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• W2051481147 abstract "Helix 9, the major structural element in the C-terminal region of class Alpha glutathione transferases, forms part of the active site of these enzymes where its dynamic properties modulate both catalytic and ligandin functions. A conserved aspartic acid N-capping motif for helix 9 was identified by sequence alignments of the C-terminal regions of class Alpha glutathione S-transferases (GSTs) and an analysis by the helix-coil algorithm AGADIR. The contribution of the N-capping motif to the stability and dynamics of the region was investigated by replacing the N-cap residue Asp-209 with a glycine in human glutathione S-transferase A1-1 (hGST A1-1) and in a peptide corresponding to its C-terminal region. Far-UV circular dichroism and AGADIR analyses indicate that, in the absence of tertiary interactions, the wild-type peptide displays a low intrinsic tendency to form a helix and that this tendency is reduced significantly by the Asp-to-Gly mutation. Disruption of the N-capping motif of helix 9 in hGST A1-1 alters the conformational dynamics of the C-terminal region and, consequently, the features of the H-site to which hydrophobic substrates (e.g. 1-chloro-2,4-dinitrobenzene (CDNB)) and nonsubstrates (e.g. 8-anilino-1-naphthalene sulfonate (ANS)) bind. Isothermal calorimetric and fluorescence data for complex formation between ANS and protein suggest that the D209G-induced perturbation in the C-terminal region prevents normal ligand-induced localization of the region at the active site, resulting in a less hydrophobic and more solvent-exposed H-site. Therefore, the catalytic efficiency of the enzyme with CDNB is diminished due to a lowered affinity for the electrophilic substrate and a lower stabilization of the transition state. Helix 9, the major structural element in the C-terminal region of class Alpha glutathione transferases, forms part of the active site of these enzymes where its dynamic properties modulate both catalytic and ligandin functions. A conserved aspartic acid N-capping motif for helix 9 was identified by sequence alignments of the C-terminal regions of class Alpha glutathione S-transferases (GSTs) and an analysis by the helix-coil algorithm AGADIR. The contribution of the N-capping motif to the stability and dynamics of the region was investigated by replacing the N-cap residue Asp-209 with a glycine in human glutathione S-transferase A1-1 (hGST A1-1) and in a peptide corresponding to its C-terminal region. Far-UV circular dichroism and AGADIR analyses indicate that, in the absence of tertiary interactions, the wild-type peptide displays a low intrinsic tendency to form a helix and that this tendency is reduced significantly by the Asp-to-Gly mutation. Disruption of the N-capping motif of helix 9 in hGST A1-1 alters the conformational dynamics of the C-terminal region and, consequently, the features of the H-site to which hydrophobic substrates (e.g. 1-chloro-2,4-dinitrobenzene (CDNB)) and nonsubstrates (e.g. 8-anilino-1-naphthalene sulfonate (ANS)) bind. Isothermal calorimetric and fluorescence data for complex formation between ANS and protein suggest that the D209G-induced perturbation in the C-terminal region prevents normal ligand-induced localization of the region at the active site, resulting in a less hydrophobic and more solvent-exposed H-site. Therefore, the catalytic efficiency of the enzyme with CDNB is diminished due to a lowered affinity for the electrophilic substrate and a lower stabilization of the transition state. As a consequence of their multifunctional capabilities, glutathione S-transferases (GSTs) 1The abbreviations used are: GST, glutathione S-transferase; ANS, 8-anilino-1-naphthalene sulfonate; CD, circular dichroism; CDNB, 1-chloro-2,4-dinitrobenzene; GSH, glutathione; G- and H-sites, glutathione and hydrophobic substrate binding sites; hGST A1-1, human glutathione transferase class Alpha with two type-1 subunits; TFE, 2,2,2-trifluoroethanol; BSP, bromosulfophthalein; ALBP, adipose lipid-binding protein. contribute toward diverse cellular processes ranging from detoxification reactions to the control of gene expression (1Sheehan D. Meade G. Foley V.M. Dowd C.A. Biochem. J. 2001; 360: 1-16Crossref PubMed Scopus (1431) Google Scholar). These proteins, ubiquitous in aerobes, form a superfamily of species-independent classes that, except for the Kappa GST (2Ladner J.E. Parsons J.F. Rife C.L. Gilliland G.L. Armstrong R.N. Biochemistry. 2004; 43: 352-361Crossref PubMed Scopus (111) Google Scholar), share a common fold. Typically, the canonical GSTs are soluble, dimeric proteins with each subunit possessing a thioredoxin-like domain 1 fused to an all-α-helical domain 2 (3Dirr H. Reinemer P. Huber R. Eur. J. Biochem. 1994; 220: 645-661Crossref PubMed Scopus (389) Google Scholar). The Kappa GST, however, is more closely related to the protein disulfide bond isomerase, dsbA, in that domain 2 is inserted in domain 1 (2Ladner J.E. Parsons J.F. Rife C.L. Gilliland G.L. Armstrong R.N. Biochemistry. 2004; 43: 352-361Crossref PubMed Scopus (111) Google Scholar). Nevertheless, the active site of GSTs consists of two adjacent regions, a G-site on the thioredoxin-like domain that supports GSH as the thiol substrate or cofactor, and a nonpolar H-site on both domains that contributes to the binding of hydrophobic substrates. Although the molecular recognition of GSH is conserved among GSTs, these enzymes employ different strategies such as the nature of the residue (Tyr, Ser, or Cys) in contact with the thiol group of GSH and the structural contribution by domain 2 to the active site to realize their diverse functionalities (1Sheehan D. Meade G. Foley V.M. Dowd C.A. Biochem. J. 2001; 360: 1-16Crossref PubMed Scopus (1431) Google Scholar, 4Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (969) Google Scholar). 2D. C. Barnwell, Y. Sayed, S. Mosebi, M. Sayed, T. Sewell, and H. W. Dirr (2005), J. Mol. Biol., manuscript in preparation. The class Alpha GSTs possess, like many other GST classes, an active site tyrosine residue for the activation of GSH but, unlike other GST classes, they possess an extended C-terminal region that forms an integral part of the active site (5Cameron A.D. Sinning I. L'Hermite G. Olin B. Board P.G. Mannervik B. Jones T.A. Structure. 1995; 3: 717-727Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 6Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dirr H.W. Huber R. Gilliland G.L. 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, 7Le T.I. Stenkamp R.E. Ibarra C. Atkins W.M. Adman E.T. Proteins. 2002; 48: 618-627Crossref PubMed Scopus (56) Google Scholar). The major structural element in the C-terminal region is the amphipathic helix 9 that, although not directly involved in the chemical mechanism of catalysis, is an important determinant of substrate selectivity (8Bruns C.M. Hubatsch I. Ridderstrom M. Mannervik B. Tainer J.A. J. Mol. Biol. 1999; 288: 427-439Crossref PubMed Scopus (156) Google Scholar, 9Allardyce C.S. McDonagh P.D. Lian L.Y. Wolf C.R. Roberts G.C. Biochem. J. 1999; 343: 525-531Crossref PubMed Google Scholar, 10Nilsson L.O. Gustafsson A. Mannervik B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9408-9412Crossref PubMed Scopus (69) Google Scholar, 11Pal A. Gu Y. Pan S.S. Ji X. Singh S.V. Biochemistry. 2001; 40: 7047-7053Crossref PubMed Scopus (5) Google Scholar), the binding of substrates (12Gustafsson A. Etahadieh M. Jemth P. Mannervik B. Biochemistry. 1999; 38: 16268-16275Crossref PubMed Scopus (47) Google Scholar), rate-determining steps (13Nilsson L.O. Edalat M. Pettersson P.L. Mannervik B. Biochim. Biophys. Acta. 2002; 1597: 157-163Crossref PubMed Scopus (14) Google Scholar), desolvation of the active site (14Nieslanik B.S. Ibarra C. Atkins W.M. Biochemistry. 2001; 40: 3536-3543Crossref PubMed Scopus (24) Google Scholar), and the pKa of the catalytic tyrosine residue (12Gustafsson A. Etahadieh M. Jemth P. Mannervik B. Biochemistry. 1999; 38: 16268-16275Crossref PubMed Scopus (47) Google Scholar, 15Bjornestedt R. Stenberg G. Widersten M. Board P.G. Sinning I. Jones T.A. Mannervik B. J. Mol. Biol. 1995; 247: 765-773Crossref PubMed Scopus (102) Google Scholar). In addition to its contributions to catalysis, the C-terminal region is an important determinant of ligandin function, i.e. the binding of nonsubstrate ligands (16Dirr H.W. Wallace L.A. Biochemistry. 1999; 38: 15631-15640Crossref PubMed Scopus (54) Google Scholar, 17Mosebi S. Sayed Y. Burke J. Dirr H.W. Biochemistry. 2003; 42: 15326-15332Crossref PubMed Scopus (24) Google Scholar, 18Sayed Y. Hornby J.A. Lopez M. Dirr H. Biochem. J. 2002; 363: 341-346Crossref PubMed Google Scholar). Given these contributions of the C-terminal region to the functions of class Alpha GSTs, the conformational dynamics of the region and their impact on enzyme function have received much attention. Although the C-terminal region in hGST A1-1 is highly dynamic and not observed in the crystal structure of the apo enzyme (5Cameron A.D. Sinning I. L'Hermite G. Olin B. Board P.G. Mannervik B. Jones T.A. Structure. 1995; 3: 717-727Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 7Le T.I. Stenkamp R.E. Ibarra C. Atkins W.M. Adman E.T. Proteins. 2002; 48: 618-627Crossref PubMed Scopus (56) Google Scholar), it is observed in the apo structure of the homologous hGST A4-4 enzyme (8Bruns C.M. Hubatsch I. Ridderstrom M. Mannervik B. Tainer J.A. J. Mol. Biol. 1999; 288: 427-439Crossref PubMed Scopus (156) Google Scholar). Rather than being completely disordered, the C-terminal region in apo-hGST A1-1 assumes helix-like conformations close to the surface of the protein (19Zhan Y. Rule G.S. Biochemistry. 2004; 43: 7244-7254Crossref PubMed Scopus (25) Google Scholar), where it experiences tertiary contacts that facilitate its folding (16Dirr H.W. Wallace L.A. Biochemistry. 1999; 38: 15631-15640Crossref PubMed Scopus (54) Google Scholar, 20Wallace L.A. Dirr H.W. Biochemistry. 1999; 38: 16686-16694Crossref PubMed Scopus (47) Google Scholar). It is only when the enzyme binds G-site and/or H-site ligands that the C-terminal region becomes fully immobilized on the protein (5Cameron A.D. Sinning I. L'Hermite G. Olin B. Board P.G. Mannervik B. Jones T.A. Structure. 1995; 3: 717-727Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 6Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dirr H.W. Huber R. Gilliland G.L. 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, 7Le T.I. Stenkamp R.E. Ibarra C. Atkins W.M. Adman E.T. Proteins. 2002; 48: 618-627Crossref PubMed Scopus (56) Google Scholar, 19Zhan Y. Rule G.S. Biochemistry. 2004; 43: 7244-7254Crossref PubMed Scopus (25) Google Scholar). However, the structural determinants that maintain the conformational stability of the region in the apo and complex forms of hGST A1-1 (and other class Alpha GSTs) are not clear. Recently, we demonstrated that a bulky, hydrophobic residue at position 219 contributes significantly toward the stability of the region in apo and complexed hGST A1-1 (17Mosebi S. Sayed Y. Burke J. Dirr H.W. Biochemistry. 2003; 42: 15326-15332Crossref PubMed Scopus (24) Google Scholar). The type of amino acid residue found at the N-terminal end of a helix has a major effect on the stability of the entire helix and is referred to as the N-capping residue (21Serrano L. Fersht A.R. Nature. 1989; 342: 296-299Crossref PubMed Scopus (352) Google Scholar). All GSTs have a conserved N-capping motif for helix 6 in domain 2 (22Aceto 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) shown to play an important role in the folding and stability of GSTs (23Cocco R. Stenberg G. Dragani B. Rossi P.D. Paludi D. Mannervik B. Aceto A. J. Biol. Chem. 2001; 276: 32177-32183Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 24Dragani 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). In this study, we identified a conserved aspartate N-capping motif for helix 9 in all class Alpha GSTs and, given the importance of N-capping motifs in the folding and stability of α-helices (25Scholtz J.M. Baldwin R.L. Annu. Rev. Biophys. Biomol. Struct. 1992; 21: 95-118Crossref PubMed Scopus (481) Google Scholar, 26Wan W.Y. Milner-White E.J. J. Mol. Biol. 1999; 286: 1633-1649Crossref PubMed Scopus (67) Google Scholar), investigated its contribution to the dynamics of the C-terminal region of hGST A1-1. Chemicals—GSH was from ICE Biomedical Inc. (Aurora, OH). TFE (>99% grade), 8-aniline-1-naphthalene sulfonate, ethacrynic acid, glutathione sulfonate, p-bromobenzyl GSH, and 1-chloro-2,4-dinitrobenzene (CDNB) were purchased from Sigma-Aldrin. All other reagents were of analytical grade. Peptides corresponding to the wild-type and D209G forms of the C-terminal region of hGST A1-1 were synthesized by Alpha Diagnostic Inc. (San Antonio, TX), and their molecular masses and purity were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The peptides have the following sequences: Ac-Tyr-Gly-Met-Asp/Gly-Glu-Lys-Ser-Leu-Glu-Glu-Ala-Arg-Lys-Ile-Phe-Arg-Phe. Residues Met3 to Phe17 correspond to the C-terminal sequence in hGST A1 (i.e. Met208 to Phe222). The underlined residues correspond to those residues located in helix 9 in the crystal structures of hGST A1-1 complexed with active site ligands (5Cameron A.D. Sinning I. L'Hermite G. Olin B. Board P.G. Mannervik B. Jones T.A. Structure. 1995; 3: 717-727Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 6Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dirr H.W. Huber R. Gilliland G.L. 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, 7Le T.I. Stenkamp R.E. Ibarra C. Atkins W.M. Adman E.T. Proteins. 2002; 48: 618-627Crossref PubMed Scopus (56) Google Scholar). The presence of a tyrosine at the N terminus of the peptide, used to quantitate the peptide, should not induce a significant aromatic band in their CD spectra because of the flexible helix-breaking glycine spacer separating the tyrosine from the rest of the peptide (27Chakrabartty A. Kortemme T. Padmanabhan S. Baldwin R.L. Biochemistry. 1993; 32: 5560-5565Crossref PubMed Scopus (333) Google Scholar). Peptide stock solutions were made in water and their concentrations determined at 275 nm using an extinction coefficient of 1450 m-1·cm-1 (28Brandts J.F. Kaplan L.J. Biochemistry. 1973; 12: 2011-2024Crossref PubMed Scopus (158) Google Scholar). Mutagenesis, Protein Expression, and Purification—The plasmid (pKHA1) used for the expression of hGST A1-1 (29Stenberg G. Bjornestedt R. Mannervik B. Protein Expr. Purif. 1992; 3: 80-84Crossref PubMed Scopus (60) Google Scholar) was a generous gift from Bengt Mannervik (Department of Biochemistry, University of Uppsala, Sweden). Oligonucleotide site-directed mutagenesis, based on the QuikChange method (Stratagene, La Jolly, CA), was performed to generate the D209G mutant of hGST A1-1 using the following primers: 5′-GCCTCCATGGGTGAGAAATCTCTAGAAGAAGC and 5′-GCTTCTTCTAGAGATTTCTCACCCATGGGAGGC. The mutations introduced for generating D209G hGST A1-1 are underlined. The presence of the mutation in the plasmid was confirmed by DNA sequencing. Wild-type and D209G hGST A1-1 were expressed in BL21(DE3) pLysS Escherichia coli cells and purified by CM-Sepharose chromatography as described previously (20Wallace L.A. Dirr H.W. Biochemistry. 1999; 38: 16686-16694Crossref PubMed Scopus (47) Google Scholar). The concentration of the dimeric proteins was determined at 280 nm using the molar extinction coefficient of 38,200 m-1·cm-1. Spectroscopic Methods—Far-UV CD measurements were done at 20 °C in a Jasco model 810 CD spectropolarimeter. Averaged CD signals, corrected for solvent, were converted to mean residue ellipticity [Θ], [Θ]=(100×Θ)/Cnl(Eq. 1) where C is the peptide concentration in millimolar, Θ is the measured ellipticity in millidegree, n is the number of residues (17 in this case), and l is the path length (cm). The percentage helical content was calculated from the mean residue ellipticity at 222 nm, using the equation, %Helix=[Θ]−[Θ]coil[Θ]helix−[Θ]coil(Eq. 2) where [Θ]helix and [Θ]coil represent the mean residue ellipticity of a helix [–42,500(1 – (3/n)] and random coil (+640), respectively (30Rohl C.A. Chakrabartty A. Baldwin R.L. Protein Sci. 1996; 5: 2623-2637Crossref PubMed Scopus (261) Google Scholar). The units of mean residue ellipticity are deg·cm2·dmol-1. Fluorescence measurements were performed at 20 °C in a Hitachi model 850 fluorescence spectrofluorometer. The intrinsic fluorescence of hGST A1-1 was monitored by selectively exciting a single tryptophan residue (Trp-21) per subunit at 295 nm. The binding of the anionic dye, ANS, to hGST A1-1 was monitored by fluorescence enhancement using an excitation wavelength of 390 nm (31Sluis-Cremer N. Naidoo N.N. Kaplan W.H. Manoharan T.H. Fahl W.E. Dirr H.W. Eur. J. Biochem. 1996; 241: 484-488Crossref PubMed Scopus (32) Google Scholar). Steady-state Kinetics—Kinetic parameters were determined at pH 6.5 for the S-conjugation reaction between GSH and CDNB (32Habig W.H. Jakoby W.B. Methods Enzymol. 1981; 77: 398-405Crossref PubMed Scopus (2098) Google Scholar). Reaction rates were measured in triplicate at 20 °C and corrected for non-enzymatic rates. Conditions for determining KmGSH were 0.05–6.5 mm GSH and 1.5 mm CDNB, and for KmCDNB they were 0.15–1.5 mm CDNB and 5 mm GSH. For kcat/KmGSH determinations, CDNB was kept at 1.5 mm, whereas GSH was varied from 0.05 to 0.15 mm. For kcat/KmCDNB determinations, GSH was kept at 5 mm, whereas CDNB was varied from 0.05 to 0.15 mm. The data were fitted by nonlinear regression analysis using Origin5 MicroCal Inc. software. Active Site Tyrosinate Formation—The ionization of the catalytic Tyr9 in the active site of hGST A1-1 was determined over the pH range 5.5–9.0 by UV absorbance difference spectroscopy as described (17Mosebi S. Sayed Y. Burke J. Dirr H.W. Biochemistry. 2003; 42: 15326-15332Crossref PubMed Scopus (24) Google Scholar). The concentration of tyrosinate ion formation was calculated using a molar extinction coefficient of 2350 m-1·cm-1 and the pKa of Tyr9 determined as described (15Bjornestedt R. Stenberg G. Widersten M. Board P.G. Sinning I. Jones T.A. Mannervik B. J. Mol. Biol. 1995; 247: 765-773Crossref PubMed Scopus (102) Google Scholar, 33Atkins W.M. Dietze E.C. Ibarra C. Protein Sci. 1997; 6: 873-881Crossref PubMed Scopus (28) Google Scholar). Isothermal Titration Calorimetry—Isothermal titration calorimetry with a VP-ITC calorimeter from MicroCal (Northampton, MA) was employed to determine the thermodynamic parameters of ANS·hGST A1-1 complex formation in 20 mm phosphate buffer, pH 6.5, as described (17Mosebi S. Sayed Y. Burke J. Dirr H.W. Biochemistry. 2003; 42: 15326-15332Crossref PubMed Scopus (24) Google Scholar, 18Sayed Y. Hornby J.A. Lopez M. Dirr H. Biochem. J. 2002; 363: 341-346Crossref PubMed Google Scholar). Total heats were obtained by titrating 0.04 mm D209G hGST A1-1 (subunit concentration) with 5-μl aliquots of 4.1 mm ANS. Heats of dilution, determined by titrating ANS into buffer alone, were subtracted from the total observed heats, and the corrected data analyzed by nonlinear regression with Origin5 (MicroCal). Molecular Docking—Two molecular docking programs, Cerius2 (Accelrys Inc., San Diego, CA) and LIGIN (34Sobolev V. Wade R.C. Vriend G. Edelman M. Proteins. 1996; 25: 120-129Crossref PubMed Scopus (163) Google Scholar), were used to predict the ANS binding site in hGST A1-1. The 1K3Y high resolution structure of wild-type hGST A1-1 without the bound ligand was used as the host apo molecule. Cerius2 treated ANS as a flexible, guest ligand and the protein host as a rigid body. Various ligand conformations and binding locations were ranked according to conformational energy minima, with the lowest being the most probable binding complex. Energy minimization was carried out with GROMOS (35van Gunsteren W.F. Berendsen H.J. Angew. Chem. Int. 1990; 29: 992-1023Crossref Scopus (1330) Google Scholar) in the WHATIF version 2.0 package (36Vriend G. J. Mol. Graph. 1990; 8: 52-56Crossref PubMed Scopus (3377) Google Scholar). LIGIN, which uses surface complementarity between guest and host as the guiding principle for predicting binding sites, was implemented as described (34Sobolev V. Wade R.C. Vriend G. Edelman M. Proteins. 1996; 25: 120-129Crossref PubMed Scopus (163) Google Scholar). Docking between ANS and hGST A1-1 was also done with the binary GSH-protein (PDB file 1GSE without the ethacrynic acid moiety) and GSO3-protein (coordinates for GSO3− obtained from PDB file 1EV4) complexes. Asp-209, the N-cap Residue of Helix 9 —The AGADIR algorithm, based on helix-coil transition theory that explicitly considers specific interactions occurring in helices devoid of tertiary interactions (37Lacroix E. Viguera A.R. Serrano L. J. Mol. Biol. 1998; 284: 173-191Crossref PubMed Scopus (378) Google Scholar), was used to identify N-capping motifs for helices in the class Alpha hGST A1-1. Two such motifs were identified; one for helix 6 and the other for helix 9, both of which are conserved in class Alpha GSTs. The N-capping motif of helix 6 (22Aceto 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) is a class 5 Ser motif (26Wan W.Y. Milner-White E.J. J. Mol. Biol. 1999; 286: 1633-1649Crossref PubMed Scopus (67) Google Scholar), which has been shown to play an important role in the folding and stability of GSTs (23Cocco R. Stenberg G. Dragani B. Rossi P.D. Paludi D. Mannervik B. Aceto A. J. Biol. Chem. 2001; 276: 32177-32183Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 24Dragani 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). The motif of helix 9 is a class 3 Asp N-capping motif (26Wan W.Y. Milner-White E.J. J. Mol. Biol. 1999; 286: 1633-1649Crossref PubMed Scopus (67) Google Scholar) and is conserved in all class Alpha GSTs (Table I). The motif together with its hydrogen-bonding pattern with the main chain of helix 9 is shown in Fig. 1.Table ISequence alignment of the C-terminal region of class Alpha GSTs The sequence alignment of the C-terminal region begins two residues preceding the first α-helical residue (N1) of α-helix 9.Gi numberTypeaType refers to the subunit type of class Alpha GSTSourcePDBSequencebThe conserved N-cap Asp residue is in boldface, and the underlined residues correspond to those located in α-helix 9 of the crystal structuresN′NN1121730A1Human1K3YMDEKSLEEARKIFRF66611A1Rat1EV9MDAKQIEEARKIFKF11514503A1Mouse1F3BMDAKQIQEARKAFKIQ1170081A1RabbitMDEKNLEKAKKIFKIP2495106A1Bos taurusTDEKKIEEARKVFKF30749486A2Mouse1ML6MDAKQIEEARKVFKF121733A2HumanMDEKSLEESRKIFRF6166190A3HumanADAKALEEARKIFRF7767212A4Human1GUMPDEIYVRTVYNIFRP3114387A4Mouse1GUKPDGPYVEVVRIVLKFa Type refers to the subunit type of class Alpha GSTb The conserved N-cap Asp residue is in boldface, and the underlined residues correspond to those located in α-helix 9 of the crystal structures Open table in a new tab Helical Content of C-terminal Region Peptides—Far-UV CD spectra, used to determine the helical content of two peptides corresponding to the sequences of the wild-type and D209G mutant C-terminal regions (WT-pep and DG-pep, respectively) in water, are shown in Fig. 2. The spectra, displaying ellipticity minima at 222 and 201 nm and ellipticity maxima at 218 and 194 nm, are characteristic of a mixture of helical and random coil conformations. The low helical content of WT-pep and DG-pep in water (10 and 4%, respectively) was substantiated by AGADIR (i.e. 20 and 8% for the wild-type and mutant sequences, respectively). AGADIR also predicted that the length of the helix in the wild-type sequence is longer (residues Asp-210 to Ile-219 with individual propensities of 21.2–37.4%) than that in the D209G mutant sequence (residues Leu-213 to Ile-219 with individual propensities of 11.4–18.6%). The residues predicted to form a helix in the wild-type sequence are in agreement with those that form helix 9 in the protein structure (6Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dirr H.W. Huber R. Gilliland G.L. 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). Capping motifs are known to increase helical propensity by initiating and/or stabilizing helix propagating tendencies at their ends (38Doig A.J. MacArthur M.W. Stapley B.J. Thornton J.M. Protein Sci. 1997; 6: 147-155Crossref PubMed Scopus (112) Google Scholar, 39Richardson J.S. Richardson D.C. Science. 1988; 240: 1648-1652Crossref PubMed Scopus (1299) Google Scholar, 40Dasgupta S. Bell J.A. Int. J. Pept. Protein Res. 1993; 41: 499-511Crossref PubMed Scopus (141) Google Scholar). Thus, the reduced CD signal of the DG-pep can also be interpreted as an increase in fraying of the helix at its N-terminal end. TFE, a co-solvent known to enhance the helical content of peptides with intrinsic helical properties (30Rohl C.A. Chakrabartty A. Baldwin R.L. Protein Sci. 1996; 5: 2623-2637Crossref PubMed Scopus (261) Google Scholar, 41Kumaran S. Roy R.P. J. Pept. Res. 1999; 53: 284-293Crossref PubMed Scopus (35) Google Scholar, 42Myers J.K. Pace C.N. Scholtz J.M. Protein Sci. 1998; 7: 383-388Crossref PubMed Scopus (61) Google Scholar), increases the helix content of both peptides, as shown in Fig. 2. The TFE-induced changes of the CD spectra exhibit an isodichroic point at 202–204 nm indicative of a two-state, helix-coil transition (43Brown J.E. Klee W.A. Biochemistry. 1971; 10: 470-476Crossref PubMed Scopus (277) Google Scholar). The helix content of both peptides reaches a maximum level of about 35% in the presence of TFE (inset in the bottom panel of Fig. 2). Although the sequence of the C-terminal region displays a preference to be disordered in aqueous solution, the flexible region in apo-GST A1-1 assumes an ensemble of native helix-like structures close to the surface of the protein where it samples multiple environments (19Zhan Y. Rule G.S. Biochemistry. 2004; 43: 7244-7254Crossref PubMed Scopus (25) Google Scholar). It is only when the active site becomes occupied by ligand that the C-terminal region becomes stabilized and localized on the surface of the protein (5Cameron A.D. Sinning I. L'Hermite G. Olin B. Board P.G. Mannervik B. Jones T.A. Structure. 1995; 3: 717-727Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 6Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dirr H.W. Huber R. Gilliland G.L. 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, 7Le T.I. Stenkamp R.E. Ibarra C. Atkins W.M. Adman E.T. Proteins. 2002; 48: 618-627Crossref PubMed Scopus (56) Google Scholar, 16Dirr H.W. Wallace L.A. Biochemistry. 1999; 38: 15631-15640Crossref PubMed Scopus (54) Google Scholar, 19Zhan Y. Rule G.S. Biochemistry. 2004; 43: 7244-7254Crossref PubMed Scopus (25) Google Scholar, 44Wallace L.A. Sluis-Cremer N. Dirr H.W. Biochemistry. 1998; 37: 5320-5328Crossref PubMed Scopus (72) Google Scholar). Tertiary interactions at the interfaces between the C-terminal region, protein, and bound ligand, therefore, play a major role in stabilizing the region (16Dirr H.W. Wallace L.A. Biochemistry. 1999; 38: 15631-15640Crossref PubMed Scopus (54) Google Scholar, 17Mosebi S. Sayed Y. Burke J. Dirr H.W. Biochemistry. 2003; 42: 15326-15332Crossref PubMed Scopus (24) Google Scholar), consistent with the helix-stabilizing effect of TFE. Disrupting the conserved N-capping motif of helix 9 by the D209G mutation substantially reduces the intrinsic ability of its sequence to form a helix. Consequently, this alters the dynamics of the C-terminal region and, because it forms part of the active site, the function of the enzyme (see below). Characteristics of D209G hGST A1-1—Helix 9 (residues 210–220) is the major structural feature in the C-terminal region (residues 210–222) of GST A1-1 (6Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dirr H.W. Huber R. Gilliland G.L. 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). As expected, replacing the N-cap residue, Asp-209, with a glycine residue had little impact on the global structural features of hGST A1-1 in that its secondary and dimeric structures were unchanged (data not shown). The tertiary environment of the lone tryptophan, Trp-21, is, however, different in the mutant, as indicated by a lower fluorescence intensity, although the maximum emission wavelength was unaltered (data not shown). Trp-21 is located at the domain-domain interface of each subunit where its indole ring is surrounded by helix 1 in domain 1 and helices 6 and 8 in domain 2. Although the Asp-210 N-capping motif of helix 9 is about 18 Å away from Trp-21, it is connected to helix 8 via a 10-amino acid linker. Not only is the fluorescence of Trp-21 sensitive to changes occurring at the domain-domain interface (45Wallace L.A. Burke J. Dirr H.W. Biochim. Biophys. Acta. 2000; 1478: 325-332Crossref PubMed Scopus (21) Google Scholar), it is also sensitive to changes in" @default.
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• W2051481147 date "2005-05-01" @default.
• W2051481147 modified "2023-09-26" @default.
• W2051481147 title "A Conserved N-capping Motif Contributes Significantly to the Stabilization and Dynamics of the C-terminal Region of Class Alpha Glutathione S-Transferases" @default.
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• W2051481147 doi "https://doi.org/10.1074/jbc.m413608200" @default.
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