Equilibrium folding of dimeric class mu glutathione transferases involves a stable monomeric intermediate.


Abstract

The conformational stabilities of two homodimeric class mu glutathione transferases (GSTM1-1 and GSTM2-2) were studied by urea- and guanidinium chloride-induced denaturation. Unfolding is reversible and structural changes were followed with far-ultraviolet circular dichroism, tryptophan fluorescence, enzyme activity, chemical cross-linking, and size-exclusion chromatography. Disruption of secondary structure occurs as a monophasic transition and is independent of protein concentration. Changes in tertiary structure occur as two transitions; the first is protein concentration dependent, while the second is weakly dependent (GSTM1-1) or independent (GSTM2-2). The second transition corresponds with the secondary structure transition. Loss in catalytic activity occurs as two transitions for GSTM1-1 and as one transition for GSTM2-2. These transitions are dependent upon protein concentration. The first deactivation transition coincides with the first tertiary structure transition. Dimer dissociation occurs prior to disruption of secondary structure. The data suggest that the equilibrium unfolding/refolding of the class mu glutathione transferases M1-1 and M2-2 proceed via a three-state process: N(2) <--> 2I <--> 2U. Although GSTM1-1 and GSTM2-2 are homologous (78% identity/94% homology), their N(2) tertiary structures are not identical. Dissociation of the GSTM1-1 dimer to structured monomers (I) occurs at lower denaturant concentrations than for GSTM2-2. The monomeric intermediate for GSTM1-1 is, however, more stable than the intermediate for GSTM2-2. The intermediates are catalytically inactive and display nativelike secondary structure. Guanidinium chloride-induced denaturation yields monomeric intermediates, which have a more loosely packed tertiary structure displaying enhanced solvent exposure of its tryptophans and enhanced ANS binding. The three-state model for the class mu enzymes is in contrast to the equilibrium two-state models previously proposed for representatives of classes alpha/pi/Sj26 GSTs. Class mu subunits appear to be intrinsically more stable than those of the other GST classes. Study holds ProTherm entries: 8608, 8609, 8610, 8611, 8612, 8613, 8614, 8615, 8616, 8617, 8618, 8619, 8620, 8621, 8622, 8623 Extra Details: additive : EDTA(1 mM),N->I,Subunit 1 structural changes; monophasic transition; dimer,dissociation; ANS binding

Submission Details

ID: T9RJU8oP4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:36 p.m.

Version: 1

Publication Details
Hornby JA;Luo JK;Stevens JM;Wallace LA;Kaplan W;Armstrong RN;Dirr HW,Biochemistry (2000) Equilibrium folding of dimeric class mu glutathione transferases involves a stable monomeric intermediate. PMID:11015213
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
6ATR 2018-09-12 1.29 Crystal structure of hGSTA1-1 complexed with two GSH analogues in each subunit
1K3Y 2002-10-30 1.3 Crystal Structure Analysis of human Glutathione S-transferase with S-hexyl glutatione and glycerol at 1.3 Angstrom
1K3L 2002-10-23 1.5 Crystal Structure Analysis of S-hexyl-glutathione Complex of Glutathione Transferase at 1.5 Angstroms Resolution
6ATO 2018-09-12 1.55 Crystal structure of hGSTA1-1 complexed with GSH and MPD in each subunit
5LD0 2016-09-21 1.6 Chimeric GST
6ATP 2018-09-12 1.7 Crystal structure of apo-hGSTA1-1 exhibiting a new conformation of C-terminal helix
1PL1 2004-06-22 1.75 Crystal structure of human glutathione transferase (GST) A1-1 in complex with a decarboxy-glutathione
3KTL 2009-12-22 1.75 Crystal Structure of an I71A human GSTA1-1 mutant in complex with S-hexylglutathione
3Q74 2011-01-12 1.79 Crystal Structure Analysis of the L7A Mutant of the Apo Form of Human Alpha Class Glutathione Transferase
1PL2 2004-06-22 1.8 Crystal structure of human glutathione transferase (GST) A1-1 T68E mutant in complex with decarboxy-glutathione
1K3O 2002-10-30 1.8 Crystal Structure Analysis of apo Glutathione S-Transferase
2R3X 2007-12-18 1.8 Crystal structure of an R15L hGSTA1-1 mutant complexed with S-hexyl-glutathione
2VCV 2008-10-28 1.8 Glutathione transferase A3-3 in complex with glutathione and delta-4- androstene-3-17-dione
1XWG 2005-11-01 1.85 Human GST A1-1 T68E mutant
3ZFB 2012-12-19 1.86 Crystal structure of the I75A mutant of human class alpha glutathione transferase in the apo form
3ZFL 2012-12-19 1.88 Crystal structure of the V58A mutant of human class alpha glutathione transferase in the apo form
5JCU 2016-10-12 1.93 Crystal Structure of hGSTA1-1 with Glutathione Adduct of Phenethyl Isothiocyanate and Cystein Adduct of Phenethyl Isothiocyanate
1YDK 2005-06-07 1.95 Crystal structure of the I219A mutant of human glutathione transferase A1-1 with S-hexylglutathione
3I6A 2009-09-01 1.98 Human GST A1-1 GIMF mutant with Glutathione
6ATQ 2018-09-12 2.0 Crystal structure of apo-hGSTA1-1 exhibiting a new conformation of C-terminal helix
1GSE 1995-09-15 2.0 GLUTATHIONE TRANSFERASE A1-1 COMPLEXED WITH AN ETHACRYNIC ACID GLUTATHIONE CONJUGATE (MUTANT R15K)
1PKW 2004-06-22 2.0 Crystal structure of human glutathione transferase (GST) A1-1 in complex with glutathione
1USB 2004-09-01 2.07 Rational design of a novel enzyme - efficient thioester hydrolysis enabled by the incorporation of a single His residue into human glutathione transferase A1-1
4HJ2 2013-03-27 2.1 Crystal Structure Analysis of GSTA1-1 in complex with chlorambucil
2VCT 2008-10-28 2.1 Glutathione transferase A2-2 in complex with delta-4-andostrene-3-17- dione
4ACS 2011-12-28 2.1 Crystal structure of mutant GST A2-2 with enhanced catalytic efficiency with azathioprine
1PKZ 2004-06-22 2.1 Crystal structure of human glutathione transferase (GST) A1-1
3L0H 2010-01-12 2.13 Crystal Structure Analysis of W21A mutant of human GSTA1-1 in complex with S-hexylglutathione
3U6V 2011-10-26 2.2 Crystal Structure Analysis of L23A mutant of human GST A1-1
3IK9 2010-06-23 2.2 Human GST A1-1-GIMF with GSDHN
2WJU 2009-06-09 2.3 Glutathione transferase A2-2 in complex with glutathione
5LCZ 2016-09-21 2.33 Chimeric GST
3I69 2009-09-01 2.38 Apo Glutathione Transferase A1-1 GIMF-helix mutant
1TDI 2005-01-18 2.4 Crystal Structure of hGSTA3-3 in Complex with Glutathione
1AGS 1995-07-10 2.5 A SURFACE MUTANT (G82R) OF A HUMAN ALPHA-GLUTATHIONE S-TRANSFERASE SHOWS DECREASED THERMAL STABILITY AND A NEW MODE OF MOLECULAR ASSOCIATION IN THE CRYSTAL
1GSD 1995-09-15 2.5 GLUTATHIONE TRANSFERASE A1-1 IN UNLIGANDED FORM
2R6K 2008-08-19 2.51 Crystal structure of an I71V hGSTA1-1 mutant in complex with S-hexylglutathione
1GUH 1993-10-31 2.6 Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the MU and PI class enzymes
1GSF 1995-09-15 2.7 GLUTATHIONE TRANSFERASE A1-1 COMPLEXED WITH ETHACRYNIC ACID

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.1 Glutathione S-transferase A1 Q7RTV2 GSTA5_HUMAN
90.5 Glutathione S-transferase A1 Q16772 GSTA3_HUMAN
95.0 Glutathione S-transferase A1 P09210 GSTA2_HUMAN
100.0 Glutathione S-transferase A1 P08263 GSTA1_HUMAN