1 Department of Biochemistry, Uppsala University, Biomedical Center,Box 576, SE-75123 Uppsala, Sweden and 3 Department of Molecular Biology MB4, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
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Abstract |
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Keywords: glutathione transferase/monomer/substrate binding/subunit interactions
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Introduction |
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Human GSTP1-1, a member of the pi class, has attracted particular interest because of its increased levels in cancer cells and possible involvement in acquired drug resistance. It is also the GST with the broadest tissue distribution. For recent reviews on GSTs, see Hayes and Pulford (Hayes and Pulford, 1995), Mannervik and Widersten (Mannervik and Widersten, 1995
), Armstrong (Armstrong, 1997
), Snyder and Maddison (Snyder and Maddison, 1997
) and Johansson and Mannervik (Johansson and Mannervik, 2001
).
GSTs are normally present as dimers and whether the single subunit can exist as a stable structured protein is not completely clear. Unfolding studies of class pi GSTs in two different laboratories have been given different interpretations. Aceto et al. (Aceto et al., 1992), in a study on human GSTP1-1, found evidence for a three-state mechanism involving the dimeric native state, stable but inactive monomers, and unfolded monomers. Dirr and Reinemer (Dirr and Reinemer, 1991
) and Erhardt and Dirr (Erhardt and Dirr, 1995
) on the other hand suggested a two-state model for the homologous porcine GSTP1-1, where the dissociation of the dimer into monomers is accompanied by their complete unfolding. There is no evidence for the existence of catalytically active monomers in the living cell. However, monomeric forms of GSTP1-1 have been detected in plasma samples (Kura et al., 1996
) and suggested to play a role in regulation of Jun N-terminal kinase signaling (Adler et al., 1999
).
The present paper describes the design, preparation and properties of a stable monomeric form of human GSTP1-1. GSTP1 was generated by the introduction of 10 site-specific mutations in the subunit interface. The monomer GSTP1 provides a new tool for studies on structureactivity relationships in GSTs. Examples of topics where the monomer could be most useful include amino acid residues involved in subunitsubunit interactions, the role of the quaternary structure for stability and catalysis, and folding of the individual subunit.
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Materials and methods |
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Site-directed mutagenesis was employed to introduce 10 mutations in the cDNA encoding human GSTP1-1. The clone pKHP1 previously described (Kolm et al., 1995) was used as template for PCR. By two rounds of inverted polymerase chain reaction (IPCR), the resulting mutant was generated in a stepwise manner. The first round yielded four mutations and the resulting DNA product was used as template for the second reaction, which added the remaining six mutations. The following primers were used (altered nucleotides are underlined): Forw1, 5'-CAG GAC GGA GAC CAG ACC GAC TAC CAG TCC AAT ACC-3'; Rev1, 5'-GAA CTT GGG GAG CTG CCC TTC CTG GCA GGA GGC TTT-3'; Forw2, 5'-AT GAC AAA GTG GAG GAC CTC CGC CAA AAA TAC ATC-3'; Rev2, 5'-T CAC CTT GTC CAC CTC TTT TTC CTC CTG CTG GTC-3'.
The primers were phosphorylated before use. The reaction mixture contained 0.2 mM dNTPs, 0.8 µM of each primer, various amounts of the template, and 2.5 U Pfu DNA polymerase (Stratagene, La Jolla, CA) in the buffer provided with the enzyme. PCR started with denaturation at 95°C for 10 min and was followed by 25 cycles of 95°C for 1 min, 70°C for 2 min and 72°C for 9 min. The program was completed with a further extension period at 72°C for 30 min. The DNA was isolated from agarose gel after electrophoresis, purified using the Geneclean kit (Bio 101 Inc., Vista, CA) and ligated. The circularized DNA was used to transform Escherichia coli JM 109 or E.coli XL-1 Blue. The entire cDNA was sequenced to verify that no unwanted mutation had been introduced.
Construction of a histidine-tagged monomer, his-GSTP
Six histidine residues were added to the N-terminus of the monomer using PCR. The plasmid containing the cDNA encoding monomeric GSTP1 was used as a template for amplification. The following primers were used: 5'-CAC CAC CAC ATG CCT CCA TAC ACA GTT GTT TAC-3' and 5'-GTG GTG GTG CAT TTT GTC ACC TTT GAA TTC TGT TTC-3'. IPCR was carried out as described above except that the annealing temperature was kept at 66°C.
Expression and preparation of a bacterial lysate
Monomeric GSTP1 was expressed in E.coli as described (Kolm et al., 1995) except that the growth temperature was decreased to 30°C. The culture was grown for
20 h, and the bacteria were collected by centrifugation for 10 min at 7000 g and re-suspended in lysis buffer, containing 20 mM potassium phosphate, pH 7.0, 5 mM EDTA, 2 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride and 1 mg/ml chicken egg-white lysozyme. For the his-monomer the lysis buffer was prepared with 10 mM 2-mercaptoethanol instead of DTT. After sonication (4x20 s), the soluble fraction was obtained by centrifugation at 30 000 g for 20 min. This process was repeated once and the pooled lysate was centrifuged at 30 000 g for 45 min.
Purification of monomeric GSTP1
All the purification steps were carried out at 4°C. The absorbance at 280 nm was followed by a UV monitor in all chromatographic steps.
Hydrophobic interaction chromatography. Ammonium sulfate was added to the lysate to a final concentration of 0.5 M. The solution was then applied to a column (8x2 cm) packed with phenyl Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with 20 mM potassium phosphate, pH 7.0, containing 2 mM DTT (buffer A) fortified with 0.5 M ammonium sulfate. After washing the column with buffer A including 0.25 M ammonium sulfate, the monomer was eluted with buffer A only. To increase the yield of monomer, an additional elution step with 50% ethylene glycol in buffer A was also performed. All steps were carried out at a flow rate of 2 ml/min. The pooled fractions were dialyzed overnight against 20 mM TrisHCl, pH 8.0, with 2 mM DTT (buffer B) and subjected to anion-exchange chromatography using Q-Sepharose (Amersham Pharmacia Biotech) equilibrated with buffer B. After washing with buffer B the monomer was eluted with the same buffer containing 150 mM NaCl at a flow rate of 1 ml/min. The pooled fraction from the ion-exchange column was concentrated using an ultra-filtration device (Amicon Inc., Beverly, MA) and applied to a gel filtration column (90x1.4 cm) of Sephadex G-75 (Amersham Pharmacia Biotech). Equilibration was with buffer B containing 100 mM NaCl and 0.02% (w/v) sodium azide. The flow rate of the column was set at 6 ml/h and fractions of 2 ml were collected.
The his-GSTP1 monomer was purified from the crude, centrifuged bacterial lysate using immobilized metal affinity chromatography (IMAC). Chelating Sepharose FF (Amersham Pharmacia Biotech) was charged with NiCl2 according to the manufacturers instructions. The column was equilibrated with 20 mM sodium phosphate containing 10 mM 2-mercaptoethanol, 500 mM NaCl and 85 mM imidazole, pH 7.5. After the lysate had been applied, the column was washed extensively with equilibration buffer. The monomer was eluted with 400 mM imidazole in equilibration buffer and dialyzed overnight against 20 mM TrisHCl containing 10 mM 2-mercaptoethanol, concentrated using ultra-filtration and then applied to the gel filtration column as previously described.
The wild-type human GSTP1-1 was expressed in E.coliXL-1 Blue and purified as described in Kolm et al. (Kolm et al., 1995) with the following modification. GlutathioneSepharose was prepared by coupling GSH via its sulfhydryl group to epoxy-activated Sepharose 6B (Amersham Pharmacia Biotech) according to Simons and Vander Jagt (Simons and Vander Jagt, 1977
). GSH was subsequently removed from the purified GSTP1-1 by filtration of the eluted protein through Sephadex G-25.
Molecular mass determination. The apparent molecular masses of the two monomeric variants were determined by size exclusion chromatography on the Sephadex G-75 column at 4°C, calibrated with the following molecular mass standard proteins: albumin (67 kDa), GSTP1-1 (46 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa). Approximately 34 mg protein of the monomeric GSTP1 and molecular mass standards were applied to the column at a flow rate of 6 ml/h.
Analysis and characterization
The purity of the protein was checked by SDSPAGE as described (Laemmli, 1970). Protein concentration was determined by the method of Bradford (Bradford, 1976
) using a kit from Bio-Rad (Hercules, CA) with bovine serum albumin (BSA) as a standard. Circular dichroism of the wild-type enzyme and the two monomeric proteins was measured in 20 mM TrisHCl, pH 8.0, at room temperature using a Cary spectropolarimeter, with a 10 mm path length. The protein concentration was 0.2 mg/ml. The enzyme activity assays were performed at 30°C with the following substrates: 1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid and 1,2-epoxy-3-(4-nitrophenoxy)propane essentially as described (Habig et al., 1974
); the GSH concentration was 10 mM in the CDNB assay. Activities with crotonaldehyde and acrolein were assayed as described (Berhane et al., 1994
). Measurements of the intrinsic fluorescence of the proteins (native dimer GSTP1-1, monomeric GSTP1 and his-GSTP1) were performed at room temperature using an Aminco SPF-500 corrected beam spectrofluorimeter. The excitation and emission band widths were both set to 5 nm. Fluorescence was measured by excitation of all aromatic residues at 280 nm or of tryptophans only at 295 nm. Fluorescence was also used for unfolding and binding experiments. The various compounds added were of different concentrations in order to keep volume and protein concentrations constant and avoid any dilution effects. The absorbance at the excitation wavelengths was measured for all additives and inner filter effects were not considered to be a problem. All fluorescence measurements were repeated at least three times.
Equilibrium unfolding/refolding studies. The protein samples (0.75 µM subunit concentration) were incubated at room temperature for 1 h in 20 mM potassium phosphate, pH 7.0, containing1 mM EDTA and 1 mM DTT, and different concentrations of urea (08 M) or guanidinium hydrochloride (Gdn-HCl) (04 M) before recording of spectra. In the refolding studies, a solution of the unfolded protein (7.5 µM of subunit incubated for 1 h in different concentrations of denaturants) was diluted 10-fold with the buffer, and the fluorescence spectra were recorded after 10 min.
Equilibrium binding of 8-anilino-1-naphthalene sulfonate (ANS) was determined by measuring the enhancement of ANS fluorescence following the addition of different concentrations of the ligand (5100 µM) to a 1 µM subunit of the monomer or the native dimer in 20 mM potassium phosphate, pH 7.0, containing 1 mM EDTA. The sample was excited at 380 nm and fluorescence intensity was measured at 480 nm. All intensities were corrected for background. The dissociation constants (Kd) of the ANSprotein complexes were determined essentially as described (Nishihira et al., 1992). Various concentrations (120 µM) of the proteins were mixed with 10 µM ANS in potassium phosphate, pH 7.0, and the fluorescence intensity was measured after 2 h of incubation.
ANS displacement experiment. Different concentrations of hydrophobic compounds [10100 µM bromosulfophthalein (BSP); 0.11.5 mM CDNB; 0.51.5 mM ethacrynic acid; 50200 µM S-hexyl-GSH or 10250 µM S-p-nitrobenzyl-GSH] were added to a mixture containing 25 µM ANS and 2 µM subunit of either monomeric GSTP1 or dimeric GSTP1-1. The change in ANS fluorescence was recorded as above.
Enzyme-linked immunosorbent assay (ELISA). A micro-titer plate was coated with the protein fractions to be analyzed. Serial dilutions of protein were made in 0.1 M sodium carbonate, pH 9.5, and the protein was allowed to bind. After washing with Tris-buffered saline (TBS)Tween, the plate was blocked with 2% BSA in TBS and washed as above. Polyclonal rabbit anti-human GSTP1-1 antibodies were added and followed by alkaline phosphatase goat anti-rabbit IgG conjugate after washing. Finally, p-nitrophenyl phosphate (50 µl per well, 0.5 mg/ml) in 25 mM diethanolamine (pH 9.5) was added and after 30 min of incubation the absorbance at 490 nm was determined using a micro-titer plate reader.
Attempts to form a heterodimer
Wild-type GSTP1-1 (0.25 mg/ml) was denatured for 1 h at room temperature in 20 mM sodium phosphate, pH 7.5, containing 6 M urea and 10 mM 2-mercaptoethanol. A 4 ml aliquot of the solution was added to 10 ml of native his-GSTP1 (0.4 mg protein/ml). After incubation for 30 min at room temperature, the mixture was further diluted 2-fold and then applied to an IMAC Ni(II) column. The column was washed with buffer containing 85 mM imidazole and the bound protein was eluted with 400 mM imidazole in buffer. The flow-through from the washing step and the eluted fractions were analyzed with SDSPAGE and assayed for CDNB conjugation activity.
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Results and discussion |
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The accessible surface area buried upon dimerization of the subunits in human GSTP1-1 is 1300 Å2 per subunit (Reinemer et al., 1992). The interface area is usually proportional to the molecular mass of the protein and hGSTP1-1 falls within the normal range. In the present work, 10 mutations were introduced at the subunits contact areas in wild-type GSTP1-1. Ten mutations grouped in three clusters (cluster 1: L49Q, Y50E; cluster 2: L61Q, L63E; and cluster 3: A87E, A88K, L89E, M92E, G96K and C102Q), were successfully incorporated into wild-type human GSTP1-1 DNA via two consecutive PCR steps. Table I
lists the mutated amino acid residues and their contributions to the subunitsubunit interactions. The targeted residues are also shown in Figure 1
.
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A particular lock-and-key motif characterizes the dimer interfaces of GSTs of classes alpha, mu and pi. Three residues out of the six that form the lock-and-key were mutated: Tyr50 into Glu, Met92 into Glu, and Gly96 into Gln. Leu49 was also changed to Gln to disrupt its hydrophobic contacts with Leu133 and Met92, residues involved in the lock-and-key interactions. The purpose of the other mutations was either to disrupt inter-monomer hydrophobic packing (Leu61 into Gln and Leu63 into Glu) or to disrupt the hydrophobic interface (Ala87 into Glu, Ala88 into Lys, and Cys102 into Gln). Together these mutations successfully disrupted the inter-subunit interactions of the wild-type enzyme and resulted in a structurally stable monomer. The mutated monomers do not associate with each other if the reduced state is maintained, or with a subunit from the native dimer.
Protein expression and purification
Expression of monomeric GSTP1 was accomplished in E.coli JM109 or E.coli XL-1 Blue at 30°C. Expression at 37°C resulted in a very low yield of soluble protein. Usually, enzymatic activity is used to monitor expression and purification of GSTs but here no activity in the bacterial lysate could be detected using CDNB as substrate. Instead, the expression was checked using antibodies raised against the wild-type enzyme GSTP1-1. It was found that the antibodies could detect the monomeric protein by means of ELISA.
The monomeric protein failed to bind to either GSHSepharose or S-hexyl-glutathioneSepharose, affinity matrices normally used for purification of GSTs, and an alternative purification strategy had to be developed involving hydrophobic interaction chromatography on phenyl-Sepharose and ion-exchange chromatography using Q-Sepharose. The final yield of monomeric GSTP1 was 4 mg from 1 l of culture, which is
10% of the expressed recombinant protein present in the bacterial lysate. Due to oxidation of cysteine residues and possibly hydrophobic interactions, the major part of the expressed protein formed inactive dimers and larger aggregates.
In order to facilitate purification, a histidine-tag was added to the N-terminus of the monomeric GSTP1. Approximately 30 mg of protein were obtained from 1 l of bacterial culture after the IMAC step and the protein was at least 99% pure as judged by SDSPAGE. A gel filtration step on Sephadex as described above was also required to eliminate oxidized products of monomeric GSTP1 and the final yield was 7 mg. Also, for the histidine-tagged variant, expression at 37°C led to a lower yield of soluble protein. Changing the growth medium to LB led to a further decrease in the protein expression level, leaving only 2 mg of GSTP1 monomer. SDSPAGE analysis of the pellet after centrifugation of the bacterial lysate showed that a substantial amount of the monomeric protein had precipitated. If the oxidized aggregates of monomeric GSTP1 were incubated with 20 mM DTT for 1 h at room temperature and then applied to the gel filtration column, approximately one third of the applied sample could be recovered in the monomeric form. To obtain a higher yield, 4 M urea in addition to DTT was required in order to dissociate the aggregates into monomeric GSTP1.
Activity measurements
No catalytic activity could be detected for GSTP1 nor with the oxidized, aggregated product (dimers and oligomers) with any of the standard substrates tested. Up to 200 µg of GSTP1 protein in 1 ml of the assay mixture were used in order to trace minute activities that could have been present (Table II), but none was found.
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The apparent molecular masses of the monomeric GSTP1 and his-GSTP1 proteins were estimated as 26.9 and 29.5 kDa, respectively, based on gel filtration. Analytical ultracentrifugation gave the result of 24.6 and 25.8 kDa for GSTP1 and his-GSTP1, respectively (data not shown).
Far-UV circular dichroism (CD) measurements showed that the secondary structure contents of the two monomers were the same and comparable to that of wild-type GSTP1-1 measured under identical conditions (data not shown).
The intrinsic fluorescence (excitation at 280 nm) for the native state of the two monomeric GSTP1 variants was the same and characterized by a maximum emission wavelength at 340 nm, whereas that of dimeric GSTP1-1 is at 335 nm. Selective excitation of tryptophan residues at 295 nm gave slightly shifted maximum emission wavelengths of 337.5 and 336 nm for the native dimer and the monomeric proteins, respectively.
The complete reversibility of unfolding of the his-GSTP1 and the dimeric wild-type enzyme was demonstrated by fluorescence measurements 10 min after 10-fold dilution of denatured protein with buffer. The unfolding transition of monomeric GSTP1 and the native dimer was measured by addition of increasing concentrations of urea (08 M) or Gdn-HCl (04 M). At equilibrium, increasing concentration of denaturant caused a red shift of the max of emission, thus indicating an increased exposure of tyrosine and tryptophan residues to the aqueous solvent. The mid-point denaturant concentration values for the monomer were 1.2 M for urea and 0.8 M for Gdn-HCl and the corresponding values for the native dimer were 4.2 and 1.5 M for urea and Gdn-HCl, respectively.
Binding of active-site ligands
The intrinsic fluorescence was also used to study the binding of different ligands to the monomer and the native dimer. It was found that 10 µM of S-p-nitrobenzylglutathione quenched the fluorescence of the native dimer to approximately two thirds of that of the unliganded enzyme (Figure 2). Under the same conditions, this compound had no effect on the fluorescence of the monomer. If higher concentrations of the ligand were used, the fluorescence of the monomer was quenched to the same extent as that of BSA, which was included for monitoring non-specific effects. In both proteins (monomer and BSA) this quenching was unaffected by the presence of up to 10 mM GSH. In contrast, the fluorescence of the native dimer in complex with S-p-nitrobenzylglutathione increased upon addition of GSH. This indicates that GSH displaces S-p-nitrobenzylglutathione from the binding site of the native dimer and that the observed quenching of the monomer and BSA at a high concentration of S-p-nitrobenzylglutathione is an artifact that may be due to the absorbance of this compound at the excitation wavelength (280 nm) used (Figure 3
).
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It was found that the monomer does not associate into a dimeric state even at high concentrations of protein (up to 20 mg/ml) if its sulfhydryl groups are maintained reduced. To find out if the constructed monomer could associate with an unmodified subunit of wild-type GSTP1-1, an experiment was designed using the difference between the his-GSTP1 variant and the wild-type enzyme for binding in the IMAC (Ni) column. No heterodimer formation could be detected.
Evolutionary aspects
Functional proteins are often oligomeric structures and several models for their formation have been proposed. In one study (Xu et al., 1998), 32 homo-dimeric proteins were analyzed from an evolutionary mechanistic point of view. The proteins were divided into three groups, each characterized by a particular evolutionary pathway. One possibility is that the functional dimer is formed directly. Proteins within this group are folded in a concerted way according to a two-state mechanism and hydrophobic interactions contribute significantly to the subunit interface. A second pathway starts with a stable monomer, which by mutations in surface residues evolves into a dimer. Here, folding proceeds via a stable intermediate, e.g. the structured monomer. The third model assumes that domain swapping is the mechanism behind dimerization. Based on work by Aceto et al. (Aceto et al., 1992
) GSTP1-1 was classified as a three-state protein. Since this indicates that GSTs have evolved from a stable monomer, the interface is likely to be relatively small and few mutations are required to restore the monomer from being a subunit in a dimer. The results of the present investigation where 10 point-mutations yielded the stable monomer support this idea.
Several questions concerning the assembly of GST subunits have been raised. Until very recently it was generally accepted that molecular recognition is strict and heterodimers can only be formed between members of the same class. However, a small amount of heterodimers between the porcine class pi enzyme and a rat mu class member has been produced in vitro and isolated after dialysis (Pettigrew and Colman, 2001). The properties of the heterodimer were not the means of those of the corresponding homodimers, indicating a role of the interface to the function of the enzyme. Still, the question remains regarding the determinants in the interface that secure the correct dimer formation. And which, if any, of the subunitsubunit interactions are needed for catalytic activity, and could there be an active monomer?
Similar to GSTs of classes alpha and mu and other members of class pi, human GSTP1-1 displays a V-shaped interface located close to the active site. These GST interfaces have a lock-and-key interaction where the key is either a Phe or Tyr and the lock is formed by five residues in the second subunit. Theta class GSTT2-2 has a somewhat simpler lock-and-key motif (Rossjohn et al., 1998) while the interface in class sigma GST is flat and hydrophilic. In human GSTP1-1 the lock-and-key function is obtained by wedging the hydrophobic side chain of Tyr50 from one subunit into the lock formed by residues Met92, Gly96, Pro129, Phe130 and Leu133 in the other subunit (Reinemer et al., 1992
). A recent study describes the effect of replacing the key residue, Tyr50 with five other amino acids with different functionalities. All mutants formed dimers but displayed largely altered catalytic and structural properties depending on the replacing residue (Stenberg et al., 2000
).
All soluble GSTs are dimers indicating the significance of this quaternary structure for function. The GSH-binding site has been characterized in detail by the crystal structures of GSTs and many residues in the G-site are conserved among classes. Bound GSH is very well accommodated in the active site and almost every functional group is involved in interactions with the protein. Particularly important is the -Glu part of the molecule (Adang et al., 1990
; Widersten et al., 1996
; Gustafsson et al., 2001
). Amino acid residues in the turn between strand ß4 and helix
3 contribute to these interactions. None of the GSH interaction residues are mutated in the monomer, but residues in ß4 are. It has also been shown through crystal structures of GSTP1-1 in complex with GSH that the key residue Tyr50 makes 15 van der Waals and hydrogen bonding contacts with the neighboring subunit, while in the apo-enzyme there are only eight such contacts (Oakley et al., 1998
). This may be a mechanism by which the dimer interface influences the conformation of the active site and binding of GSH. Tyr50 is located in a flexible part of the protein molecule, consisting of helix
2 and the following loop. It is thought that the flexibility may have a role in regulating catalysis (Caccuri et al., 1996
). The only inter-subunit contact to GSH is by Asp99. Several studies, three on human GSTP1-1, on the mutagenesis of this residue have been reported. The effect is largely dependent on the replacing residue. A leucine in position 99 causes almost complete loss of activity and <6% of binding to GSH remains (Manoharan et al., 1992
). Widersten et al. (Widersten et al., 1992
) replaced Asp99 with Asn and concluded that the aspartic acid contributed mainly to catalysis and not to binding of GSH. Kong et al. (Kong et al., 1993
) on the other hand reported an effect on binding and a smaller impact on catalysis based on studies of mutant D99A. Mutation of the corresponding residue in rat GSTA1-1, an alpha class enzyme, to glutamate and asparagine showed a minor effect by the former mutation and a binding effect of the latter substitution (Wang et al., 1992
).
The present results indicate that loss of catalytic activity of the monomer may be due to a structural change in the G-site while the H-site remains substantially structurally unchanged. The monomer in this case shows similarities to a dimer formed from two proteolytic fragments of hGSTP1-1 of 17 kDa each (mostly constituted by domain II) (Aceto et al., 1995). All amino acid residues needed for formation of the interface were present in the polypeptide and apparently a stable dimeric structure could be established. Although this truncated dimer was found to have lost the ability to bind to GSHSepharose, it could still bind a number of hydrophobic compounds such as TNS, CDNB, hemin and bilirubin (Aceto et al., 1995
).
All together, it is unlikely that a single-point mutation alone is responsible for the lost affinity for GSH by the monomer. Several factors must contribute to the structural change of the G-site, which may as a result lead to an inactive monomer. Earlier work addressing the significance of subunitsubunit interactions to structure and activity in human GSTP1-1 further emphasizes the sensitivity of the G-site to structural modifications also in parts distant from the active site (Stenberg et al., 2000).
The successful design and preparation of the monomeric GSTP1 is of value not only for studies on the folding and assembly of the dimeric GSTP1-1, but also for investigations of the structural requirements for catalytic activity. Phage display has been employed for the redesign of GSTs with novel functional properties (Widersten and Mannervik, 1995; Hansson et al., 1997
), and the monomeric protein has distinct advantages over a dimeric structure, since it avoids the dimerization step in the expression of the protein. BSP has previously been used as an affinity chromatography ligand for the purification of GSTs (Clark et al., 1977
; McLellan and Hayes, 1989
). Possibly, the findings reported here that BSP can displace ANS from the monomer can be further explored. The monomeric GSTP1-1 could be used as part of fusion proteins that need to be maintained in a monomeric state. Finally, it has been proposed that GSTs could be employed as binding proteins, glubodies (Napolitano et al., 1996
) and for certain applications a monomeric structure may be particularly useful.
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Notes |
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4 To whom correspondence should be addressed. E-mail: gun.stenberg{at}biokem.uu.se
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Acknowledgments |
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References |
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Received March 25, 2002; revised June 27, 2002; accepted July 3, 2002.