Search

HIV protease is essential for processing the Gag polyprotein to produce infectious virions and is a major target in antiretroviral therapy. We have identified an unusual HIV-1 subtype C variant that contains insertions of leucine and asparagine (L38↑N↑L) in the hinge region of protease at position 38. This was isolated from a protease inhibitor naïve infant. Isothermal titration calorimetry showed that 10% less of L38↑N↑L protease was in the active conformation as compared with a reference strain. L38↑N↑L protease displayed a ±50% reduction in KM and kcat. The catalytic efficiency (kcat/KM) of L38↑N↑L protease was not significantly different from that of wild type although there was a 42% reduction in specific activity for the variant. An in vitro phenotypic assay showed the L38↑N↑L protease to be susceptible to lopinavir (LPV), atazanavir (ATV) and darunavir in the context of an unrelated Gag. However, in the presence of the related Gag, L38↑N↑L showed reduced susceptibility to darunavir while remaining susceptible to LPV and ATV. Furthermore, a reduction in viral replication capacity (RC) was observed in combination with the related Gag. The reduced susceptibility to darunavir and decrease in RC may be due to PTAPP duplication in the related Gag. The present study shows the importance of considering the Gag region when looking at drug susceptibility of HIV-1 protease variants.

Plasmodium falciparum, the main agent of malaria expresses six members of the heat shock protein 70 (Hsp70) family. Hsp70s serve as protein folding facilitators in the cell. Amongst the six Hsp70 species that P. falciparum expresses, Hsp70‐x (PfHsp70‐x), is partially exported to the host red blood cell where it is implicated in host cell remodeling. Nearly 500 proteins of parasitic origin are exported to the parasite‐infected red blood cell (RBC) along with PfHsp70‐x. The role of PfHsp70‐x in the infected human RBC remains largely unclear. One of the defining features of PfHsp70‐x is the presence of EEVN residues at its C‐terminus. In this regard, PfHsp70‐x resembles canonical eukaryotic cytosol‐localized Hsp70s which possess EEVD residues at their C‐termini in place of the EEVN residues associated with PfHsp70‐x. The EEVD residues of eukaryotic Hsp70s facilitate their interaction with co‐chaperones. Characterization of the role of the EEVN residues of PfHsp70‐x could provide insights into the function of this protein. In the current study, we expressed and purified recombinant PfHsp70‐x (full length) and its EEVN minus form (PfHsp70‐xT). We then conducted structure‐ function assays towards establishing the role of the EEVN motif of PfHsp70‐x. Our findings suggest that the EEVN residues of PfHsp70‐x are important for its ATPase activity and chaperone function. Furthermore, the EEVN residues are crucial for the direct interaction between PfHsp70‐x and human Hsp70‐Hsp90 organizing protein (hHop) in vitro. Hop facilitates functional cooperation between Hsp70 and Hsp90. However, it remains to be established if PfHsp70‐x and hHsp90 cooperate in vivo.

In recent years, various strategies have been used to overexpress and purify HIV-1 protease because it is an essential drug target in anti-retroviral therapy. Obtaining sufficient quantities of the enzyme, however, remains challenging. Overexpression of large quantities is prevented due to the enzyme's autolytic nature and its inherent cytotoxicity in Escherichia coli cells. Here, we describe a novel HIV-1 protease purification method using a thioredoxin-hexahistidine fusion system for the wild-type and two variant proteases. The fusion proteases were overexpressed in E. coli and recovered by immobilised metal ion affinity chromatography. The proteases were cleaved from the fusion constructs using thrombin. When compared to the standard overexpression and purification protocol in use in our laboratory, the expression of the fusion-derived wild-type protease was increased from 0.83 to 2.5 mg/l of culture medium. The expression levels of the two variant proteases ranged from 1.5 to 2 mg/l of culture medium. The fusion wild-type and variant proteases were inactive before the cleavage of the thioredoxin-hexahistidine fusion tag as no enzymatic activity was observed. The proteases were, however, active after cleavage of the tag. The novel thioredoxin-hexahistidine fusion system, therefore, enables the successful overexpression and purification of catalytically active HIV-1 proteases.

FOXP2 is a transcription factor expressed in multiple tissues during embryonic development. FOXP2 regulates transcription by binding to DNA at its DNA binding domain, the forkhead domain (FHD) through the recognition helix. Ser557 is a residue located within the recognition helix that has the potential to become phosphorylated posttranslationally. In this study we investigated whether phosphorylation of Ser557 can influence the structure and DNA binding of the FOXP2 FHD. We did this by constructing S557E, a phosphomimetic mutant, and comparing its behaviour to the wild type. The mutation did not affect the secondary or tertiary structure of the protein although it did decrease the propensity of the FOXP2 FHD to form dimers. Most notably, the mutation showed significantly reduced DNA binding compared to the wild type as detected using electrophoretic mobility shift assays. Molecular docking was also performed in which the wild type, phosphomimetic mutant and phosphorylated wild-type were docked to DNA and their interactions with DNA were compared. These results indicated that the wild type forms more interactions with the DNA and that the phosphomimetic mutant as well as the phosphorylated wild type did not associate as favourably with the DNA. This indicates that phosphorylation of Ser557 could disrupt DNA binding likely due to electrostatic and steric hindrance. This suggests that phosphorylation of Ser557 in the FOXP2 FHD could act as a control mechanism for FOXP2 and ultimately could be involved in regulation of transcription.

The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival.

FOXP2 is a transcriptional repressor involved in development of the human brain and is the first gene product to be linked to the evolution of human speech. FOXP2 belongs to the FOX superfamily of proteins that share a common winged helix DNA binding domain - the forkhead domain. A divalent cation (Mg2+ or Ca2+) has been identified bound to a group of highly conserved residues in a number of FOX forkhead domain crystal structures. This work aims to investigate the role of the conserved divalent cation binding site by studying both the structure and DNA-binding function of the FOXP2 forkhead domain when in the presence and absence of either cation (Mg2+or Ca2+). The presence of the cations does not significantly alter the structure of the apo-FOXP2 forkhead domain. However, when in the presence of a cognate oligonucleotide sequence, differences are observed upon addition of divalent cation. These differences occur both in the structure and in the thermodynamic DNA binding signature of the FOXP2 forkhead domain. The incorporation of molecular dynamics simulations together with the experimental data provides us with sufficient insight so as to propose a possible role for divalent cations in the regulation of DNA binding to FOX transcription factors.

Forkhead box (FOX) proteins are a ubiquitously expressed family of transcription factors that regulate the development and differentiation of a wide range of tissues in animals. The FOXP subfamily members are the only known FOX proteins capable of forming domain-swapped forkhead domain (FHD) dimers. This is proposed to be due to an evolutionary mutation (P539A) that lies in the FHD hinge loop, a key region thought to fine-tune DNA sequence specificity in the FOX transcription factors. Considering the importance of the hinge loop in both the dimerisation mechanism of the FOXP FHD and its role in tuning DNA binding, a detailed investigation into the implications of mutations within this region could provide important insight into the evolution of the FOX family. Isothermal titration calorimetry and hydrogen exchange mass spectroscopy were used to study the thermodynamic binding signature and changes in backbone dynamics of FOXP2 FHD DNA binding. Dual luciferase reporter assays were performed to study the effect that the hinge-loop mutation has on FOXP2 transcriptional activity in vivo. We demonstrate that the change in dynamics of the hinge-loop region of FOXP2 alters the energetics and mechanism of DNA binding highlighting the critical role of hinge loop mutations in regulating DNA binding characteristics of the FOX proteins.

The forkhead box (FOX) proteins are a family of transcription factors that interact with DNA via a winged helix motif that forms part of the forkhead domain. The FOXP (FOXP1-4) subfamily is unique in the family in that the forkhead domains of these proteins are able to dimerise via domain swapping. In this event, structural elements are exchanged via extension of the hinge loop region. Despite the high sequence homology among the FOXP subfamily members, the stability of their forkhead domain dimers varies, with FOXP3 forming the most stable dimer. An amino acid difference is observed in the hinge region of the FOXP subfamily where a tyrosine in all members is replaced with a phenylalanine in FOXP3. In this work, the role of phenylalanine at this position in the hinge region was investigated. This was done by creating the Y540F variant of the FOXP2 forkhead domain. The effect of the Y540F mutation on the structure, dimerisation propensity and DNA binding ability of the FOXP subfamily was investigated. The mutation altered the structure of the protein by decreasing the disorder of the backbone as measured by circular dichroism spectroscopy and by altering the local environment of the hinge region as measured by tryptophan fluorescence. The propensity of the forkhead domain to form a dimer was improved ~9.5 fold by the mutation. This was attributed to increased hydrophobicity at the dimer interface as well as altered tension in the hinge loop region. DNA binding assays indicated that the affinity for DNA was decreased by the mutation. Taken together, these findings suggest that domain swapping may modulate DNA binding.

Since its identification, HIV has continued to have a detrimental impact on the lives of millions of people throughout the world. The protease of HIV is a major target in antiviral treatment. The South African HIV-1 subtype C (C-SA) protease displays weaker binding affinity for some clinically approved protease inhibitors in comparison with the HIV-1 subtype B protease. The heavy HIV burden in sub-Saharan Africa, where subtype C HIV-1 predominates, makes this disparity a topic of great interest. In light of this, the enzyme activity and affinity of protease inhibitors for the subtype B and C-SA proteases were determined. The relative vitality, indicating the selective advantage of polymorphisms, of the C-SA protease relative to the subtype B protease in the presence of ritonavir and darunavir was four- and tenfold greater, respectively. Dynamic differences that contribute to the reduced drug susceptibility of the C-SA protease were investigated by performing hydrogen-deuterium exchange/mass spectrometry (HDX/MS) on unbound subtype B and C-SA proteases. The reduced propensity to form the E35-R57 salt bridge, and alterations in the hydrophobic core of the C-SA protease, are proposed to affect the anchoring of the flexible flaps, resulting in an increased proportion of the fully open flap conformation. HDX/MS data suggested that the N-terminus of both proteases is less stable than the C-terminus of the proteases, thus explaining the increased efficacy of dimerization inhibitors targeted toward the C-terminus of HIV proteases. As far as we are aware, this is the first report on assessment of HIV protease dynamics using HDX/MS.

The seemingly simple proton abstraction reactions underpin many chemical transformations, including isomerization reactions, and are thus of immense biological significance. Despite the energetic cost, enzyme-catalyzed proton abstraction reactions show remarkable rate enhancements. The pathways leading to these accelerated rates are numerous and on occasion partly enigmatic. The isomerization of the steroid Δ(5)-androstene-3,17-dione by the glutathione transferase A3-3 in mammals was investigated to gain insight into the mechanism. Particular emphasis was placed on the nature of the transition state, the intermediate suspected of aiding this process, and the hydrogen bonds postulated to be the stabilizing forces of these transient species. The UV-visible detection of the intermediate places this species in the catalytic pathway, whereas fluorescence spectroscopy is used to obtain the binding constant of the analog intermediate, equilenin. Solvent isotope exchange reveals that proton abstraction from the substrate to form the intermediate is rate-limiting. Analysis of the data in terms of the Marcus formalism indicates that the human glutathione transferase A3-3 lowers the intrinsic kinetic barrier by 3 kcal/mol. The results lead to the conclusion that this reaction proceeds through an enforced concerted mechanism in which the barrier to product formation is kinetically insignificant.

Chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. The oligomerization of the transmembrane domain (TMD) remains speculative despite it being implicated in pore formation. The extent to which electrostatic and van der Waals interactions drive folding and association of the dimorphic TMD is unknown and is complicated by the requirement of interactions favorable in both aqueous and membrane environments. Here we report a putative Lys37-Trp35 cation-π interaction and show that it stabilizes the dimeric form of the CLIC1 TMD in membranes. A synthetic 30-mer peptide comprising a K37M TMD mutant was examined in 2,2,2-trifluoroethanol, sodium dodecyl sulfate micelles, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes using far-ultraviolet (UV) circular dichroism, fluorescence, and UV absorbance spectroscopy. Our data suggest that Lys37 is not implicated in the folding, stability, or membrane insertion of the TMD peptide. However, removal of this residue impairs the formation of dimers and higher-order oligomers. This is accompanied by a 30-fold loss of chloride influx activity, suggesting that dimerization modulates the rate of chloride conductance. We propose that, within membranes, individual TMD helices associate via a Lys37-mediated cation-π interaction to form active dimers. The latter findings are also supported by results of modeling a putative TMD dimer conformation in which Lys37 and Trp35 form cation-π pairs at the dimer interface. Dimeric helix bundles may then associate to form fully active ion channels. Thus, within a membrane-like environment, aromatic interactions involving a polar lysine side chain provide a thermodynamic driving force for helix-helix association.

Protein S-nitrosation is a post-translational modification that regulates the function of more than 500 human proteins. Despite its apparent physiological significance, S-nitrosation is poorly understood at a molecular level. Here, we investigated the effect of S-nitrosation on the activity, structure, stability, and dynamics of human glutathione transferase P1-1 (GSTP1-1), an important detoxification enzyme ubiquitous in aerobes. S-Nitrosation at Cys47 and Cys101 reduces the activity of the enzyme by 94%. Circular dichroism spectroscopy, acrylamide quenching, and amide hydrogen-deuterium exchange mass spectrometry experiments indicate that the loss of activity is caused by the introduction of local disorder at the active site of GSTP1-1. Furthermore, the modification destabilizes domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. In contrast, S-nitrosation at Cys101 alone introduces a refolding defect in domain 1 but compensates by stabilizing the domain kinetically. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation and provide general insight into the consequences of S-nitrosation on protein stability and dynamics.

Glutathione S-transferase A3-3 is the most catalytically efficient steroid isomerase enzyme known in humans, transforming Δ5-androstene-3-17-dione into Δ4-androstene-3-17-dione. GSTA3-3 catalyzes this reaction with ten-fold greater efficiency than GSTA1-1, its closest competitor in the Alpha class of GSTs. In order to examine the differences between Alpha class GSTs and to better elucidate the mechanism of GSTA3-3 the roles of Tyr9 and Arg15 were examined. Tyr9 is the major catalytic residue of Alpha class GSTs and Arg15 is proposed to be catalytically important to GSTA3-3 but never before experimentally examined. While the structure and stability of the Alpha class enzymes are highly comparable, subtle differences at the G-site of the enzymes account for GSTA3-3 having a ten-fold greater affinity for the substrate GSH. Y9F and R15L mutations, singly or together, have no effect on the structure and stability of GSTA3-3 (the same effect they have on GSTA1-1) despite the R15L mutation removing an interdomain salt-bridge at the active site. Hydrogen-deuterium exchange mass spectrometry also revealed that neither mutation had a significant effect on the conformational dynamics of GSTA3-3. The R15L and Y9F mutations are equally important to the specific activity of the steroid isomerase reaction; however, Arg15 is more important for lowering the pKa of GSH. Lowering the pKa of GSH being how GSTs catalyze their reactions. Additionally, there is evidence to suggest that Arg15 is integral to allowing GSTA3-3 to differentiate between Δ5-androstene-3-17-dione and Δ4-androstene-3-17-dione, indicating that Arg15 is a more important active-site residue than previously known.

The energetics of ligand binding to human eukaryotic elongation factor 1 gamma (heEF1γ) was investigated using reduced glutathione (GSH), oxidised glutathione (GSSG), glutathione sulfonate and S-hexylglutathione as ligands. The experiments were conducted using isothermal titration calorimetry, and the findings were supported using computational studies. The data show that the binding of these ligands to heEF1γ is enthalpically favourable and entropically driven (except for the binding of GSSG). The full length heEF1γ binds GSSG with lower affinity (K d = 115 μM), with more hydrogen-bond contacts (ΔH = −73.8 kJ/mol) and unfavourable entropy (−TΔS = 51.7 kJ/mol) compared to the glutathione transferase-like N-terminus domain of heEF1γ, which did not show preference to any specific ligand. Computational free binding energy calculations from the 10 ligand poses show that GSSG and GSH consistently bind heEF1γ, and that both ligands bind at the same site with a folded bioactive conformation. This study reveals the possibility that heEF1γ is a glutathione-binding protein.

Chloride intracellular channel proteins exist in both a soluble cytosolic form and a membrane-bound form. The mechanism of conversion between the two forms is not properly understood, although one of the contributing factors is believed to be the variation in pH between the cytosol (~7.4) and the membrane (~5.5). We systematically mutated each of the three histidine residues in CLIC1 to an alanine at position 74 and a phenylalanine at positions 185 and 207. We examined the effect of the histidine-mediated pH dependence on the structure and global stability of CLIC1. None of the mutations were found to alter the global structure of the protein. However, the stability of H74A-CLIC1 and H185F-CLIC1, as calculated from the equilibrium unfolding data, is no longer dependent on pH because similar trends are observed at pH 7.0 and 5.5. The crystal structures show that the mutations result in changes in the local hydrogen bond coordination. Because the mutant total free energy change upon unfolding is not different from that of the wild type at pH 7.0, despite the presence of intermediates that are not seen in the wild type, we propose that it may be the stability of the intermediate state rather than the native state that is dependent on pH. On the basis of the lower stability of the intermediate in the H74A and H185F mutants compared to that of the wild type, we conclude that both His74 and His185 are involved in triggering the pH changes to the conformational stability of wild-type CLIC1 via their protonation, which stabilizes the intermediate state.

A C-terminal helix (α9) adjacent to the active site on each subunit is a structural feature unique to the alpha isoform of glutathione transferases which contributes to the catalytic and ligandin functions of the enzyme. The ionisation state of Tyr-9, a residue critical to catalysis, influences α9 dynamics, although the mechanism is poorly understood. In this study, isothermal titration calorimetry was used to probe the binding energetics of G-site (glutathione and glutathione sulfonate) and H-site (ethacrynic acid) ligands to wild-type and a Y9F mutant of human glutathione transferase A1-1. Although previous studies have reported a favourable entropic component to the binding of conjugates occupying both sites, our data reveal that ligand binding is enthalpically driven when either the G- or H-site is occupied independently. Also, heat capacity changes demonstrate that α9 is fully localised by H-site but not G-site occupation. The Tyr-9 hydroxyl group contributes significantly to ligand binding energetics, although the effect differs between the two binding sites. G-site binding is made slightly enthalpically more favourable and entropically less favourable by the Y9F mutation. Binding to the H-site is more dramatically affected, with the K d for ethacrynic acid increasing 5 fold despite a more favourable Δ S . The heat capacity change is more negative for G-site binding in the absence of the Tyr-9 hydroxyl (ΔΔ C p = − 0.73 kJ mol −1 K −1 ), but less negative for H-site binding to the Y9F mutant (ΔΔ C p = 0.63 kJ mol −1 K −1 ). This suggests that the relationship between Tyr-9 and α9 is not independent of the ligand. Rather, Tyr-9 appears to function in orienting the ligand optimally for α9 closure.

Rabies is an ancient and neglected zoonotic disease caused by the rabies virus, a neurotropic RNA virus that belongs to the Rhabdoviridae family, genus Lyssavirus. It remains an important public health problem as there are cost and health concerns imposed by the current human post exposure prophylaxis therapy. The use of monoclonal antibodies (mAbs) is therefore an attractive alternative. Rabies mostly affects people that reside in resource-limited areas where there are occasional failures in the cold-chain. These environmental changes may upset the stability of the mAbs. This study focused on mAbs 62-71-3 and E559; their structures, responses to freeze/thaw (F/T) and exposure to reactive oxygen species were therefore studied with the aid of a wide range of biophysical and in silico techniques in order to elucidate their stability and identify aggregation prone regions. E559 was found to be less stable than 62-71-3. The complementarity determining regions (CDR) contributed the most to its instability, more specifically: peptides (EIWD102)-E-99 and (92)ATSPYT(97) found in CDR3, Trp33 found in CDR1 and the oxidised Met34. The constant region "(158)SWNSGALTGHTFPAVL(175)" was also flagged by the special aggregation propensity (SAP) tool and F/T experiments to be highly prone to aggregation. The E559 peptides "(4)LQESGSVL(11) from the heavy chain and (4)LTQSPSSL(11) from the light chain, were also highly affected by F/T. These residues may serve as good candidates for mutation, in the aim to bring forward more stable therapeutic antibodies, thus paving a way to a more safe and efficacious antibody-based cocktail treatment against rabies.

Six Hsp70-like genes are represented on the genome of Plasmodium falciparum. Of these two occur in the cytosol: P. falciparum Hsp70-z (PfHsp70-z) and PfHsp70-1. PfHsp70-1 is a well characterised canonical Hsp70 that facilitates protein quality control and is crucial for the development of malaria parasites. There is very little known about PfHsp70-z. However, PfHsp70-z is known to be essential and is implicated in suppressing aggregation of asparagine-rich proteins of P. falciparum. In addition, its expression at the clinical stage of malaria correlates with disease prognosis. Based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins. Since Hsp110 proteins have been described as nucleotide exchange factors (NEFs) of their canonical Hsp70 counterparts, it has been speculated that PfHsp70-z may serve as a NEF of PfHsp70-1. In the current study, P. falciparum cells cultured in vitro were subjected to heat stress, triggering the enhanced expression of PfHsp70-z. Biochemical assays conducted using recombinant PfHsp70-z protein demonstrated that the protein is heat stable and possesses ATPase activity. Furthermore, we observed that PfHsp70-z is capable of self-association. The structural-functional features of PfHsp70-z provide further evidence for its role as a chaperone and possible nucleotide exchange factor of PfHsp70-1.

Canonical glutathione (GSH) transferases are dimeric proteins with subunits composed of an N-terminal GSH binding region (domain 1) and a C-terminal helical region (domain 2). The stabilities of several GSH transferase dimers are dependent upon two groups of interactions between domains 1 and 2 of opposing subunits: a hydrophobic ball-and-socket motif and a buried charge cluster motif. In rGSTM1-1, these motifs involve residues F56 and R81, respectively. The structural basis for the effects of mutating F56 to different residues on dimer stability and function has been reported (Codreanu et al. (2005) Biochemistry 44, 10605-10612). Here, we show that the simultaneous disruption of both motifs in the F56S/R81A mutant causes complete dissociation of the dimer to a monomeric protein on the basis of gel filtration chromatography and multiple-angle laser light scattering. The fluorescence and far-UV CD properties of the double mutant as well as the kinetics of amide H/D exchange along the polypeptide backbone suggest that the monomer has a globular structure that is similar to a single subunit in the native protein. However, the mutant monomer has severely impaired catalytic activity, suggesting that the dimer interface is vital for efficient catalysis. Backbone amide H/D exchange kinetics in the F56S and F56S/R81A mutants indicate that a reorganization of the loop structure between helix alpha2 and strand beta3 near the active site is responsible for the decreased catalytic activity of the monomer. In addition, the junction between the alpha4 and alpha5 helices in F56S/R81R shows decreased H/D exchange, indicating another structural change that may affect catalysis. Although the native subunit interface is important for dimer stability, urea-induced unfolding of the F56S/R81A mutant suggests that the interface is not essential for the thermodynamic stability of individual subunits. The H/D exchange data reveal a possible molecular basis for the folding cooperativity observed between domains 1 and 2.

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.

The C-terminal region in class alpha glutathione transferases (GSTs) modulates the catalytic and nonsubstrate ligand binding functions of these enzymes. Except for mouse GST A1-1 (mGST A1-1), the structures of class alpha GSTs have a bulky aliphatic side chain topologically equivalent to Ile219 in human GST A1-1 (hGST A1-1). In mGST A1-1, the corresponding residue is an alanine. To investigate the role of Ile219 in determining the conformational dynamics of the C-terminal region in hGST A1-1, the residue was replaced by alanine. The substitution had no effect on the global structure of hGST A1-1 but did reduce the conformational stability of the C-terminal region of the protein. This region could be stabilized by ligands bound at the active site. The catalytic behavior of hGST A1-1 was significantly compromised by the I219A mutation as demonstrated by reduced enzyme activity, increased K(m) for the substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB), and reduced catalytic efficiencies. Inhibition studies also indicated that the binding affinities for product and substrate analogues were dramatically decreased. The affinity of the mutant for GSH was, however, only slightly increased, indicating that the G-site was unaltered by the mutation. The binding affinity and stoichiometry for the anionic dye 8-anilino-1-naphthalene sulfonate (ANS) was also not significantly affected by the I219A mutation. However, the lower DeltaC(p) for ANS binding to the mutant (-0.34 kJ/mol per K compared with -0.84 kJ/mol per K for the wild-type protein) suggests that ANS binding to the mutant results in the burial of less hydrophobic surface area. Fluorescence data also indicates that ANS bound to the mutant is more prone to quenching by water. Overall, the data from this study, together with the structural details of the C-terminal region in mGST A1-1, show that Ile219 is an important structural determinant of the stability and dynamics of the C-terminal region of hGST A1-1.

The thioredoxin-like fold has a betaalphabetaalphabetabetaalpha topology, and most proteins/domains with this fold have a topologically conserved cis -proline residue at the N-terminus of beta-strand 3. This residue plays an important role in the catalytic function and stability of thioredoxin-like proteins, but is reported not to contribute towards the stability of glutathione S-transferases (GSTs) [Allocati, Casalone, Masulli, Caccarelli, Carletti, Parker and Di Ilio (1999) FEBS Lett. 445, 347-350]. In order to further address the role of the cis -proline in the structure, function and stability of GSTs, cis -Pro-56 in human GST (hGST) A1-1 was replaced with a glycine, and the properties of the P56G mutant were compared with those of the wild-type protein. Not only was the catalytic function of the mutant dramatically reduced, so was its conformational stability, as indicated by equilibrium unfolding and unfolding kinetics experiments with urea as denaturant. These findings are discussed in the context of other thioredoxin-like proteins.

Heat shock proteins (Hsps), amongst them, Hsp70 and Hsp90 families, serve mainly as facilitators of protein folding (molecular chaperones) of the cell. The Hsp70 family of proteins represents one of the most important molecular chaperones in the cell. Plasmodium falciparum, the main agent of malaria, expresses six Hsp70 isoforms. Two (PfHsp70-1 and PfHsp70-z) of these localize to the parasite cytosol. PHsp70-1 is known to occur in a functional complex with another chaperone, PfHsp90 via a co-chaperone, P. falciparum Hsp70-Hsp90 organising protein (PfHop). (−)-Epigallocatechin-3-gallate (EGCG) is a green tea constituent that is thought to possess antiplasmodial activity. However, the mechanism by which EGCG exhibits antiplasmodial activity is not fully understood. A previous study proposed that EGCG binds to the N-terminal ATPase domain of Hsp70. In the current study, we overexpressed and purified recombinant forms of two P. falciparum cytosol localized Hsp70s (PfHsp70-1 and PfHsp70-z), and PfHop, a co-chaperone of PfHsp70-1. Using the surface plasmon resonance approach, we demonstrated that EGCG directly binds to the two Hsp70s. We further observed that binding of EGCG to the two proteins resulted in secondary and tertiary conformational changes. In addition, EGCG inhibited the ATPase and chaperone function of the two proteins. Furthermore, EGCG abrogated association of the two Hsp70s with their functional partners. Using parasites cultured in vitro at the blood stages, we observed that 2.9 µM EGCG suppressed 50% P. falciparum parasite growth (IC50). Our findings demonstrate that EGCG directly binds to PfHsp70-1 and PfHsp70-z to inhibit both the ATPase and chaperone functions of the proteins. Our study constitutes the first direct evidence suggesting that the antiplasmodial activity of EGCG is at least in part accounted for by its inhibition of Hsp70 function.

The eukaryotic elongation factor 1 gamma (eEF1γ) is a multi-domain protein, which consist of a glutathione transferase (GST)-like N-terminus domain. In association with α, β and δ subunits, eEF1γ forms part of the eukaryotic elongation factor complex, which is mainly involved in protein biosynthesis. The N-terminus GST domain of eEF1γ interacts with the β subunit. eEF1γ subunit is over-expressed in human carcinoma. The role of human eEF1γ (heEF1γ) is poorly understood. A successful purification of recombinant heEF1γ is the first step towards determining unknown properties of the protein, including putative GST-like activities and the structure of the protein. This paper describes the over-expression, purification and characterisation of recombinant full-length, and the N- and C-terminus domains of heEF1γ. All three recombinant heEF1γ constructs over-expressed in the soluble Escherichia coli cell fraction and were purified to homogeneity. Secondary structure analysis indicates that the heEF1γ constructs have high α-helical structural character. The full-length and N-terminus domain are dimeric, while the C-terminus is monomeric. Both full-length and N-terminus domain interact with 8-anilino-1-naphthalene sulfonate (ANS) with KD = 70.0 (±5.7) μM and with reduced glutathione (GSH). Glutathione sulfonate displaced ANS bound to hydrophobic binding sites in the recombinant N-terminus domain. Using the standard GSH-1-chloro-2,4-dinitrobenzene conjugation assay, the N-domain showed some enzyme activity (0.03 μmol min−1 mg−1 protein), while the full-length heEF1γ did not catalyse the GSH-CDNB conjugation. Consequently, we hypothesize the presence of a presumed GST-like active site structure in the heEF1γ, which comprises a glutathione binding site and a hydrophobic substrate binding site.

The chloride intracellular channel protein 1 (CLIC1) is unique among eukaryotic ion channels in that it can exist as either a soluble monomer or an integral membrane channel. CLIC1 contains no known membrane-targeting signal sequences and the environmental factors which promote membrane binding of the transmembrane domain (TMD) are poorly understood. Here we report a positively charged motif at the C-terminus of the TMD and show that it enhances membrane partitioning and insertion. A 30-mer TMD peptide was synthesized in which the positively charged motif was replaced by three glutamate residues. The peptide was examined in 2,2,2-trifluoroethanol (TFE), sodium dodecyl sulfate micelles and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes using size-exclusion chromatography, far-UV CD, and fluorescence spectroscopy. The motif appears to enhance membrane interaction via electrostatic contacts and functions as an electrostatic plug to anchor the TMD in membranes. In addition, the motif is also involved in orientating the TMD with respect to the cis and trans faces of the membrane. These findings shed light on the intrinsic and environmental factors that promote the spontaneous conversion of CLIC1 from a water-soluble to a membrane-bound protein.

The folding and assembly of the dimeric glutathione transferases (GST) involves the association of two structurally distinct domains per subunit. A prominent and conserved domain–domain interaction in class α GSTs is formed by the packing of the indole side chain of Trp-20 from domain I into a hydrophobic pocket in domain II. Stability studies have shown that partial dissociation of the domains near Trp-20 occurs as an initial fast event during the unfolding kinetics of human GSTA1-1 (Wallace et al., Biochemistry 37 (1998) 5320–5328; Wallace et al., Biochem. J. 336 (1998) 413–418). The contribution of Trp-20 toward stabilising the domain–domain interface was investigated by mutating it to either a phenylalanine (W20F) or alanine (W20A) and determining the functionality (catalysis and non-substrate ligand binding) and stability (thermal- and urea-induced denaturation) of the mutant proteins. The replacement of Trp-20 did not impact on the protein’s gross structural properties. Functionally, the W20F was non-disruptive, whereas the cavity-creating W20A mutation was. Both mutants destabilised the native state with W20A exerting the greatest effect. Reduced m -values as well as the protein concentration dependence of the urea unfolding transitions for W20F GSTA1-1 suggest the presence of a dimeric intermediate at equilibrium that is not observed with wild-type protein. Unfolding kinetics monitored by stopped-flow tyrosine fluorescence was mono-exponential and corresponded to the global unfolding of the protein during which the dimeric intermediate unfolds to two unfolded monomers. The similar unfolding kinetics data for wild-type and W20F A1-1 indicates that the global unfolding event was not affected by amino acid replacement. We propose that the packing interactions at the conserved Trp-20 plays an important role in stabilising the intrasubunit domain I–domain II interface of class α GSTs.

We have shown previously that the solvent-induced equilibrium unfolding mechanism of class Sigma glutathione S-transferase (GST) is strongly affected by ionic strength [Stevens, Hornby, Armstrong and Dirr (1998) Biochemistry 37, 15534-15541]. The protein is dimeric and has a hydrophilic subunit interface. Here we show that ionic strength alone has significant effects on the conformation of the protein, in particular at the active site. With the use of NaCl at up to 2 M under equilibrium conditions, the protein lost 60% of its catalytic activity and the single tryptophan residue per subunit became partly exposed. The effect was independent of protein concentration, eliminating the dissociation of the dimer as a possibility for the conformational changes. This was confirmed by size-exclusion HPLC. There was no significant change in the secondary structure of the protein according to far-UV CD data. Manual-mixing and stopped-flow kinetics experiments showed a slow single-exponential salt-induced change in protein fluorescence. For equilibrium and kinetics experiments, the addition of an active-site ligand (S-hexylglutathione) completely protected the protein from the ionic-strength-induced conformational changes. This suggests that the change occurs at or near the active site. Possible structural reasons for these novel effects are proposed, such as the flexibility of the α-helix 2 region as well as the hydrophilic subunit interface, highlighting the importance of electrostatic interactions in maintaining the structure of the active site of this GST.

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone homologous with the human heat shock cognate protein 70 (hsc70)/heat shock protein 90 (hsp90)-organizing protein (Hop). The concomitant interaction of mSTI1 with hsp70 and hsp90 at its N- and C-termini respectively is mediated by the tetratricopeptide repeat (TPR) motifs in these regions. With the use of co-precipitation assays, we show here that the N-terminal TPR domain of mSTI1 without extensive flanking regions is both necessary and sufficient to mediate a specific interaction with hsc70. In contrast, other TPR-containing co-chaperones require TPR flanking regions for target substrate recognition, suggesting different mechanisms of TPR-mediated chaperone-co-chaperone interactions. Furthermore, the interaction between mSTI1 and hsc70 was analysed to ascertain the effect of replacing or deleting conserved amino acid residues and sequences within the three TPR motifs constituting the N-terminal TPR domain of full-length mSTI1. Replacement of a bulky hydrophobic residue in TPR1 disrupted the interaction of mSTI1 with hsc70. A highly conserved sequence in TPR2 was altered by deletion or single amino acid replacement. These derivatives retained a specific interaction with hsc70. These results are consistent with a model in which conserved residues within the N-terminal TPR region of mSTI1 contribute differentially to the interaction with hsc70, and in which TPR1 has a significant role in targeting mSTI1 to hsc70. The contribution of the TPR domain mutations and deletions are discussed with respect to their effect on target substrate interactions.

Glutathione transferases function as detoxification enzymes and ligand-binding proteins for many hydrophobic endogenous and xenobiotic compounds. The molecular mechanism of folding of urea-denatured homodimeric human glutathione transferase A1-1 (hGSTA1-1) was investigated. The kinetics of change were investigated using far-UV CD, Trp20 fluorescence, fluorescence-detected ANS binding, acrylamide quenching of Trp20 fluorescence, and catalytic reactivation. The very early stages of refolding (millisecond time range) involve the formation of structured monomers with native-like secondary structure and exposed hydrophobic surfaces that have a high binding capacity for the amphipathic dye ANS. Dimerization of the monomeric intermediates was detected using Trp fluorescence and occurs as fast and intermediate events. The intermediate event was distinguished from the fast event because it is limited by a preceding slow trans-to-cis isomerization reaction (optically silent in this study). At high concentrations of hFKBP, dimerization is not limited by the isomerization reaction, and only the fast event was detected. The fast (tau = 200 ms) and intermediate (tau = 2.5 s) events show similar urea-, temperature-, and ionic strength-dependent properties. The dimeric intermediate has a partially functional active site ( approximately 20%). Final reorganization to form the native tertiary and quaternary structures occurs during a slow, unimolecular, urea- and ionic strength-independent event. During this slow event (tau = 250 s), structural rearrangements at the domain interface occur at/near Trp20 and result in burial of Trp20. The slow event results in the regain of the fully functional dimer. The role of the C-terminus helix 9 (residues 210-221) as a structural determinant for this final event is proposed.

Helix 9 at the C-terminus of class alpha glutathione transferase (GST) polypeptides is a unique structural feature in the GST superfamily. It plays an important structural role in the catalytic cycle. Its contribution toward protein stability/folding as well as the binding of nonsubstrate ligands was investigated by protein engineering, conformational stability, enzyme activity, and ligand-binding methods. The helix9 sequence displays an unfavorable propensity toward helix formation, but tertiary interactions between the amphipathic helix and the GST seem to contribute sufficient stability to populate the helix on the surface of the protein. The helix's stability is enhanced further by the binding of ligands at the active site. The order of ligand-induced stabilization increases from H-site occupation, to G-site occupation, to the simultaneous occupation of H- and G-sites. Ligand-induced stabilization of helix9 reduces solvent accessible hydrophobic surface by facilitating firmer packing at the hydrophobic interface between helix and GST. This stabilized form exhibits enhanced affinity for the binding of nonsubstrate ligands to ligandin sites (i.e., noncatalytic binding sites). Although helix9 contributes very little toward the global stability of hGSTA1-1, its conformational dynamics have significant implications for the protein's equilibrium unfolding/refolding pathway and unfolding kinetics. Considering the high concentration of reduced glutathione in human cells (about 10 mM), the physiological form of hGSTA1-1 is most likely the thiol-complexed protein with a stabilized helix9. The C-terminus region (including helix9) of the class alpha polypeptide appears not to have been optimized for stability but rather for catalytic and ligandin function.

The binding interactions between dimeric human class alpha glutathione S-transferase A1-1 (GST A1-1) and aflatoxin B1 or sulphobromophthalein (BSP) were characterised. Aflatoxin B1 binds to GST A1-1 with a stoichiometry of 1.1 mol/mol of dimeric enzyme. The binding interaction, which can be described by a hyperbolic saturation isotherm (Kd = 8+/-2 microM), does not induce major structural changes in the enzyme, nor does it inhibit enzymatic activity. The average distance between the single tryptophan residue (Trp20) of GST A1-1 and protein-bound aflatoxin B1 was calculated to be 22.7 A by means of fluorescence resonance energy transfer. The aflatoxin-binding region, according to this calculated distance, was determined to be located in the dimer interface cleft near the crystallographic two-fold axis. Hill-plot analyses suggest that a positive co-operative interaction exists between BSP and the dimeric GST A1-1 (h = 1.6+/-0.1; K' = 14+/-0.6 microM). The binding of BSP induces a conformational change in the enzyme which is accompanied by a decrease in the molecular flexibility and in the solvent-accessible properties of the enzyme's Trp20 residue. Site-directed mutagenesis of Trp20 (Trp20-->Phe) confirms that this residue is situated in the binding environment and although it is not essential for BSP binding, it is involved in the interaction. Furthermore, the structural change associated with BSP binding alters the hyperbolic character of the glutathione saturation curve. This indicates that there may also be a cooperative interaction between glutathione and BSP or that BSP binding induces asymmetric functioning of the two enzyme subunits so that they become unequal in catalytic activity.

A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a delta G degree (H2O) = 26.0 +/- 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol.

HIV-1 protease (PR) is an obligate homodimer which plays a pivotal role in the maturation and hence propagation of HIV. Although successful developments on PR active site inhibitors have been achieved, the major limiting factor has been the emergence of HIV drug-resistant strains. Disruption of the dimer interface serves as an alternative mechanism to inactivate the enzyme. The terminal residue, F99, was mutated to an alanine to investigate its contribution to dimer stability in the South African HIV-1 subtype C (C-SA) PR. The F99A PR and wild-type C-SA PR were overexpressed and purified. The activities of the PRs and their ability to bind an active site inhibitor, acetyl-pepstatin, were determined in vitro. The F99A PR showed no activity and the inability to bind to the inhibitor. Secondary and quaternary structure analysis were performed and revealed that the F99A PR is monomeric with reduced β-sheet content. The mutation of F99 to alanine disrupted the presumed ‘lock-and-key’ motif at the terminal dimer interface, in turn creating a cavity at the N- and C-terminal antiparallel β-sheet. These findings support the design of inhibitors targeting the C-terminus of the C-SA PR, centered on interactions with the bulky F99.

S-Nitrosation is a post-translational modification of protein cysteine residues, which occurs in response to cellular oxidative stress. Although it is increasingly being linked to physiologically important processes, the molecular basis for protein regulation by this modification remains poorly understood. We used transient kinetic methods to determine a minimal mechanism for spontaneous S-nitrosoglutathione (GSNO)-mediated transnitrosation of human glutathione transferase (GST) P1-1, a major detoxification enzyme and key regulator of cell proliferation. Cys47 of GSTP1-1 is S-nitrosated in two steps, with the chemical step limited by a pre-equilibrium between the open and closed conformations of helix α2 at the active site. Cys101, in contrast, is S-nitrosated in a single step but is subject to negative cooperativity due to steric hindrance at the dimer interface. Despite the presence of a GSNO binding site at the active site of GSTP1-1, isothermal titration calorimetry as well as nitrosation experiments using S-nitrosocysteine demonstrate that GSNO binding does not precede S-nitrosation of GSTP1-1. Kinetics experiments using the cellular reductant glutathione show that Cys101-NO is substantially more resistant to denitrosation than Cys47-NO, suggesting a potential role for Cys101 in long term nitric oxide storage or transfer. These results constitute the first report of the molecular mechanism of spontaneous protein transnitrosation, providing insight into the post-translational control of GSTP1-1 as well as the process of protein transnitrosation in general. Background: S-Nitrosation is an emerging post-translational modification that is not yet well understood on a molecular level. Results: We propose a mechanism for S-nitrosation of glutathione transferase P1-1 by S-nitrosoglutathione. Conclusion: Cys101 is nitrosated in a single step, but Cys47 nitrosation is limited by the rate of helix 2 opening. Significance: Detailing the mechanism of spontaneous transnitrosation is crucial to understanding how protein S-nitrosation is controlled.

Chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. The transmembrane domain (TMD), implicated in membrane penetration and pore formation, comprises helix α1 and strand β2 of the N-domain of soluble CLIC1. The mechanism by which the TMD binds, inserts, and oligomerizes in membranes to form a functional chloride channel is unknown. Here we report the secondary, tertiary, and quaternary structural changes of the CLIC1 TMD as it partitions between an aqueous and membrane-mimicking environment. A synthetic 30-mer peptide comprising the TMD was examined in 2,2,2-trifluoroethanol, sodium dodecyl sulfate (SDS) micelles, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes using far-ultraviolet circular dichroism and fluorescence spectroscopy. Data obtained in the presence of SDS micelles and POPC liposomes show that Trp35 and Cys24 have reduced solvent accessibility, indicating that the peptide adopts an inserted orientation. The peptide assumes a helical structure in the presence of these mimetics, consistent with its predicted membrane conformation. This acquisition of secondary structure is concentration-dependent, suggesting an oligomerization event. Stable dimeric and trimeric species were subsequently identified using SDS-polyacrylamide gel electrophoresis. We propose that, in the vicinity of membranes, the mixed α/β TMD in CLIC1 rearranges to form a helix that then likely dimerizes via noncovalent helix-helix interactions to form a membrane-competent protopore complex. Such oligomerization would be essential for forming a functional ion channel, given that each CLIC1 monomer possesses only a single TMD. This work highlights the central role of the TMD in CLIC1 function: It is capable of promoting membrane insertion and dimerization in the absence of the C-domain and large portions of the N-domain.

FOXP2 is a member of the P subfamily of FOX transcription factors, the DNA-binding domain of which is the winged helix forkhead domain (FHD). In this work we show that the FOXP2 FHD is able to bind to various DNA sequences, including a novel sequence identified in this work, with different affinities and rates as detected using surface plasmon resonance. Combining the experimental work with molecular docking, we show that high-affinity sequences remain bound to the protein for longer, form a greater number of interactions with the protein and induce a greater structural change in the protein than low-affinity sequences. We propose a binding model for the FOXP2 FHD that involves three types of binding sequence: low affinity sites which allow for rapid scanning of the genome by the protein in a partially unstructured state; moderate affinity sites which serve to locate the protein near target sites and high-affinity sites which secure the protein to the DNA and induce a conformational change necessary for functional binding and the possible initiation of downstream transcriptional events.

JNK1 is activated by phosphorylation of the canonical T183 and Y185 residues, modifications that are catalysed typically by the upstream eukaryotic kinases MKK4 and MKK7. Nonetheless, the exact sites at which the most abundant JNK variant, JNK1β1, is further modified by MKK4 for phospho-regulation has not been previously investigated. Aiming to characterise the nature of JNK1β1 phosphorylation by active MKK4 using mass spectrometry, a recognised yet uncharacterised phospho-site (S377) as well as two novel phospho-residues (T228 and S284) were identified. Interestingly, the identical sites were phosphorylated during overexpression of JNK1β1 in Escherichia coli, raising important questions that have significant implications for heterologous protein expression.

Solvent-induced unfolding of porcine class pi glutathione S-transferase (pGST P1-1), a homodimeric protein, was monitored under equilibrium conditions using different physicochemical parameters (tryptophan fluorescence, anisotropy, degree of tyrosine exposure, binding of 8-anilino-1- naphthalenesulphonic acid, size-exclusion HPLC). The coincidence of unfolding curves obtained with functional (enzyme activity) and structural probes (anisotropy), the absence of thermodynamically stable intermediates such as a folded monomer (determined by binding of 8-anilino-1- naphthalenesulphonic acid and size-exclusion HPLC), and the dependence of pGST P1-1 stability upon protein concentration (measured with structural and functional probes), indicate a cooperative and concerted two-state unfolding transition between native dimeric pGST P1-1 and unfolded monomeric enzyme. [References: 48]

The c-Jun N-terminal kinase (JNK) pathway forms part of the mitogen-activated protein kinase (MAPK) signaling pathways comprising a sequential three-tiered kinase cascade. Here, an upstream MAP3K (MEKK1) phosphorylates and activates a MAP2K (MKK4 and MKK7), which in turn phosphorylates and activates the MAPK, JNK. The C-terminal kinase domain of MEKK1 (MEKK-C) is constitutively active, while MKK4/7 and JNK are both activated by dual phosphorylation of S/Y, and T/Y residues within their activation loops, respectively. While improvements in the purification of large quantities of active JNKs have recently been made, inadequacies in their yield, purity, and the efficiency of their phosphorylation still exist. We describe a novel and robust method that further improves upon the purification of large yields of highly pure, phosphorylated JNK1β1, which is most suitable for biochemical and biophysical characterization. Codon harmonization of the JNK1β1 gene was used as a precautionary measure toward increasing the soluble overexpression of the kinase. While JNK1β1 and its substrate ATF2 were both purified to >99% purity as GST fusion proteins using GSH-agarose affinity chromatography and each cleaved from GST using thrombin, constitutively-active MEKK-C and inactive MKK4 were separately expressed in E. coli as thioredoxin-His(6)-tagged proteins and purified using urea refolding and Ni(2+)-IMAC, respectively. Activation of JNK1β1 was then achieved by successfully reconstituting the JNK MAPK activation cascade in vitro; MEKK-C was used to activate MKK4, which in turn was used to efficiently phosphorylate and activate large quantities of JNK1β1. Activated JNK1β1 was thereafter able to phosphorylate ATF2 with high catalytic efficiency.

The ion channel protein CLIC1 exists in both a soluble conformation in the cytoplasm and a membrane-bound conformation. The conformational stability of soluble CLIC1 demonstrates pH sensitivity which may be attributable to very specific residues that function as pH sensors. These sensors could be histidine or glutamate residues with pK(a) values that fall within the physiological pH range. The role of Glu81, a member of a topologically conserved buried salt bridge in CLIC1, as a pH sensor was investigated here. The mutants E81M, R29M, and E81M/R29M were designed to break the salt bridge between Glu81 and Arg29 and examine the effect of each member on the stability of the protein. Spectroscopic studies and the solved crystal structures indicated that the global structure of CLIC1 was not affected by the mutations. Urea-induced equilibrium unfolding unexpectedly showed E81M to stabilize CLIC1 at pH 7. This was due to stabilizing hydrophobic interactions with Met81 and a water-mediated compensatory H-bond between Met81 and Arg29. R29M and E81M/R29M destabilized CLIC1 at pH 7, and the unfolding transition changed from two-state to three-state, mimicking the wild type at pH 5.5. This observation points out the significance of the salt bridge in stabilizing the native state. The total unfolding free energy change of E81M CLIC1 does not change with pH, implying that Glu81 forms one of a network of pH-sensor residues in CLIC1 responsible for destabilization of the native state. This allows detachment of the N-domain from the C-domain at low pH.

Crystal structures of cytosolic glutathione S-transferases (EC 2.5.1.18), complexed with glutathione or its analogues, are reviewed. The atomic models define protein architectural relationships between the different gene classes in the superfamily, and reveal the molecular basis for substrate binding at the two adjacent subsites of the active site. Considerable progress has been made in understanding the mechanism whereby the thiol group of glutathione is destabilized (lowering its pKa) at the active site, a rate-enhancement strategy shared by the soluble glutathione S-transferases.

The canonical glutathione transferase (GST) fold found in many monomeric and dimeric proteins consists of two domains that differ in structure and conformational dynamics. However, no evidence exists that the two domains unfold/fold independently at equilibrium, indicating the significance of interdomain interactions in governing cooperativity between domains. Bioinformatics analyses indicate the interdomain interface of the GST fold is large, predominantly hydrophobic with a high packing density explaining cooperative interdomain behavior. Structural alignments reveal a topologically conserved lock-and-key interaction across the domain interface in which a bulky hydrophobic residue ("key") protrudes from the surface of the N-domain and inserts into a pocket ("lock") in the C-domain. To better understand the molecular basis for the contribution of interdomain interactions toward cooperativity within the GST fold in the absence of any influence from quaternary interactions, studies were done with two monomeric GST proteins: Escherichia coli Grx2 (EcGrx2) and human CLIC1 (hCLIC1). Replacing the methionine "key" residue with alanine is structurally nondisruptive, whereas it significantly diminishes the folding cooperativity of both proteins. The loss in cooperativity between domains in the mutants is reflected by a change in the equilibrium folding mechanism from a wild-type two-state process to a three-state process, populating a stable folding intermediate.

Cytosolic glutathione transferases (GSTs) are major detoxification enzymes in aerobes. Each subunit has two distinct domains and an active site consisting of a G-site for binding GSH and an H-site for an electrophilic substrate. While the active site is located at the domain interface, the role of the stability of this interface in the catalytic function of GSTs is poorly understood. Domain 1 of class alpha GSTs has a conserved tryptophan (Trp21) in helix 1 that forms a major interdomain contact with helices 6 and 8 in domain 2. Replacing Trp21 with an alanine is structurally non-disruptive but creates a cavity between helices 1, 6 and 8 thus reducing the packing density and van der Waals contacts at the domain interface. This results in destabilization of the protein and a marked reduction in catalytic activity. While functionality at the G-site is not adversely affected by the W21A mutation, the H-site becomes more accessible to solvent and less favorable for the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB). Not only does the mutation result in a reduction in the energy for stabilizing the transition state formed in the SNAr reaction between the substrates GSH and CDNB, it also compromises the ability of the enzyme to form and stabilize a transition state analogue (Meisenheimer complex) formed between GSH and 1,3,5-trinitrobenzene (TNB). The study demonstrates that the stability of the domain–domain interface plays a role in mediating the catalytic functionality of the active site, particularly the H-site, of class alpha GSTs.

Cytosolic class pi glutathione transferase P1-1 (GSTP1-1) is associated with drug resistance and proliferative pathways because of its catalytic detoxification properties and ability to bind and regulate protein kinases. The native wild-type protein is homodimeric, and whereas the dimeric structure is required for catalytic functionality, a monomeric and not dimeric form of class pi GST is reported to mediate its interaction with and inhibit the activity of the pro-apoptotic enzyme c-Jun N-terminal kinase (JNK) [Adler, V., et al. (1999) EMBO J. 18, 1321-1334]. Thus, the existence of a stable monomeric form of wild-type class pi GST appears to have physiological relevance. However, there are conflicting accounts of the subunit's intrinsic stability since it has been reported to be either unstable [Dirr, H., and Reinemer, P. (1991) Biochem. Biophys. Res. Commun. 180, 294-300] or stable [Aceto, A., et al. (1992) Biochem. J. 285, 241-245]. In this study, the conformational stability of GSTP1-1 was re-examined by equilibrium folding and unfolding kinetics experiments. The data do not demonstrate the existence of a stable monomer but that unfolding of hGSTP1-1 proceeds via an inactive, nativelike dimeric intermediate in which the highly dynamic helix 2 is unfolded. Furthermore, molecular modeling results indicate that a dimeric GSTP1-1 can bind JNK. According to the available evidence with regard to the stability of the monomeric and dimeric forms of GSTP1-1 and the modality of the GST-JNK interaction, formation of a complex between GSTP1-1 and JNK most likely involves the dimeric form of the GST and not its monomer as is commonly reported.

The common fold shared by members of the glutathione-transferase (GST) family has a topologically conserved isoleucine residue at the N-terminus of helix 3 which is involved in the packing of helix 3 against two beta-strands in domain 1. The role of the isoleucine residue in the structure, function and stability of GST was investigated by replacing the Ile71 residue in human GSTA1-1 by alanine or valine. The X-ray structures of the I71A and I71V mutants resolved at 1.75 and 2.51 A, respectively, revealed that the mutations do not alter the overall structure of the protein compared with the wild type. Urea-induced equilibrium unfolding studies using circular dichroism and tryptophan fluorescence suggest that the mutation of Ile71 to alanine or valine reduces the stability of the protein. A functional assay with 1-chloro-2,4-dinitrobenzene shows that the mutation does not significantly alter the function of the protein relative to the wild type. Overall, the results suggest that conservation of the topologically conserved Ile71 maintains the structural stability of the protein but does not play a significant role in catalysis and substrate binding.

Arg15, conserved in class Alpha GSTs (glutathione transferases), is located at the interface between the G- and H-sites of the active site where its cationic guanidinium group might play a role in catalysis and ligand binding. Arg15 in human GSTA1-1 was replaced with a leucine and crystallographic, spectroscopic, thermodynamic and molecular docking methods were used to investigate the contribution made by Arg15 towards (i) the binding of glutathione (GSH) to the G-site, (ii) the p K a of the thiol group of GSH, (iii) the stabilization of an analog of the anionic transition state of the S N Ar reaction between 1-chloro-2,4-dinitrobenzene (CDNB) and GSH, and, (iv) the binding of the anionic non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to the H-site. While the R15L mutation substantially diminishes the CDNB–GSH conjugating activity of the enzyme, it has little effect on protein structure and stability. Arg15 does not contribute significantly towards the enzyme's affinity for GSH but does determine the reactivity of GSH by reducing the thiol's p K a from 7.6 to 6.6. The anionic σ -complex formed between GSH and 1,3,5-trinitrobenzene is stabilized by Arg15, suggesting that it also stabilizes the transition state formed in the S N Ar reaction between GSH and CDNB. The trinitrocyclohexadienate moiety of the σ -complex binds the H-site where the catalytic residue, Tyr9, was identified to hydrogen bond to an o -nitro group of the σ -complex. The affinity for ANS at the H-site is decreased about 3-fold by the R15L mutation implicating the positive electrostatic potential of Arg15 in securing the organic anion at this site.

The equilibrium unfolding transition of class pi glutathione S-transferase, a homodimeric protein, from porcine lung was monitored by spectroscopic methods (fluorescence emission and ultraviolet absorption), and by enzyme activity changes. Solvent (guanidine hydrochloride and urea)-induced denaturation is well described by a two-state model involving significant populations of only the folded dimer and unfolded monomer. Neither a folded, active monomeric form nor stable unfolding intermediates were detected. The conformational stability, delta Gu (H2O), of the native dimer was estimated to be about 25.3 +/- 2 kcal/mol at 20 degrees C and pH6.5.

CLIC proteins function as anion channels when their structures convert from a soluble form to an integral membrane form. While very little is known about the mechanism of the conversion process, channel formation and activity are highly pH-dependent. In this study, the structural properties and conformational stability of CLIC1 were determined as a function of pH in the absence of membranes to improve our understanding of how its conformation changes when the protein encounters the acidic environment at the surface of a membrane. Although the global conformation and size of CLIC1 are not significantly altered by pH in the range of 5.5-8.2, equilibrium unfolding studies reveal that the protein molecule becomes destabilized at low pH, resulting in the formation of a highly populated intermediate with a solvent-exposed hydrophobic surface. Unlike the intermediates formed by many soluble pore-forming proteins for their insertion into membranes, the CLIC1 intermediate is not a molten globule. Acid-induced destabilization and partial unfolding of CLIC1 involve helix alpha1 which is the major structural element of the transmembrane region. We propose that the acidic environment encountered by CLICs at the surface of membranes primes the transmembrane region in the N-domain, thereby lowering the energy barrier for the conversion of soluble CLICs to their membrane-inserted forms.

Glutaredoxin 2 (Grx2) from Escherichia coli is monomeric and an atypical glutaredoxin that takes part in the monothiol deglutathionylation of proteins. Unlike its orthologs, Grx2 is a larger molecule with a canonical glutathione transferase (GST) fold that consists of two structurally distinct domains, an N-terminal glutaredoxin domain and a C-terminal alpha-helical domain. While GSTs are dimeric proteins, the conformational stability and unfolding kinetics of Grx2 were investigated to establish the contribution made by the domain interface to the stability of the tertiary structure of GST-like proteins without any influence from quaternary interactions. Equilibrium unfolding transitions for Grx2, using urea as a denaturant, are monophasic and exhibit coincidence of the fluorescence and CD data indicative of a concerted loss or formation of tertiary and secondary structure. The data fit well to a two-state N U model with no evidence that an intermediate is being formed. The experimental m value [2.7 kcal mol (-1) (M urea) (-1)] is in excellent agreement with a predicted value of 2.5 kcal mol (-1) (M urea) (-1) based on the amount of surface area expected to become exposed during unfolding. These findings provide evidence that the two structurally distinct domains of Grx2 behave as a single cooperative folding unit. The unfolding kinetics are complex which, as a result of native-state heterogeneity, are characterized by two observable unfolding reactions that occur in parallel. A major population representing one distinct nativelike form unfolds on a fast track to denatured Grx2 with cis-Pro49. This is followed by a spectroscopically silent cis-trans proline isomerization reaction as determined by interrupted unfolding experiments. A minor population representing the other distinct nativelike form unfolds slowly with its rate being limited by an undetermined structural isomerization reaction. Further, there is no evidence indicating that unfolding proceeds via a high-energy intermediate that might suggest independent unfolding of the two nonidentical domains in Grx2. The kinetics data are, therefore, consistent with the existence of cooperativity between the domains, in agreement with the equilibrium data.

Molecular docking and ANS-displacement experiments indicated that 8-anilinonaphthalene sulfonate (ANS) binds the hydrophobic site (H-site) in the active site of dimeric class Mu rGST M1-1. The naphthalene moiety provides most of the van der Waals contacts at the ANS-binding interface while the anilino group is able to sample different rotamers. The energetics of ANS binding were studied by isothermal titration calorimetry (ITC) over the temperature range of 5–30 °C. Binding is both enthalpically and entropically driven and displays a stoichiometry of one ANS molecule per subunit (or H-site). ANS binding is linked to the uptake of 0.5 protons at pH 6.5. Enthalpy of binding depends linearly upon temperature yielding a Δ C p of − 80 ± 4 cal K − 1 mol − 1 indicating the burial of solvent-exposed nonpolar surface area upon ANS-protein complex formation. While ion-pair interactions between the sulfonate moiety of ANS and protein cationic groups may be significant for other ANS-binding proteins, the binding of ANS to rGST M1-1 is primarily hydrophobic in origin. The binding properties are compared with those of other GSTs and ANS-binding proteins.