., Moomaw,J.F., Moomaw,C.R. and Casey,P.J. (1996) J. Biol. Chem., 271, 28541–28548.[Abstract/Free Full Text]

Furfine,E., Leban,J., Landavazo,A., Moomaw,J. and Casey,P. (1995) Biochemistry, 34, 6857–6862.[ISI][Medline]

Gibbs,J.B., Oliff,A. and Kohl,N.E. (1994) Cell, 77, 175–178.[ISI][Medline]

Hart,H.E. and Greenwald,E.B. (1978) J. Nucl. Med., 19, 681.

He,B., Chen,P., Chen,S.-Y., Vancura,K.L., Michaelis,S. and Powers,S. (1991) Proc. Natl Acad. Sci. USA, 88, 11373–11377.[Abstract]

James,G.L., Goldstein,J.L., Brown,M.S., Rawson,T.E., Somers,T.C., McDowell,R.S., Crowley,C.W., Lucas,B.K., Levinson,A.D. and Marsters,J.C. (1993) Science, 260, 1937–1942.[ISI][Medline]

Kinsella,B.T. and Maltese,W.A. (1991) J. Biol. Chem., 266, 8540–8544.[Abstract/Free Full Text]

Kohl,N.E., Mosser,S.D., Desolms,S.J., Giuliani,E.A., Pompliano,D.L., Graham,S.L., Smith,R.L., Scolnick,E.M., Oliff,A. and Gibbs,J.B. (1993) Science, 260, 1934–1937.[ISI][Medline]

Kohl,N.E. et al. (1995) Nature Med., 1, 792–797.[ISI][Medline]

Laemmli,U.K. (1970) Nature, 227, 680–685.[ISI][Medline]

Leonard,D.M. (1997) J. Med. Chem., 40, 2971–2990.[ISI][Medline]

Long,S.B., Casey,P.J. and Beese,L.S. (1998) Biochemistry, 37, 9612–9618.[ISI][Medline]

Lowy,D.R. and Willumsen,B.M. (1993) Annu. Rev. Biochem., 62, 851–891.[ISI][Medline]

Mathis,J. and Poulter,C. (1997) Biochemistry, 36, 6367–6376.[ISI][Medline]

Mayer,M.P., Prestwich,G.D., Dolence,J.M., Bond,P.D., Wu,H.-y. and Poulter, C.D. (1993) Gene (Amsterdam), 132, 41–47.[ISI][Medline]

Moores,S.L., Schaber,M.D., Mosser,S.D., Rands,E., O'Hara,M.B., Garsky, V.M., Marshall,M.S., Pompliano,D.L. and Gibbs,J.B. (1991) J. Biol. Chem., 266, 14603–14610.[Abstract/Free Full Text]

Omer,C.A., Kral,A.M., Diehl,R.E., Prendergast,G.C., Powers,S., Allen,C.M., Gibbs,J.B. and Kohl,N.E. (1993) Biochemistry, 32, 5167–5176.[ISI][Medline]

Otwinowski,Z. and Minor,W. (1997) Methods Enzymol., 276, 307–326.[ISI]

Park,H.-W., Boduluri,S.R., Moomaw,J.F., Casey,P.J. and Beese,L.S. (1997) Science, 275, 1800–1804.[Abstract/Free Full Text]

Pompliano,D., Rands,E., Schaber,M., Mosser,S., Anthony,N. and Gibbs,J. (1992) Biochemistry, 31, 3800–3807.[ISI][Medline]

Pompliano,D., Schaber,M., Mosser,S., Omer,C., Shafer,J. and Gibbs,J. (1993) Biochemistry, 32, 8341–8347.[ISI][Medline]

Reiss,Y., Goldstein,J., Seabra,M., Casey,P. and Brown,M. (1990a) Cell, 62, 81–88.[ISI][Medline]

Reiss,Y., Seabra,M.C., Goldstein,J.L. and Brown,M.S. (1990b) Methods: Companion Methods Enzymol., 1, 241–245.

Reiss,Y., Stradley,S.J., Gierasch,L.M., Brown,M.S. and Goldstein,J.L. (1991) Proc. Natl Acad. Sci. USA, 88, 732–736.[Abstract]

Sack,J. (1988) J. Mol. Graphics, 6, 224–225.

Schoner,B.E., Belagajie,R.M. and Schoner,R.G. (1990) Methods Enzymol., 185, 94–103.[Medline]

Seabra,M.C., Goldstein,J.L., Sudhof,T.C. and Brown,M.S. (1992) J. Biol. Chem., 267, 14497–14503.[Abstract/Free Full Text]

Shi,Y., Brown,E.D. and Walsh,C.T. (1994) Proc. Natl Acad. Sci. USA, 91, 2767–2771.[Abstract]

Strickland,C., Windsor,W., Syto R., Wang,L., Bond,R., Wu,Z., Schwartz,J., Le,H., Beese,L. and Weber,P. (1998) Biochemistry, 37, 16601–16611.[ISI][Medline]

Tsao,K.-L. and Waugh,D.S. (1997) Protein Express. Purif., 11, 233–240.[ISI][Medline]

Yokoyama,K., Goodwin,G.W., Ghomashchi,F., Glomset,J.A. and Gelb,M.H. (1991) Proc. Natl Acad.. Sci. USA, 88, 5302–5306.[Abstract]

Yokoyama,K., McGeady,P. and Gelb,M.H. (1995) Biochemistry, 34, 1344–1354.[ISI][Medline]

Zhang,F.L. and Casey,P.J. (1996) Annu. Rev. Biochem., 65, 241–269.[ISI][Medline]

Zhang,F.L., Diehl,R.E., Kohl,N.E., Gibbs,J.B., Giros,B., Casey,P.J. and Omer,C.A. (1994) J. Biol. Chem., 269, 3175–3180.[Abstract/Free Full Text]

Received August 31, 1998; revised December 2, 1998; accepted December 16, 1998.