Overexpression of Par‐4 sensitizes TRAIL‐induced apoptosis via inactivation of NF‐κB and Akt signaling pathways in renal cancer cells

The prostate‐apoptosis‐response‐gene‐4 (Par‐4) is up‐regulated in prostate cells undergoing programmed cell death. Furthermore, Par‐4 protein has been shown to function as an effector of cell death in response to various apoptotic stimuli that trigger mitochondria and membrane receptor‐mediated cell death pathways. In this study, we investigated how Par‐4 modulates TRAIL‐mediated apoptosis in TRAIL‐resistant Caki cells. Par‐4 overexpressing cells were strikingly sensitive to apoptosis induced by TRAIL compared with control cells. Par‐4 overexpressing Caki cells treated with TRAIL showed an increased activation of the initiator caspase‐8 and the effector caspase‐3, together with an enforced cleavage of XIAP and c‐FLIP. TRAIL‐induced reduction of XIAP and c‐FLIP protein levels in Par‐4 overexpressing cells was prevented by z‐VAD pretreatment. In addition, the surface DR5 protein level was increased in TRAIL‐treated Par‐4 overexpressing cells. Interestingly, even though a deletion of leucine zipper domain in Par‐4 recovered Bcl‐2 level to basal level induced by wild type Par‐4, it partly decreased sensitivity to TRAIL in Caki cells. In addition, exposure of Caki/Par‐4 cells to TRAIL led to reduction of phosphorylated Akt levels, but deletion of leucine zipper domain of Par‐4 did not affect these phosphorylated Akt levels. In conclusion, we here provide evidence that ectopic expression of Par‐4 sensitizes Caki cells to TRAIL via modulation of multiple targets, including DR5, Bcl‐2, Akt, and NF‐κB. J. Cell. Biochem. 109: 885–895, 2010. © 2010 Wiley‐Liss, Inc.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin‐binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

A 34‐residue α/β peptide [IG(28–61)], derived from the C‐terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C‐terminal part (a 16‐residue‐long fragment) of this peptide, which corresponds to the sequence of the β‐hairpin in the native structure, forms structure similar to the β‐hairpin only at T = 313 K, and the structure is stabilized by non‐native long‐range hydrophobic interactions (Val47–Val59). On the other hand, the N‐terminal part of IG(28–61), which corresponds to the middle α‐helix in the native structure, is unstructured at low temperature (283 K) and forms an α‐helix‐like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long‐range connectivities which would have supported packing between the C‐terminal (β‐hairpin) and the N‐terminal (α‐helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726–736), based on Monte‐Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 469–480, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The membrane localization domains of two distinct bacterial toxins form a 4‐helix‐bundle in solution

Membrane localization domain (MLD) was first proposed for a 4‐helix‐bundle motif in the crystal structure of the C1 domain of Pasteurella multocida toxin (PMT). This structure motif is also found in the crystal structures of several clostridial glycosylating toxins (TcdA, TcdB, TcsL, and TcnA). The Ras/Rap1‐specific endopeptidase (RRSP) module of the multifunctional autoprocessing repeats‐in‐toxins (MARTX) toxin produced by Vibrio vulnificus has sequence homology to the C1‐C2 domains of PMT, including a putative MLD. We have determined the solution structure for the MLDs in PMT and in RRSP using solution state NMR. We conclude that the MLDs in these two toxins assume a 4‐helix‐bundle structure in solution.     

Solution structure of a novel T‐cell adhesion inhibitor derived from the fragment of ICAM‐1 receptor: Cyclo(1,8)‐Cys‐Pro‐Arg‐Gly‐Gly‐Ser‐Val‐Cys

This study is aimed at elucidating the structure of a novel T‐cell adhesion inhibitor, cyclo(1,8)‐CPRGGSVC using one‐ and two‐dimensional (2D) ¹H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)‐PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule‐1 (ICAM‐1). Our previous results show that the cyclo(1,8)‐CPRGGSVC peptide binds to the LFA‐1 I‐domain and inhibits heterotypic T‐cell adhesion, presumably by blocking the LFA‐1/ICAM‐1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type‐I β‐turn conformation at the Pro2‐Arg3‐Gly4‐Gly5 (PRGG) sequence. The β‐turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM‐1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)‐CPRGGSVC peptide. Meanwhile, the Gly5‐Ser6‐Val7‐Cys8‐Cys1 (GSVCC) sequence forms a “turn‐like” random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)‐CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA‐1 I‐domain. The conserved β‐turn conformation of the PRGG motif in ICAM‐1, cIBR, and cyclo(1,8)‐CPRGGSVC peptides can potentially be used to design peptidomimetics. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 633–641, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Proteomic,functional and motif‐based analysis of C‐terminal Src kinase‐interacting proteins

C‐terminal Src kinase (Csk) that functions as an essential negative regulator of Src family tyrosine kinases (SFKs) interacts with tyrosine‐phosphorylated molecules through its Src homology 2 (SH2) domain, allowing it targeting to the sites of SFKs and concomitantly enhancing its kinase activity. Identification of additional Csk‐interacting proteins is expected to reveal potential signaling targets and previously undescribed functions of Csk. In this study, using a direct proteomic approach, we identified 151 novel potential Csk‐binding partners, which are associated with a wide range of biological functions. Bioinformatics analysis showed that the majority of identified proteins contain one or several Csk‐SH2 domain‐binding motifs, indicating a potentially direct interaction with Csk. The interactions of Csk with four proteins (partitioning defective 3 (Par3), DDR1, SYK and protein kinase C iota) were confirmed using biochemical approaches and phosphotyrosine 1127 of Par3 C‐terminus was proved to directly bind to Csk‐SH2 domain, which was consistent with predictions from in silico analysis. Finally, immunofluorescence experiments revealed co‐localization of Csk with Par3 in tight junction (TJ) in a tyrosine phosphorylation‐dependent manner and overexpression of Csk, but not its SH2‐domain mutant lacking binding to phosphotyrosine, promoted the TJ assembly in Madin‐Darby canine kidney cells, implying the involvement of Csk‐SH2 domain in regulating cellular TJs. In conclusion, the newly identified potential interacting partners of Csk provided new insights into its functional diversity in regulation of numerous cellular events, in addition to controlling the SFK activity.     

The flexible C‐terminal arm of the Lassa arenavirus Z‐protein mediates interactions with multiple binding partners

The arenavirus genome encodes for a Z‐protein, which contains a RING domain that coordinates two zinc ions, and has been identified as having several functional roles at various stages of the virus life cycle. Z‐protein binds to multiple host proteins and has been directly implicated in the promotion of viral budding, repression of mRNA translation, and apoptosis of infected cells. Using homology models of the Z‐protein from Lassa strain arenavirus, replica exchange molecular dynamics (MD) was used to refine the structures, which were then subsequently clustered. Population‐weighted ensembles of low‐energy cluster representatives were predicted based upon optimal agreement of the chemical shifts computed with the SPARTA program with the experimental NMR chemical shifts. A member of the refined ensemble was indentified to be a potential binder of budding factor Tsg101 based on its correspondence to the structure of the HIV‐1 Gag late domain when bound to Tsg101. Members of these ensembles were docked against the crystal structure of human eIF4E translation initiation factor. Two plausible binding modes emerged based upon their agreement with experimental observation, favorable interaction energies and stability during MD trajectories. Mutations to Z are proposed that would either inhibit both binding mechanisms or selectively inhibit only one mode. The C‐terminal domain conformation of the most populated member of the representative ensemble shielded protein‐binding recognition motifs for Tsg101 and eIF4E and represents the most populated state free in solution. We propose that C‐terminal flexibility is key for mediating the different functional states of the Z‐protein. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Calculation of symmetric multimer structures from NMR data using a priori knowledge of the monomer structure, co-monomer restraints, and interface mapping: The case of leucine zippers

Summary NMR studies of symmetric multimers are problematic due to the difficulty in distinguishing between intra-, inter-, and co-monomer (mixed) NOE signals. Previously, one of us described a general calculation strategy called dynamic assignment by which this difficulty can be overcome [Nilges, M. (1993) Proteins, 17, 297–309]. Here we describe extensions to the method for handling many co-monomer NOEs and for taking advantege of prior knowledge of monomer structures. The new protocol was developed for the particularly difficult case of leucine zipper (LZ) homodimers, for which the previous protocol proved inefficient. In addition to the problem of dimer symmetry, LZs have a particularly high proportion of co-monomer NOE signals and a high degree of repetition in sequence and structure, leading to significant spectral overlap. Furthermore, the leucine zipper is a rather extended (as opposed to globular) protein domain; accurately determining such a structure based only on the very short distances obtainable by NMR is clearly a challenge to the NMR structure determination method. We have previously shown that, for LZ homodimers, many of the backbone-backbone NOESY cross peaks can be unambiguously assigned as intra-monomer, enabling approximate monomer structures to be calculated. Using model and experimental data sets, we verified that the new protocol converges to the correct dimer structure. The results show that short-range NMR distance data can be sufficient to define accurately the extended LZ. The protocol has been used to derive a novel solution structure of the c-Jun LZ domain. Based on these calculations, we propose the protocol as a prototype for the general case of symmetric multimers where the monomer structure is known.Abbreviations 3D three-dimensional - GCN4-c crystal structure of the GCN4 LZ homodimer - GCN4-s solution structures of GCN4 - GSYM global symmetry - Jun-m model structure of the Jun LZ homodimer - Jun-s solution structure of Jun - LZ leucine zipper - MFP mean force potential - MDSA molecular dynamical simulated annealing - NCS noncrystallographic symmetry - NOE nuclear Overhauser enhancement - rmsd root-mean-square deviation - vdW van der Waals     

Expression, purification, and characterization of coiled coil and leucine zipper domains of C-terminal myosin binding subunit of myosin phosphatase for solution NMR studies

Protein-protein interactions between MBS and PKG are mediated by the involvement of C-terminal domain of MBS, MBS(CT180) and N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKG-Iα, PKG-Iα1(-59). MBS(CT180) is comprised of three structurally variant domains of non-CC, CC, and LZ nature. Paucity of three-dimensional structural information of these MBS domains precludes atomic level understanding of MBS-PKG contractile complex structure. Here we present data on cloning, expression, and purification of CC, LZ, and CCLZ domains of MBS(CT180) and their biophysical characterization using size exclusion chromatography (SEC), circular dichroism (CD), and two-dimensional (1)H-(15)N HSQC NMR. The methods as detailed resulted in high level protein expression and high milligram quantities of purified isotopically ((15)N and (13)C) enriched polypeptides. SEC, CD, and (1)H-(15)N HSQC NMR experiments demonstrated that recombinantly expressed MBS CC domain is well folded and exists as a dimer within physiologic pH range, which is supported by our previous findings. The dimerization of CC MBS is likely mediated through formation of coiled coil conformation. In contrast, MBS LZ domain was almost unfolded that exists as non-stable low structured monomer within physiologic pH range. Protein folding and stability of MBS LZ was improved as a function of decrease in pH that adopts a folded, stable, and structured conformation at acidified pH 4.5. SEC and NMR analyses of LZ vs. CCLZ MBS domains indicated that inclusion of CC domain partially improves protein folding of LZ domain.     

Probing the interaction between the coiled coil leucine zipper of cGMP-dependent protein kinase Ialpha and the C terminus of the myosin binding subunit of the myosin light chain phosphatase

Nitric oxide and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase I (PKG-Ialpha)-mediated activation of myosin phosphatase (MLCP). Mechanistically it has been proposed that protein-protein interactions between the N-terminal leucine zipper (LZ) domain of PKG-Ialpha ((PKG-Ialpha(1-59)) and the LZ and/or coiled coil (CC) domain of the myosin binding subunit (MBS) of MLCP are localized in the C terminus of MBS. Although recent studies have supported these interactions, the critical amino acids responsible for these interactions have not been identified. Here we present structural and biophysical data identifying that the LZ domain of PKG-Ialpha(1-59) interacts with a well defined 42-residue CC motif (MBS(CT42)) within the C terminus of MBS. Using glutathione S-transferase pulldown experiments, chemical cross-linking, size exclusion chromatography, circular dichroism, and isothermal titration calorimetry we identified a weak dimer-dimer interaction between PKG-Ialpha(1-59) and this C-terminal CC domain of MBS. The K(d) of this non-covalent complex is 178.0+/-1.5 microm. Furthermore our (1)H-(15)N heteronuclear single quantum correlation NMR data illustrate that this interaction is mediated by several PKG-Ialpha residues that are on the a, d, e, and g hydrophobic and electrostatic interface of the C-terminal heptad layers 2, 4, and 5 of PKG-Ialpha. Taken together these data support a role for the LZ domain of PKG-Ialpha and the CC domain of MBS in this requisite contractile complex.     

Comparison of the folding mechanism of highly homologous proteins in the lipid‐binding protein family

The folding mechanism of two closely related proteins in the intracellular lipid‐binding protein family, human bile acid‐binding protein (hBABP), and rat bile acid‐binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence. Both of these single domain proteins fit well to a two‐state model for unfolding by fluorescence and circular dichroism at equilibrium. Three phases were observed during the unfolding of rBABP by fluorescence but only one phase was observed during the unfolding of hBABP, suggesting that at least two kinetic intermediates accumulate during the unfolding of rBABP that are not observed during the unfolding of hBABP. Fluorine NMR was used to examine the equilibrium unfolding behavior of the W49 side chain in 6‐fluorotryptophan‐labeled rBABP and hBABP. The structure of rBABP appears to be more dynamic than that of hBABP in the vicinity of W49 in the absence of denaturant, and urea has a greater effect on this dynamic behavior for rBABP than for hBABP. As such, the folding behavior of highly sequence related proteins in this family can be quite different. These differences imply that moderately sized proteins with high sequence and structural similarity can still populate quite different structures during folding. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Axially chiral N‐(o‐aryl)‐4‐hydroxy‐2‐oxazolidinone derivatives from diastereoselective reduction of N‐(o‐aryl)‐2,4‐oxazolidinediones: Thermally interconvertible atropisomers via ring‐chain‐ring tautomerization

The reduction of the axially chiral N‐(o‐aryl)‐5,5‐dimethyl‐2,4‐oxazolidinediones by NaBH₄ yielded axially chiral N‐(o‐aryl)‐4‐hydroxy‐5,5‐dimethyl‐2‐oxazolidinone enantiomers having a chiral center at C‐4, with 100% diastereoselectivity as has been shown by their ¹H and ¹³C NMR spectra and by enantioselective HPLC analysis. The resolved enantiomeric isomers were found to interconvert thermally through an aldehyde intermediate formed upon ring cleavage via a latent ring‐chain‐ring tautomerization. It was found that the rate of enantiomerization depended on the size and the electronic effect of the ortho substituent present on the aryl ring bonded to the nitrogen of the heterocycle. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

The unique N‐terminal zinc finger of synaptotagmin‐like protein 4 reveals FYVE structure

Synaptotagmin‐like protein 4 (Slp4), expressed in human platelets, is associated with dense granule release. Slp4 is comprised of the N‐terminal zinc finger, Slp homology domain, and C2 domains. We synthesized a compact construct (the Slp4N peptide) corresponding to the Slp4 N‐terminal zinc finger. Herein, we have determined the solution structure of the Slp4N peptide by nuclear magnetic resonance (NMR). Furthermore, experimental, chemical modification of Cys residues revealed that the Slp4N peptide binds two zinc atoms to mediate proper folding. NMR data showed that eight Cys residues coordinate zinc atoms in a cross‐brace fashion. The Simple Modular Architecture Research Tool database predicted the structure of Slp4N as a RING finger. However, the actual structure of the Slp4N peptide adopts a unique C₄C₄‐type FYVE fold and is distinct from a RING fold. To create an artificial RING finger (ARF) with specific ubiquitin‐conjugating enzyme (E2)‐binding capability, cross‐brace structures with eight zinc‐ligating residues are needed as the scaffold. The cross‐brace structure of the Slp4N peptide could be utilized as the scaffold for the design of ARFs.     

Sequence determinants of thermodynamic stability in a WW domain—An all‐β‐sheet protein

The stabilities of 66 sequence variants of the human Pin1 WW domain have been determined by equilibrium thermal denaturation experiments. All 34 residues composing the hPin1 WW three‐stranded β‐sheet structure could be replaced one at a time with at least one different natural or non‐natural amino acid residue without leading to an unfolded protein. Alanine substitutions at only four positions within the hPin1 WW domain lead to a partially or completely unfolded protein—in the absence of a physiological ligand. The side chains of these four residues form a conserved, partially solvent‐inaccessible, continuous hydrophobic minicore comprising the N‐ and C‐termini. Ala mutations at five other residues, three of which constitute the ligand binding patch on the concave side of the β‐sheet, significantly destabilize the hPin1 WW domain without leading to an unfolded protein. The remaining mutations affect protein stability only slightly, suggesting that only a small subset of side chain interactions within the hPin1 WW domain are mandatory for acquiring and maintaining a stable, cooperatively folded β‐sheet structure.     

The possible roles of B‐cell novel protein‐1 (BCNP1) in cellular signalling pathways and in cancer

B‐cell novel protein‐1 (BCNP1) or Family member of 129C (FAM129C) was identified as a B‐cell‐specific plasma‐membrane protein. Bioinformatics analysis predicted that BCNP1 might be heavily phosphorylated. The BCNP1 protein contains a pleckstrin homology (PH) domain, two proline‐rich (PR) regions and a Leucine Zipper (LZ) domain suggesting that it may be involved in protein‐protein interactions. Using The Cancer Genome Atlas (TCGA) data sets, we investigated the correlation of alteration of the BCNP1 copy‐number changes and mutations in several cancer types. We also investigated the function of BCNP1 in cellular signalling pathways. We found that BCNP1 is highly altered in some types of cancers and that BCNP1 copy‐number changes and mutations co‐occur with other molecular alteration events for TP53 (tumour protein P53), PIK3CA (Phosphatidylinositol‐4,5‐Bisphosphate 3‐Kinase, Catalytic Subunit Alpha), MAPK1 (mitogen‐activated protein kinase‐1; ERK: extracellular signal regulated kinase), KRAS (Kirsten rat sarcoma viral oncogene homolog) and AKT2 (V‐Akt Murine Thymoma Viral Oncogene Homolog 2). We also found that PI3K (Phoshoinositide 3‐kinase) inhibition and p38 MAPK (p38 mitogen‐activated protein kinase) activation leads to reduction in phosphorylation of BCNP1 at serine residues, suggesting that BCNP1 phosphorylation is PI3K and p38MAPK dependent and that it might be involved in cancer. Its degradation depends on a proteasome‐mediated pathway.     

Structural and biochemical characterization of a metagenome‐derived esterase with a long N‐terminal extension

The genes encoding six novel esterolytic/lipolytic enzymes, termed LC‐Est1～6, were isolated from a fosmid library of a leaf‐branch compost metagenome by functional screening using tributyrin agar plates. These enzymes greatly vary in size and amino acid sequence. The highest identity between the amino acid sequence of each enzyme and that available from the database varies from 44 to 73%. Of these metagenome‐derived enzymes, LC‐Est1 is characterized by the presence of a long N‐terminal extension (LNTE, residues 26–283) between a putative signal peptide (residues 1–25) and a C‐terminal esterase domain (residues 284–510). A putative esterase from Candidatus Solibacter usitatus (CSu‐Est) is the only protein, which shows the significant amino acid sequence identity (46%) to the entire region of LC‐Est1. To examine whether LC‐Est1 exhibits activity and its LNTE is important for activity and stability of the esterase domain, LC‐Est1 (residues 26–510), LC‐Est1C (residues 284–510), and LC‐Est1C* (residues 304–510) were overproduced in E. coli, purified, and characterized. LC‐Est1C* was only used for structural analysis. The crystal structure of LC‐Est1C* highly resembles that of the catalytic domain of Thermotoga maritima esterase, suggesting that LNTE is not required for folding of the esterase domain. The enzymatic activity of LC‐Est1C was lower than that of LC‐Est1 by 60%, although its substrate specificity was similar to that of LC‐Est1. LC‐Est1C was less stable than LC‐Est1 by 3.3°C. These results suggest that LNTE of LC‐Est1 rather exists as an independent domain but is required for maximal activity and stability of the esterase domain.     

Identification and characterization of peptides that bind the PPIase domain of Parvulin17

Peptidyl‐prolyl cis‐trans isomerases (PPIases) are the enzymes that increase the rate of isomerization of the peptide bond N‐terminal to the proline substrate. Par14 and its isoform Par17 belong to the Parvulin family of PPIases. Par14 can bind AT‐rich double‐stranded DNA and was shown to be part of the pre‐ribosomal ribonucleoprotein (pre‐rRNP) complexes, where it functions as an RNA processing factor that is involved in ribosome biogenesis. Its longer isoform Par17 is expressed only in cells of hominids, where it is targeted to the mitochondria. To find binding partners (peptides or proteins) for Par17, we applied the phage display technology. We panned 7‐mer and 12‐mer peptide libraries against Par17. The consensus sequence XHSXVHØ, where X can be any amino acid and Ø is a hydrophobic amino acid, was enriched from both libraries. We demonstrate the binding of this motif to the PPIase domain of Par17 using phage ELISA and NMR spectroscopy. We propose that residues Met90, Val91, Phe94, Gln95, Glu96, and Ala98 of Par17 are involved in substrate recognition, and that the phage display‐selected motif XHSXVHØ can be recognized by Par17 PPIase domain in vivo. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Thermal‐ and urea‐induced unfolding processes of glutathione S‐transferase by molecular dynamics simulation

The Schistosoma juponicum 26 kDa glutathione S‐transferase (sj26GST) consists of the N‐terminal domain (N‐domain), containing three alpha‐helices (named H1‐H3) and four anti‐parallel beta‐strands (S1‐S4), and the C‐terminal domain (C‐domain), comprising five alpha‐helices (named H4‐H8). In present work, molecular dynamics simulations and fluorescence spectroscopic were used to gain insights into the unfolding process of sj26GST. The molecular dynamics simulations on sj26GST subunit both in water and in 8 M urea were carried out at 300 K, 400 K and 500 K, respectively. Spectroscopic measurements were employed to monitor structural changes. Molecular dynamics simulations of sj26GST subunit induced by urea and temperature showed that the initial unfolding step of sj26GST both in water and urea occurred on N‐domain, involving the disruption of helices H2, H3 and strands S3 and S4, whereas H6 was the last region exposed to solution and was the last helix to unfold. Moreover, simulations analyses combining with fluorescence and circular dichroism spectra indicated that N‐domain could not fold independent, suggesting that correct folding of N‐domain depended on its interactions with C‐domain. We further proposed that the folding of GSTs could begin with the hydrophobic collapse of C‐domain whose H4, H5, H6 and H7 could move close to each other and form a hydrophobic core, especially H6 wrapped in the hydrophobic center and beginning spontaneous formation of the helix. S3, S4, H3, and H2 could form in the wake of the interaction between C‐domain and N‐domain. The paper can offer insights into the molecular mechanism of GSTs unfolding. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 247–259, 2015.