Babbitt Lab > Resources > Supplementary data from "Glutathione transferases are structural and functional outliers in the thioredoxin fold"

Glutathione transferases are structural and functional outliers in the thioredoxin fold

Atkinson, HJ, and Babbitt, PC. "Glutathione transferases are structural and functional outliers in the thioredoxin fold." 2009, in preparation.

Glutathione transferases (GSTs) are ubiquitous scavengers of toxic compounds that fall, structurally and functionally, within the thioredoxin fold suprafamily. The fundamental catalytic capability of GSTs is catalysis of the nucleophilic addition or substitution of glutathione at electrophilic centers in a wide range of xenobiotic electrophilic substrates. However, little else is known about the structural and functional relationships between different subgroups of GSTs. Through a global analysis of sequence and structural similarity, it was determined that variation in the binding of glutathione between the two major classes of cytosolic GSTs results in a different mode of coenzyme activation. Additionally, the convergent features of glutathione binding between cytosolic GSTs and mitochondrial GST kappa are described. The identification of these structural and functional themes help to illuminate some of the fundamental contributions of the thioredoxin fold to catalysis, and clarify how the thioredoxin fold can be modified to enable new functions.

Available files:

  1. Datafiles generated in the analysis
  2. Movie illustrating the similarity of the GST kappa and tau GSH binding site
  3. Tutorials on sequence similarity networks
    1. How to view SSN files
    2. Basic Cytoscape tutorial

1. Datafiles generated in the analysis


Fig. 1 - GST structure network

File Description
[tab-separated text file]
gstStrucs_16.0.tsv

Information about the 40 structures present in the networks in Fig. 1.
Column Description
IDPDB ID_Chain ID
SPFamilySwissProt classification from SIMILARITY line if exists
sp_IDassociated SwissProt sequence ID
exp_methodNMR or X-ray
resolutionif X-ray, resolution; 0.0 if NMR

[network]
gstStrucs_16.0.xgmml [290K]

Structure similarity network shown in Fig. 1 with a FAST score cutoff of 16.0

Network contains 40 structures that are a maximum of 60% identical by chain sequence and span the cytosolic GSTs. Edges at the limiting threshold of 16.0 threshold represent alignments with a median 2.5A RMSD over 177 aligned positions, while the rest of the edges represent better alignments.

This file can be viewed in Cytoscape using File: Import network

Attributes are defined for each structure in the network
Attribute:
  • PDB chain
  • Associated SwissProt sequence info
  • Annotation of PDB chain sequence: PFAM Trx Clan HMMs
  • Description
    PDB chain
    IDPDB ID_Chain ID
    pdb_chainIDPDB ID_Chain ID
    pdbIDPDB ID
    commentPDB structure "title"
    exp_methodNMR or X-ray
    resolutionif X-ray, resolution; 0.0 if NMR
    yeardate of deposition in the PDB
    het_namelist of non-standard residues (often ligands)
    chain_seqamino acid sequence of PDB chain
    chain_seq_lengthlength of 'chain_seq'
    chain_seq_rangeseq indices corresponding to SwissProt sequence
    chainslist of chain IDs found in entire PDB structure
    Associated SwissProt sequence info
    sp_IDassociated SwissProt sequence ID
    chain_seq_rangeSwissProt sequence indices indicating coverage by chain seq
    ACUniProt accession
    DEUniProt definition line
    speciesspecies
    taxIDNCBI taxonomy ID
    SPFamilySwissProt classification from SIMILARITY line if exists; Fig. 1 coloring
    SPSequenceFull length SwissProt sequence assoc w/ sp_ID
    dbsourcesequences is in SwissProt (sp) or TrEMBL (tr) database
    seq_lengthSwissProt sequence length
    strucIDscolon-separated list of PDB IDs associated with this SwissProt ID in the PDB database
    struc_ctnumber of PDB IDs associated with this SwissProt ID in the PDB database
    Annotation of PDB chain sequence: PFAM Trx Clan HMMs
    1stModelBest hit to PFAM Trx Clan member, if aligned better than gathering threshold
    1stLEval-log10(E-value) [1stModel]
    domain_lenlength of 'domain_seq'
    domain_seqsequence region aligning to '1stModel' HMM
    seq_ststart index in ID:sequence for 'domain_seq'
    seq_endend index

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    Fig. 2 - GST sequence network

    File Description
    [sequences]
    gsts40nr1e-12.fa [174K]
    622 sequences from the GST network in Fig. 2
    [network]
    gsts40nr1e-12.xgmml [6M]

    Sequence similarity network shown in Fig. 2 with a BLAST E-value cutoff of 1x10-12

    Network contains 622 sequences that are a maximum of 40% identical and span the cytosolic GSTs. Similarity is defined by pairwise BLAST alignments better than an E-value of 1x10-12; edges at this threshold represent alignments with a median 27% identity over 200 residues, while the rest of the edges represent better alignments.

    This file can be viewed in Cytoscape using File: Import network

    Attributes are defined for each sequence in the network
    Attribute:
    1. General
    2. Domains and PFAM Trx Clan HMMs
    3. Structures
    4. Sequence motifs
    5. Taxonomic
    Description
    General
    IDUniProt:SwissProt/TrEMBL ID
    nameUniProt:SwissProt/TrEMBL ID
    ACUniProt accession
    DEUniProt definition line
    SPFamilySwissProt classification from SIMILARITY line if exists; Fig 2 coloring
    dbsourcesequences is in SwissProt (sp) or TrEMBL (tr) database
    sequencefull length sequence
    seq_lengthsequence length
    Domains and PFAM Trx Clan HMMs
    1stModelBest hit to PFAM Trx Clan member, if aligned better than gathering threshold
    1stLEval-log10(E-value) [1stModel]
    domain_seqsequence region aligning to '1stModel' HMM
    domain_lenlength of 'domain_seq'
    seq_ststart index in ID:sequence for 'domain_seq'
    seq_endend index
    domOrdercolon-separated list of Trx domains present in sequence, ordered N to C
    doE-valsHMM alignment E-vals for each of the above domains
    do_num_domainsnumber of Trx clan domains present in sequence
    Structures
    has_strucyes if is associated with a structure in the PDB database
    strucIDscolon-separated list of PDB IDs associated with this sequence in the PDB database
    struc_ctnumber of PDB IDs associated with this sequence in the PDB database
    idsFrom60nrPdbNetList of PDB chains with >= 40% identity to this sequence in the 159-structure net from Fig 1 (ie, chain sequences are also < 60% identity to each other)
    nStrucsFrom60nrPdbNet= count(idsFrom60nrPdbNet)
    60nrPDB_IDPDB ID for representative of this sequence in structure-based network (Fig. 1) (ie, member of the >= 40% identity sequence cluster containing the sequence associated with that PDB structure chain -- nearest by sequence from 'idsFrom60nrPdbNet')
    Sequence motifs
    catTypeCxxC, Cxxc, cxxC, loopC_C3, other;
    C0Amino acid at Cxxc position
    X1Amino acid at cXxc position
    X2Amino acid at cxXc position
    C3Amino acid at cxxC position
    CXXC'C0'+'X1'+'X2'+'C3'
    cPorRis there a Pro or Arg at the N-term of the third beta strand?
    Taxonomic
    speciesspecies
     Note that standard hierarchy is:
    superkingdom, kingdom, phylum, class, order, family, genus, species
    kingToSuperkingIf species is associated with a taxonomic kingdom, this is the attribute value. Otherwise, superkingdom value is used
    phylumToClassIf species is associated with a taxonomic phylum, this is the attribute value. Otherwise, class value is used.
    orderToFamilyIf species is associated with a taxonomic order, this is the attribute value. Otherwise, family value is used
    taxIDNCBI taxonomy ID

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    Fig. 3 - GST structure-based sequence alignment

    File Description
    [sequence alignment]
    Fig3_17strucAlignTrxDomain.afa
    Sequence alignment derived from the structural alignment of 17 GST structures (Trx domain region), as shown in Fig. 3
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    Fig. 5 - Structure alignment featuring GSH sulfur position

    File Description
    [tab-separated text file]
    GST_sulfurs.tsv
    List of 32 GST structures with GSH or analogue sulfur visible in the alignment in Fig. 5 (does not include Grx 3: 3GRX). Text file includes info on associated SwissProt sequence, GST subgroup, ligand, etc.
    [sequence alignment]
    2cz2_1tu7.afa
    Structure-based sequence alignment of pi GST 1TU7 and zeta GST 2CZ2 as displayed in Fig. 5. Keys the alignment of 32 structures listed in GST_sulfurs.tsv.
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    Fig. 6 - Mitochondrial GST kappa

    File Description
    [structure network]
    SwissProtPfamTrxClanOnly60nr.pdb.tsv.ids_4.5.fc.xgmml [3M]
    Fig.6B — structure similarity network including 159 examples spanning the full Trx fold class, thresholded at a FAST score of 4.5.

    For structure attributes included in the network, see network from Fig. 1 above.

    [sequences]
    1. Kappa-like: A.ids.fa
    2. DsbA-like: B.ids.fa
    3. S/C GST-like: J.ids.fa
    Fig.6C — sequences used to tabulate catalytic motifs at the "CxxC" position.
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    2. Movie illustrating the similarity of the GST kappa and tau GSH binding site

    File Description
    [Quicktime format movie]
    kappa_tau_dsbG.mov [83M]
    *Note: movie is pretty large; may take a while to [down]load

    The movie linked at left details the similarities and differences between GST kappa (1r4w), DsbA-like DsbG (1v58), thioredoxin (1thx), and cytosolic S/C-GST tau (1oyj).

    1. It begins with a view of the functional GST kappa dimer.

    2. Next, thioredoxin is aligned to kappa to show where the Trx domain is located within the structure.

    3. An alignment between kappa and DsbA-like DsbG is shown. While DsbG has an additional dimerization domain, there are significant similarities in the tertiary structure between kappa and DsbG; they both share an interruption at the same point within the Trx domain, and the extended "helix 2" is similar between the two enzymes. There is also sequence similarity between kappa and DsbA-like proteins within the Trx-like domain.

    4. An alignment between kappa and cytosolic GST tau is shown. The overall structures are quite different.

    5. However, zooming in on the bound GSH from each structure, when residues interacting with GSH are displayed, there is a surprising amount of similarity in conformation and biophysical character.

    Movie created using UCSF Chimera

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