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• W2066864373 abstract "In human glutathione transferase P1-1 (hGSTP1-1) position 146 is occupied by a glycine residue, which is located in a bend of a long loop that together with the α6-helix forms a substructure (GST motif II) maintained in all soluble GSTs. In the present study G146A and G146V mutants were generated by site-directed mutagenesis in order to investigate the function played by this conserved residue in folding and stability of hGSTP1-1. Crystallographic analysis of the G146V variant, expressed at the permissive temperature of 25 °C, indicates that the mutation causes a substantial change of the backbone conformation because of steric hindrance. Stability measurements indicate that this mutant is inactivated at a temperature as low as 32 °C. The structure of the G146A mutant is identical to that of the wild type with the mutated residue having main-chain bond angles in a high energy region of the Ramachandran plot. However even this Gly → Ala substitution inactivates the enzyme at 37 °C. Thermodynamic analysis of all variants confirms, together with previous findings, the critical role played by GST motif II for overall protein stability. Analysis of reactivation in vitro indicates that any mutation of Gly-146 alters the folding pathway by favoring aggregation at 37 °C. It is hypothesized that the GST motif II is involved in the nucleation mechanism of the protein and that the substitution of Gly-146 alters this transient substructure. Gly-146 is part of the buried local sequence GXXh(T/S)XXDh (X is any residue and h is a hydrophobic residue), conserved in all GSTs and related proteins that seems to behave as a characteristic structural module important for protein folding and stability. In human glutathione transferase P1-1 (hGSTP1-1) position 146 is occupied by a glycine residue, which is located in a bend of a long loop that together with the α6-helix forms a substructure (GST motif II) maintained in all soluble GSTs. In the present study G146A and G146V mutants were generated by site-directed mutagenesis in order to investigate the function played by this conserved residue in folding and stability of hGSTP1-1. Crystallographic analysis of the G146V variant, expressed at the permissive temperature of 25 °C, indicates that the mutation causes a substantial change of the backbone conformation because of steric hindrance. Stability measurements indicate that this mutant is inactivated at a temperature as low as 32 °C. The structure of the G146A mutant is identical to that of the wild type with the mutated residue having main-chain bond angles in a high energy region of the Ramachandran plot. However even this Gly → Ala substitution inactivates the enzyme at 37 °C. Thermodynamic analysis of all variants confirms, together with previous findings, the critical role played by GST motif II for overall protein stability. Analysis of reactivation in vitro indicates that any mutation of Gly-146 alters the folding pathway by favoring aggregation at 37 °C. It is hypothesized that the GST motif II is involved in the nucleation mechanism of the protein and that the substitution of Gly-146 alters this transient substructure. Gly-146 is part of the buried local sequence GXXh(T/S)XXDh (X is any residue and h is a hydrophobic residue), conserved in all GSTs and related proteins that seems to behave as a characteristic structural module important for protein folding and stability. human glutathione transferase P1-1 1-chloro-2,4-dinitrobenzene glutathione transferase 2-[N-morpholino]ethanesulfonic acid first residue of the N-capping box motif located at the beginning of an α-helix non-crystallographic symmetry root mean-square Numerous investigations over the last 40 years have focused on the folding and structural determinants that govern how a polypeptide adopts its native structure. Most of our knowledge on protein folding derives from studies on small monomeric proteins. However, the majority of native proteins are more complex structures, composed of several subunits that in turn consist of domains. The extrapolation of results obtained in the study of small proteins to larger ones is not always appropriate, and it is therefore important to investigate proteins composed of more than one subunit. Human glutathione transferase P1-1 (hGSTP1-1),1 a homodimeric enzyme and thus representing the simplest type of oligomeric structure, has been the subject of recent studies on protein folding (1Aceto A. Dragani B. Melino S. Allocati N. Masulli M., Di Ilio C. Petruzzelli R. Biochem. J. 1997; 322: 229-234Crossref PubMed Scopus (37) Google Scholar, 2Dragani B. Stenberg G. Melino S. Petruzzelli R. Mannervik B. Aceto A. J. Biol. Chem. 1997; 272: 25518-25523Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 3Stenberg G. Dragani B. Cocco R. Mannervik B. Aceto A. J. Biol. Chem. 2000; 275: 10421-10428Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The aim of these investigations was to identify sequence patterns of importance to folding kinetics and/or structure of the final native state. hGSTP1-1 is a Pi class member of a multifunctional superfamily of enzymes, the GSTs. The role of GSTs is considered to be the detoxication of a large number of hydrophobic compounds by catalyzing the conjugation to glutathione (GSH) and thus increase their water solubility (4Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google Scholar). The cytosolic GSTs have been grouped into a number of different evolutionary classes denoted by the names of Greek letters such as Alpha, Mu, Pi, and so on (4Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google Scholar, 5Mannervik B. Ålin P. Guthenberg C. Jensson H. Tahir M.K. Warholm M. Jörnvall H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7202-7206Crossref PubMed Scopus (1077) Google Scholar). The classification was originally based on primary structure similarities, substrate specificities, and immunologic properties (5Mannervik B. Ålin P. Guthenberg C. Jensson H. Tahir M.K. Warholm M. Jörnvall H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7202-7206Crossref PubMed Scopus (1077) Google Scholar), but sequence similarities are currently the overriding criterion. The mass of the dimeric GSTs is ∼50 kDa, and subunits within the same class can combine to form either homo- or heterodimers (6Mannervik B. Jensson H. J. Biol. Chem. 1982; 257: 9909-9912Abstract Full Text PDF PubMed Google Scholar). Each subunit contains an active site, and two domains form the subunit. The smaller N-terminal domain (domain I) adopts an α/β topology and provides most of the contacts with GSH. The larger C-terminal domain (domain II) is completely helical and contains most of the residues that form the hydrophobic binding site (10Reinemer P. Dirr H.W. Ladenstein R. Schaffer J. Gallay O. Huber R. EMBO J. 1991; 10: 1997-2005Crossref PubMed Scopus (332) Google Scholar, 11Ji X. Zhang P. Armstrong R.N. Gilliland G.L. Biochemistry. 1992; 31: 10169-10184Crossref PubMed Scopus (378) Google Scholar, 12Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dirr H.W. Huber R. Gilliland G. Armstrong R.N., Ji, X. Board P.G. Olin B. Mannervik B. Jones T.A. J. Mol. Biol. 1993; 232: 192-212Crossref PubMed Scopus (415) Google Scholar, 13Wilce M.C.J. Board P.G. Feil S.C. Parker M.W. EMBO J. 1995; 14: 2133-2143Crossref PubMed Scopus (218) Google Scholar). Alignment of all known GST structures (more than 100) shows that only 6–7 residues, which is less than 5% of the entire polypeptide chain, are strictly conserved. Despite this limited sequence similarity, all GSTs adopt the same native fold. GST structure analysis with special emphasis on the few conserved residues led to the identification of two local structural motifs in a characteristic position, the N-terminal region of the α6-helix (1Aceto A. Dragani B. Melino S. Allocati N. Masulli M., Di Ilio C. Petruzzelli R. Biochem. J. 1997; 322: 229-234Crossref PubMed Scopus (37) Google Scholar). The N-capping box ((S/T)XXD), and the hydrophobic staple motif (14Richardson J.S. Richardson D.C. Science. 1988; 240: 1648-1652Crossref PubMed Scopus (1299) Google Scholar, 15Presta L.G. Rose G.D. Science. 1988; 240: 1632-1641Crossref PubMed Scopus (631) Google Scholar, 16Seale J.W. Srinivasan R. Rose D.G. Protein Sci. 1994; 3: 1741-1745Crossref PubMed Scopus (124) Google Scholar, 17Munoz V. Blanco F.L. Serrano L. Nat. Struct. Biol. 1995; 2: 380-385Crossref PubMed Scopus (130) Google Scholar, 18Munoz V. Serrano L. Biochemistry. 1995; 34: 15301-15306Crossref PubMed Scopus (35) Google Scholar, 19Aurora R. Rose G.D. Protein Sci. 1998; 7: 21-38Crossref PubMed Scopus (657) Google Scholar) in which two hydrophobic residues flanking the N-capping box are present in all GSTs known today (1Aceto A. Dragani B. Melino S. Allocati N. Masulli M., Di Ilio C. Petruzzelli R. Biochem. J. 1997; 322: 229-234Crossref PubMed Scopus (37) Google Scholar, 2Dragani B. Stenberg G. Melino S. Petruzzelli R. Mannervik B. Aceto A. J. Biol. Chem. 1997; 272: 25518-25523Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 3Stenberg G. Dragani B. Cocco R. Mannervik B. Aceto A. J. Biol. Chem. 2000; 275: 10421-10428Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar,20Snyder M.J. Maddison D.R. DNA Cell Biol. 1997; 16: 1373-1384Crossref PubMed Scopus (53) Google Scholar). Previous investigations demonstrated that single mutations of residues forming the capping box ((S/T)XXD) and the hydrophobic staple motifs have a dramatic effect on the protein stability (1Aceto A. Dragani B. Melino S. Allocati N. Masulli M., Di Ilio C. Petruzzelli R. Biochem. J. 1997; 322: 229-234Crossref PubMed Scopus (37) Google Scholar, 2Dragani B. Stenberg G. Melino S. Petruzzelli R. Mannervik B. Aceto A. J. Biol. Chem. 1997; 272: 25518-25523Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 3Stenberg G. Dragani B. Cocco R. Mannervik B. Aceto A. J. Biol. Chem. 2000; 275: 10421-10428Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The same amino acid substitutions also have significant effects on protein folding, generating temperature-sensitive folding mutants unable to refold at the physiological temperature 37 °C (1Aceto A. Dragani B. Melino S. Allocati N. Masulli M., Di Ilio C. Petruzzelli R. Biochem. J. 1997; 322: 229-234Crossref PubMed Scopus (37) Google Scholar, 2Dragani B. Stenberg G. Melino S. Petruzzelli R. Mannervik B. Aceto A. J. Biol. Chem. 1997; 272: 25518-25523Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 3Stenberg G. Dragani B. Cocco R. Mannervik B. Aceto A. J. Biol. Chem. 2000; 275: 10421-10428Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Mutations corresponding to those made in hGSTP1-1 have also been constructed in hGSTA1-1, a member of the Alpha class showing 31.6% sequence identity with hGSTP1-1 (21Cocco R. Stenberg G. Dragani B. Rossi Principe D. Paludi D. Mannervik B. Aceto A. J. Biol. Chem. 2001; 276: 32177-32183Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The results obtained were in accordance with those found for hGSTP1-1, further emphasizing that the highly conserved N-capping box and hydrophobic staple motifs play critical and universal roles in GST folding and stability. The α6-helix and preceding loop form a substructure, named GST motif II, which is conserved in the core of all GSTs and related proteins. The analysis of the protein crystal structure indicates that GST motif II is stabilized by a buried network of eight hydrogen bonds, half of which involve water-mediated contacts (23Rossjohn J. McKinstry W.J. Oakley A.J. Parker M.W. Stenberg G. Mannervik B. Dragani B. Cocco R. Aceto A. J. Mol. Biol. 2000; 302: 295-302Crossref PubMed Scopus (17) Google Scholar). Crystallographic studies of the capping mutants, expressed at the permissive temperature of 25 °C, indicated that these amino acid substitutions locally destabilize GST motif II through a partial or complete loss of the hydrogen bond network (23Rossjohn J. McKinstry W.J. Oakley A.J. Parker M.W. Stenberg G. Mannervik B. Dragani B. Cocco R. Aceto A. J. Mol. Biol. 2000; 302: 295-302Crossref PubMed Scopus (17) Google Scholar). All these results indicate that a local destabilization of GST motif II has a critical effect on the overall protein stability and strongly support the hypothesis that this buried region might be involved in the nucleation mechanism of protein folding. A strictly conserved glycine residue, Gly-146, in hGSTP1-1 is located four residues before Ser or Thr of the Ncap motif in all known GSTs (1Aceto A. Dragani B. Melino S. Allocati N. Masulli M., Di Ilio C. Petruzzelli R. Biochem. J. 1997; 322: 229-234Crossref PubMed Scopus (37) Google Scholar). In particular Gly-146 is part of a buried local sequence GXXh(T/S)XXDh (X is any residue and h is a hydrophobic residue), which is maintained in all GSTs and, as a more general folding module, in other proteins such as EF1γ and URE2. Its role has until now remained unexplored. This amino acid residue is located in a bend of the long loop preceding the α6-helix and does not make any specific contacts with other structural parts of the molecule but only with neighboring residues in the polypeptide. Its strict conservation through evolution implicates the absolute necessity of a small amino acid residue in this position. The present investigation addresses the function of the strictly conserved Gly-146 in folding, and its significance for structural stability. By a combination of protein engineering and x-ray structure-function analysis we have obtained evidence that Gly-146 is part of a conserved folding module. As a universal motif it plays a critical role in the refolding and stability of all GSTs and, probably, of other structurally related proteins such as EF1γ and URE2. Wild-type human GSTP1-1 was obtained by expression of a cloned cDNA in Escherichia coli XL-1 Blue (Stratagene, La Jolla, CA) as previously described (24Kolm R.H. Stenberg G. Widersten M. Mannervik B. Protein Expression Purif. 1995; 6: 265-271Crossref PubMed Scopus (50) Google Scholar). GSH and 1-chloro-2,4-dinitrobenzene (CDNB) were purchased from Sigma. Oligonucleotides and dNTPs were obtained from Amersham Biosciences. Site-directed mutagenesis was employed to generate single-point mutants. The following oligonucleotides were used: 5′-CC TTC ATT GTG A/GC/TA GAC CAG-3′ and 5′-TCT TGC CTC CCT GGT TCT G-3′. The altered codon is underlined. The oligonucleotides were phosphorylated and then used in inverted polymerase chain reaction. The expression clone pKHP1 (24Kolm R.H. Stenberg G. Widersten M. Mannervik B. Protein Expression Purif. 1995; 6: 265-271Crossref PubMed Scopus (50) Google Scholar) was used as a template. The polymerase chain reaction mixture contained 0.8 μm each primer, 0.2 mm dNTPs, 2.5 units ofPfu DNA polymerase (Stratagene, La Jolla, CA); the buffer supplied with the enzyme and various amounts of DNA template. The temperature program started at 94 °C for 10 min and was followed by 25 cycles of 94 °C for 1 min, 70 °C for 1 min, and 72 °C for 9 min. The program terminated with a reaction at 72 °C for 30 min. After electrophoresis the DNA product from the reaction was recovered from the agarose gel. The DNA was ligated and used for transformation of competent E. coli XL-1 Blue cells. The cDNAs encoding the isolated GSTP1-1 mutants were sequenced in their entirety to verify that no undesired mutations had been introduced in the polymerase chain reaction. Cultures of E. coli XL-1 Blue-containing plasmids were grown in 300 ml of LB broth in a 1-liter Erlenmeyer flask at 37 °C. At an OD555 of 0.35, isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 0.2 mm. From the time of addition the culture was grown for about 20 h at 25 or 37 °C. The subsequent purification of wild-type and mutant proteins was performed as described previously (24Kolm R.H. Stenberg G. Widersten M. Mannervik B. Protein Expression Purif. 1995; 6: 265-271Crossref PubMed Scopus (50) Google Scholar) with the only modification that the enzymes were purified on a GSH-Sepharose affinity column (25Aceto A. Caccuri A.M. Sacchetta P. Bucciarelli T. Dragani B. Rosato N. Federici G. Di Ilio C. Biochem. J. 1992; 285: 241-245Crossref PubMed Scopus (68) Google Scholar). The kinetic parameters,k cat and K m, were determined at 25 °C as previously described (26Aceto A., Di Ilio C., Lo Bello M. Bucciarelli T. Angelucci S. Federici G. Carcinogenesis. 1990; 11: 2267-2269Crossref PubMed Scopus (31) Google Scholar). The concentrations of GSH and CDNB were 2 and 1 mm, respectively. Spectroscopic properties of the mutants and the wild-type enzyme were also studied. A Jasco-600 spectropolarimeter was used for CD measurements in the far-ultraviolet region from 200 to 250 nm. Spectra were recorded using a protein concentration of 0.3 mg/ml with cuvettes of 0.1 cm path length in a thermostat-controlled cell holder. Intrinsic fluorescence emission spectra were measured with a Spex (model Fluoromax) spectrofluorometer. The excitation wavelength was 280 nm and the λmax and all emission spectra were analyzed at the same protein concentration (0.1 mg/ml). Enzyme was incubated at each temperature for 10 min at a protein concentration of 0.05 mg/ml in 0.01m potassium phosphate (pH 7.0) containing 1 mmEDTA and 5 mm dithiothreitol to prevent oxidative inactivation. The enzyme was heat-inactivated in sealed Eppendorf tubes, and the temperature was monitored with a Cryson telethermometer. The inactivation time courses were determined by withdrawing suitable aliquots at different time points from the denaturation mixture for assay of remaining activity. The activity was assayed in 0.1m potassium phosphate (pH 6.5) with 2 mm GSH and 1 mm CDNB at 25 °C. Lowering the temperature of incubation could not reverse the thermal inactivation for any of the proteins. The denaturation of the wild-type and glycine mutants was monitored at different temperatures. The enzymes were incubated in 10 mm potassium phosphate, pH 7.0, 1 mm EDTA, 5 mm dithiothreitol, and their activity was monitored for 120 min taking the first value as 100% native protein. An equation describing a single exponential decay with a rate constant of thermal unfolding k u was fitted to the data according to Equation 1. −ln(%native/100%)=kutEquation 1 The free energy of activation of thermal unfolding (ΔG u) was calculated according to Eyring theory (27Johnson F.H. Eyring H. Polissar M.J. The Kinetic Basis of Molecular Biology. John Wiley and Sons, New York1954Google Scholar) as Equation 2,lnku=lnKkbTh−ΔGuRTEquation 2 where kb is the Boltzmann constant; T, the absolute temperature in Kelvin; h, Plank's constant; R, the gas constant; and K is the transmission factor, which was set to unity. The difference of free energy of activation of thermal denaturation between wild-type and each mutant protein (ΔΔG u) was calculated according to Equation3.ΔΔGu=ΔGu,wt−ΔGu,mut=−RT ln(ku,wt/ku,mut)Equation 3 Substitution of Equation 4,ΔGu=ΔHu−TΔSuEquation 4 into Equation 2 yields Equation 5.lnkuT=lnKkbh−ΔHuR1T+ΔSuREquation 5 Both activation enthalpy ΔH u and entropy ΔS u were determined from the temperature dependence of k u. When the refolding of human GSTP1-1 and its mutants was to be monitored, 10 μm enzyme was first denatured in 4 m guanidinium chloride (0.2 mpotassium phosphate, 1 mm EDTA, 5 mmdithiothreitol, pH 7.0) at 25, 33, and 40 °C for 30 min and then diluted (defining time 0) 1:40 into renaturation buffer (0.2m potassium phosphate, 1 mm EDTA, 5 mm dithiothreitol, pH 7.0) at the same temperature. The final guanidinium chloride concentration was 0.1 m during refolding. All refolding experiments were carried out by rapid addition of the denatured enzyme to renaturation buffer. Activity recovered as a function of time was monitored by withdrawal of appropriate aliquots of the renaturation mixture followed immediately by dilution into 2.0 ml of assay buffer. The kinetic parameters of refolding were determined by non-linear regression analysis by fitting equations with one or two exponentials to the experimental data using the KaleidaGraph 3.0.5 program (Abelbek Software). The values reported in this study represent the means of at least three different experimental data sets. The influence of concentration on folding was analyzed by refolding the wild-type and Gly-146 mutants as described in the previous section. Six different protein concentrations were used for denaturation ranging from 0.2 to 6.0 mg/ml. Refolding was monitored as described above by assaying for recovered activity after 2 h. A second dilution during refolding was performed at different time points after the initiation of reactivation. Enzymes at an optimized concentration (1 mg/ml) were denatured and allowed to start to refold as described above. After 1 min (wild-type) and after 5 and 20 min (all enzymes), the refolding mixture was diluted 10-fold and analyzed for reactivation by measuring increase of activity with time. Coordinates of GST x-ray structures were derived from the Protein Data Bank (www.rcsb.org/pdb/) via the anonymous file-transfer protocol. The crystal structures were analyzed by using Hyperchem (Autodesk, Sausalito, CA) and MolView 1.4.6 (Purdue University) programs. The figures were generated using the RasMol (version 2.6) program. A PHI-BLAST (28Zhang Z. Schäffer A.A. Miller W. Madden T.L. Lipman D.J. Koonin E.V. Altschul S.F. Nucleic Acids Res. 1998; 26: 3986-3990Crossref PubMed Scopus (256) Google Scholar) (www.ncbi.nlm.nih.gov/blast/psiblast.cgi) search was performed using the BLOSUM62 matrix. The pattern used as a query was GX(2)-[LIVY]-[ST]-X(2)-[D]-[LYIVA] and the threshold value was set to 0.001. Crystallization was performed by the hanging drop vapor diffusion method as described elsewhere (29Oakley A.J., Lo Bello M. Battistoni A. Ricci G. Rossjohn J. Villar H.O. Parker M.W. J. Mol. Biol. 1997; 274: 84-100Crossref PubMed Scopus (154) Google Scholar). Briefly, a 2-μl drop of a protein solution containing the GST mutant (4.2 mg/ml for G146V, 2 mg/ml for G146A) in 1 mm EDTA, 1 mm dithiothreitol, and 10 mm HEPES buffer (pH 7.0) was mixed with an equal volume of reservoir solution, which consisted of 15–25% (w/v) polyethylene glycol (PEG) 8000, 20 mm CaCl2, 1 mm GSH, 10 mm dithiothreitol, and 100 mm MES buffer (pH range 5.2–5.8). All trials were carried out at a constant temperature of 22 °C. Crystals took between 3 and 5 days to appear and grew to their final size within 2 weeks. The x-ray diffraction data were collected using a MARResearch area detector with CuKα X-rays generated by a Rigaku RU-200 rotating anode x-ray generator. The data were collected at 100 K. The diffraction data were processed and analyzed using programs in the HKL (30Otwinowski Z. Sawyer L. Isaacs N. Bailey S. Data Collection and Processing. SERC Daresbury Laboratory, Warrington, UK1993Google Scholar) and CCP4 suites (31CCP4 Acta Crystallogr. Sect. D. 1994; 50: 750-763Google Scholar). The G146A and G146V mutants crystallized in the same space group and cell as wild type. For both mutants the refinement began with wild-type Pi class GST in the C2 space group (10GS; Ref. 29Oakley A.J., Lo Bello M. Battistoni A. Ricci G. Rossjohn J. Villar H.O. Parker M.W. J. Mol. Biol. 1997; 274: 84-100Crossref PubMed Scopus (154) Google Scholar) that had inhibitor and water molecules omitted. In addition residues between 144 and 150 in each monomer were removed from the starting model. Rigid body refinement in CNS (32Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16978) Google Scholar) was used to compensate for any possible changes in crystal packing. As the asymmetric unit of the crystal contained two GST monomers, use was made of the non-crystallographic symmetry (NCS) restraints on all non-hydrogen atoms throughout the course of the positional refinements. The 2F obs − F calc map of the G146A mutant showed clear and continuous density for residues between 144 and 150 that had been omitted from the search model. These residues were then built into the map. Among the significant features in the subsequent F obs − F calcmap were positive peaks (close to 4× the r.m.s. error of the map) within 2 Å of the Cα of Gly-146, thus confirming the mutation. The residue at position 146 was changed from glycine to alanine and the model refined. The 2F obs −F calc and F obs −F calc maps of the G146V mutant clearly indicated a change in the backbone conformation of the region between residues 144 and 146. After the backbone alterations were made, there was additional density next to the Cα position of residue 146 sufficient to accommodate a valine side-chain (close to 3× the r.m.s. error of the F obs − F calc map), and hence the glycine residue was changed to a valine residue. These residues were then built into the map of the G146V mutant. For both mutants, a number of rounds of positional refinement were performed followed by model building and then by rounds of positional and individual NCS-restrained B-factor refinement. In the final stages of refinement a bulk solvent correction was employed. The correctness of the final structures in the regions around the mutation were confirmed by calculating omit maps between residues 144 and 148. A stereochemical analysis of the refined structure with the program PROCHECK (33Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) gave values either similar or better than expected for structures refined at similar resolutions. The coordinates for the G146A and G146V models have been deposited in the Protein Data Bank with accession numbers1MD3 and 1MD4, respectively. To investigate the role of the buried and conserved glycine residue (Gly-146, in hGSTP1-1) this amino acid was replaced with alanine and valine by oligonucleotide-directed mutagenesis producing the mutants G146A and G146V. Mutant and wild-type enzymes were expressed in E. coli XL-1 Blue and purified by affinity chromatography on immobilized GSH. The purified enzymes gave a single band on SDS-PAGE (not shown). 65–90% of the total activity was recovered showing that the affinity for GSH-Sepharose was essentially unaffected by the mutations. Considering that the above substitutions could represent temperature-sensitive mutations, protein expression was performed both at 25 and 37 °C of host cell growth. No dependence of protein yield on temperature could be observed (not shown). The yields of the G146A and G146V mutants, in percentage of total cytosolic proteins, were 2.4 and 1.7% respectively after purification, both slightly lower than that (4.0%) of the wild-type enzyme. Table Isummarizes the kinetic parameters for the conjugation of CDNB to GSH catalyzed by the wild-type hGSTP1-1 and Gly-146 mutants. The replacement of Gly-146 with either alanine or valine significantly increases the k cat values of the corresponding mutants as compared with that of the parental enzyme. TheK m values of the mutants were 1.5–2-fold higher for both substrates than those of the wild type. This suggests that the above substitutions slightly but significantly decrease the affinity for GSH and CDNB in both mutants. The physical properties of the Gly-146 mutants and the wild-type enzyme were very similar. The far-UV CD spectra as well as their gel filtration retention times (not shown) were the same, suggesting that all enzyme variants are, in terms of secondary structure and dimeric state of the molecule, essentially identical. The λmax values of the intrinsic fluorescence spectra were 338, 338, and 339 nm for the wild-type enzyme and the G146V and G146A mutants, respectively (not shown), suggesting that a similar polarity characterizes the environment of the tryptophan residues of all enzyme variants. The normalized intensities of the fluorescence of the mutants were slightly higher than that of the wild-type enzyme. This indicates that limited conformational changes distinguish the final structure of the mutants from that of the parent enzyme. These differences involve the environment of one or both tryptophan residues located in GST domain I, far from the mutation site.Table IKinetic parameters of wild-type and glycine mutants of hGSTP1-1 heterologously expressed at 25 °C in E. coliSpecific activityk catCDNBK mGSHK mCDNBk cat/K mCDNBunits/mgs −1mmmms −1 mm −1Wild-type35 ± 534 ± 20.15 ± 0.020.94 ± 0.136G146A43 ± 688 ± 40.31 ± 0.021.81 ± 0.0149G146V85 ± 755 ± 40.25 ± 0.041.65 ± 0.1833 Open table in a new tab The structure of helix α6 and the preceding loop is well defined in the wild-type 1.9 Å resolution electron density maps (29Oakley A.J., Lo Bello M. Battistoni A. Ricci G. Rossjohn J. Villar H.O. Parker M.W. J. Mol. Biol. 1997; 274: 84-100Crossref PubMed Scopus (154) Google Scholar). The region encompassing the loop (residues 141–149) and the N-terminal end of the helix is characterized by a network of eight hydrogen bonds between the two, half of which involving a water-mediated contact (Fig. 1 A). In addition to these contacts, other significant polar contacts involving the loop include the backbone carbonyl moiety of Gly-146 forming hydrogen-bonding interactions with the side-chains of Asn-137 and Thr-142 and the side-chain of Gln-148 forming water-mediated contacts with the backbone of Ile-149, Gly-78, and Gly-81. Gly-146 adopts φ/ψ angles of 70°, −163° lying within an allowed region for glycine residues in the Ramachandran plot (33Laskowski R.A. Mac" @default.
• W2066864373 created "2016-06-24" @default.
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• W2066864373 date "2003-01-01" @default.
• W2066864373 modified "2023-10-09" @default.
• W2066864373 title "Contribution of Glycine 146 to a Conserved Folding Module Affecting Stability and Refolding of Human Glutathione Transferase P1-1" @default.
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