Molecular dynamics simulations of human glutathione transferase P1-1: conformational fluctuations of the apo-structure.

We have investigated by molecular dynamics simulations the conformational fluctuations of the monomer of human apo-glutathione transferase P1-1. After attainment of steady-state dynamics, the structural fluctuations involve mainly the protein segments that participate also in the holo-apo transition discussed in the accompanying article (Stella et al., 1999:37:1-9.). The most mobile region is the C-terminal segment of helix 2. In contrast, helices 1, 6, 7, and 8 constitute a relatively rigid protein core. An "essential dynamics" analysis of the simulation shows that the largest fluctuations involve specific regions of glutathione transferases. In such regions, atomic motions are correlated. Motions of helix 2 are accounted for by the second most prominent principal component, which reveals a fluctuation between two distinct conformations. The residues that constitute the H-site undergo a breathing motion, possibly relevant during the binding of hydrophobic cosubstrates. Based on our simulation, several experimental findings can be rationalized, including the viscosity-dependent reactivity of Cys 47 and Cys 101 as well as the selective proteolysis of the peptide bond between Lys 44 and Ala 45. We have also modeled the structural changes that lead to the formation of an intrachain disulfide bridge between cysteines 47 and 101 and to the inactivation of the enzyme. The resulting structure maintains essentially the native fold except for helix 2, which closes the G-site. Proteins 1999;37:10-19.     

S-Nitrosation of Glutathione Transferase P1-1 Is Controlled by the Conformation of a Dynamic Active Site Helix

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.     

Role of the C-terminal helix 9 in the stability and ligandin function of class alpha glutathione transferase A1-1

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.     

Recognition and Detoxification of the Insecticide DDT by Drosophila melanogaster Glutathione S-Transferase D1

GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 Å resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model of the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional ¹H,¹⁵N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.     

Ceramide-1-phosphate transfer protein promotes sphingolipid reorientation needed for binding during membrane interaction

Lipid transfer proteins acquire and release their lipid cargoes by interacting transiently with source and destination biomembranes. In the GlycoLipid Transfer Protein (GLTP) superfamily, the two-layer all-α-helical GLTP-fold defines proteins that specifically target sphingolipids (SLs) containing either sugar or phosphate headgroups via their conserved but evolutionarily-modified SL recognitions centers. Despite comprehensive structural insights provided by X-ray crystallography, the conformational dynamics associated with membrane interaction and SL uptake/release by GLTP superfamily members have remained unknown. Herein, we report insights gained from molecular dynamics (MD) simulations into the conformational dynamics that enable ceramide-1-phosphate transfer proteins (CPTPs) to acquire and deliver ceramide-1-phosphate (C1P) during interaction with 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. The focus on CPTP reflects this protein’s involvement in regulating pro-inflammatory eicosanoid production and autophagy-dependent inflammasome assembly that drives interleukin (IL-1β and IL-18) production and release by surveillance cells. We found that membrane penetration by CPTP involved α-6 helix and the α-2 helix N-terminal region, was confined to one bilayer leaflet, and was relatively shallow. Large-scale dynamic conformational changes were minimal for CPTP during membrane interaction or C1P uptake except for the α-3/α-4 helices connecting loop, which is located near the membrane interface and interacts with certain phosphoinositide headgroups. Apart from functioning as a shallow membrane-docking element, α-6 helix was found to adeptly reorient membrane lipids to help guide C1P hydrocarbon chain insertion into the interior hydrophobic pocket of the SL binding site.These findings support a proposed ‘hydrocarbon chain-first’ mechanism for C1P uptake, in contrast to the ‘lipid polar headgroup-first’ uptake used by most lipid-transfer proteins.     

Calorimetric and structural studies of the nitric oxide carrier S-nitrosoglutathione bound to human glutathione transferase P1-1

The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S-nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1-1 (hGSTP1-1) at 1.4 A resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild-type hGSTP1-1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild-type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S-nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix alpha2 of hGST P1-1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild-type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix alpha2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S-nitrosylation of Cys47 by GSNO is a likely physiological scenario.     

Localization of the C-terminus of rat glutathione S-transferase A1-1: crystal structure of mutants W21F and W21F/F220Y

Twelve C-terminal residues of human glutathione S-transferase A1-1 form a helix in the presence of glutathione-conjugate, or substrate alone, and partly cover the active site. According to X-ray structures, the helix is disordered in the absence of glutathione, but it is not known if it is helical and delocalized, or in a random-coil conformation. Mutation to a tyrosine of residue 220 within this helix was previously shown to affect the pK(a) of Tyr-9 at the active site, in the apo form of the enzyme, and it was proposed that an on-face hydrogen bond between Tyr-220 and Tyr-9 provided a means for affecting this pK(a). In the current study, X-ray structures of the W21F and of the C-terminal mutation, W21F/F220Y, with glutathione sulfonate bound, show that the C-terminal helix is disordered (or delocalized) in the W21F crystal but is visible and ordered in a novel location, a crystal packing crevice, in one of three monomers in the W21F/F220Y crystal, and the proposed hydrogen bond is not formed. Fluorescence spectroscopy studies using an engineered F222W mutant show that the C-terminus remains delocalized in the absence of glutathione or when only the glutathione binding site is occupied, but is ordered and localized in the presence of substrate or conjugate, consistent with these and previous crystallographic studies. Proteins 2001;42:192-200.     

Molecular and structural insight into the role of key residues of thrombospondin-1 and calreticulin in thrombospondin-1-calreticulin binding

Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on the cell surface signals focal adhesion disassembly, leading to the intermediate adhesive phenotype, cell migration, anoikis resistance, and collagen stimulation. Residues Lys 24 and 32 in TSP1 and amino acids 24-26 and 32-34 in CRT have been shown through biochemical and cell-based approaches to be critical for TSP1-CRT binding and signaling. This study investigated the molecular and structural basis for these key TSP1 and CRT residues in TSP1-CRT binding. On the basis of a validated TSP1-CRT complex structure, we adopted steered molecular dynamics simulations to determine the effect of mutation of these key residues on TSP1-CRT binding and validated the simulation results with experimental observations. We further performed 30 ns molecular dynamics simulations for wild-type TSP1, CRT, K24A/K32A mutant TSP1, and mutant CRT (residues 24-26 and 32-34 mutated to Ala) and studied the conformational and structural changes in TSP1 and CRT as the result of mutation of these critical residues. Results showed that mutation of residues 24 and 32 to Ala in TSP1 and of amino acids 24-26 and 32-34 to Ala in CRT results in a shortened β-strand in the binding site, decreased hydrogen bond occupancy for β-strand pairs that are located within or near the binding site, increased conformational flexibility of the binding site, a changed degree of dynamically correlated motion between the residues in the binding site and the other residues in protein, and a changed degree of overall correlated motion between the residues in the protein. These changes could directly contribute to the loss or weakened binding between TSP1 and CRT and the resultant effects on TSP1-CRT binding-induced cellular activities. Results from this study provide a molecular and structural insight into the role of these critical residues of TSP1 and CRT in TSP1-CRT binding.     

Engineering a new C-terminal tail in the H-site of human glutathione transferase P1-1: structural and functional consequences

We have sought the structural basis for the differing substrate specificities of human glutathione transferase P1-1 (class Pi) and human glutathione transferase A1-1 (class Alpha) by adding an extra helix (helix 9), found in the electrophilic substrate-binding site (H-site) of the human class Alpha enzyme, at the C terminus of the human class Pi enzyme. This class Pi-chimera (CODA) was expressed in Escherichia coli, purified and characterized by kinetic and crystallographic approaches. The presence of the newly engineered tail in the H-site of the human Pi enzyme alters its catalytic properties towards those exhibited by the human Alpha enzyme, as assessed using cumene hydroperoxide (diagnostic for class Alpha enzymes) and ethacrynic acid (diagnostic for class Pi) as co-substrates. There is a change of substrate selectivity in the latter case, as the k(cat)/K(m)(EA) value decreases about 70-fold, compared to that of class Pi. With 1-chloro-2,4-dinitrobenzene as co-substrate there is a loss of catalytic activity to about 2% with respect to that of the Pi enzyme. Crystallographic and kinetic studies of the class Pi-chimera provide important clues to explain these altered catalytic properties. The new helix forms many complimentary interactions with the rest of the protein and re-models the original electrophilic substrate-binding site towards one that is more enclosed, albeit flexible. Of particular note are the interactions between Glu205 of the new tail and the catalytic residues, Tyr7 and Tyr108, and the thiol moiety of glutathione (GSH). These interactions may provide an explanation of the more than one unit increase in the pK(a) value of the GSH thiolate and affect both the turnover number and GSH binding, using 1-chloro-2,4-dinitrobenzene as co-substrate. The data presented are consistent with the engineered tail adopting a highly mobile or disordered state in the apo form of the enzyme.     

Molecular Basis for the Dissociation Dynamics of Protein A-Immunoglobulin G1 Complex

Staphylococcus aureus protein A (SpA) is the most popular affinity ligand for immunoglobulin G1 (IgG1). However, the molecular basis for the dissociation dynamics of SpA-IgG1 complex is unclear. Herein, coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field were used to study the dissociation dynamics of the complex. The CG-MD simulations were first verified by the agreement in the structural and interactional properties of SpA and human IgG1 (hIgG1) in the association process between the CG-MD and all-atom MD at different NaCl concentrations. Then, the CG-MD simulation studies focused on the molecular insight into the dissociation dynamics of SpA-hIgG1 complex at pH 3.0. It is found that there are four steps in the dissociation process of the complex. First, there is a slight conformational adjustment of helix II in SpA. This is followed by the phenomena that the electrostatic interactions provided by the three hot spots (Glu143, Arg146 and Lys154) of helix II of SpA break up, leading to the dissociation of helix II from the binding site of hIgG1. Subsequently, breakup of the hydrophobic interactions between helix I (Phe132, Tyr133 and His137) in SpA and hIgG1 occurs, resulting in the disengagement of helix I from its binding site of hIgG1. Finally, the non-specific interactions between SpA and hIgG1 decrease slowly till disappearance, leading to the complete dissociation of the SpA-hIgG1 complex. This work has revealed that CG-MD coupled with the Martini force field is an effective method for studying the dissociation dynamics of protein-protein complex.     

Three-dimensional structure of class pi glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 A resolution.

The three-dimensional structure of human class pi glutathione S-transferase from placenta (hGSTP1-1), a homodimeric enzyme, has been solved by Patterson search methods and refined at 2.8 A resolution to a final crystallographic R-factor of 19.6% (8.0 to 2.8 A resolution). Subunit folding topology, subunit overall structure and subunit association closely resembles the structure of porcine class pi glutathione S-transferase. The binding site of a competitive inhibitor, S-hexylglutathione, is analyzed and the locations of the binding regions for glutathione (G-site) and electrophilic substrates (H-site) are determined. The specific interactions between protein and the inhibitor's glutathione peptide are the same as those observed between glutathione sulfonate and the porcine isozyme. The H-site is located adjacent to the G-site, with the hexyl moiety lying above a segment (residues 8 to 10) connecting strand beta 1 and helix alpha A where it is in hydrophobic contact with Tyr7, Phe8, Val10, Val35 and Tyr106. Catalytic models are discussed on the basis of the molecular structure.     

The structure of the complexes of DNA with non-histone chromosomal protein HMGB1 in the presence of manganese ions

The complexes of DNA - HMGB1 protein - manganese ions have been studied using circular dichroism (CD) technique. It was shown that in such three-component system the interactions of both the protein and metal ions with DNA differ from those in two-component complexes. The manganese ions do not affect the CD spectrum of free HMGB1 protein. However, Mn2+ ions induce considerable changes in the CD spectrum of free DNA in the spectral range of 260-290 nm. The presence of Mn2+ ions prevents formation of the ordered supramolecular structures specific for the HMGB1-DNA complexes. The interaction of manganese ions with DNA has a marked influence on the local DNA structure changing the properties of protein-binding sites. This results in the serious decrease in cooperativity of the DNA-protein binding. Such changes in the mode of the DNA-protein interactions occur at concentrations as small as 0.01 mM Mn2+. Moreover, the changes in local DNA structure induced by manganese ions promote the appearance of new HMGB1 binding sites on the DNA double helix. At the same time interactions with HMGB1 protein induce alterations in the structure of the DNA double helix which increase with a growth of the protein/DNA ratio. These alterations make the DNA/protein complex especially sensitive to manganese ions. Under these conditions the Mn2+ ions strongly affect the DNA structure that reflects in abrupt changes of the CD spectra of DNA in the complex in the range of 260-290 nm. Thus, structural changes of the DNA double helix in the three-component DNA-HMGB1-Mn2+ complexes come as a result of the combined and interdependent interactions of DNA with Mn2+ ions and the molecules of HMGB1.     

A molecular dynamics study of the ligand release path in yeast cytosine deaminase

Yeast cytosine deaminase, a zinc metalloenzyme, catalyzes the deamination of cytosine to uracil. Experimental and computational evidence indicates that the rate-limiting step is product release, instead of the chemical reaction step. In this work, we use molecular dynamics to suggest ligand exit paths. Simulation at 300 K shows that the active site is well protected by the C-terminal helix (residues 150-158) and F-114 loop (residues 111-117) and that on the molecular dynamics timescale water does not flow in or out of the active site. In contrast, simulation at 320 K shows a significant increase in flexibility of the C-terminal helix and F-114 loop. The motions of these two regions at 320 K open the active site and permit water molecules to diffuse into and out of the active site through two paths with one much more favored than the other. Cytosine is pushed out of the active site by a restraint method in two directions specified by these two paths. In path 1 the required motion of the protein is local-involving only the C-terminal helix and F-114 loop-and two residues, F-114 and I-156, are identified that have to be moved away to let cytosine out; whereas in path 2, the protein has to rearrange itself much more extensively, and the changes are also much larger compared to the path 1 simulation.     

Energetics of ligand binding to human glutathione transferase A1-1: Tyr-9 associated localisation of the C-terminal helix is ligand-dependent

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 ΔS. The heat capacity change is more negative for G-site binding in the absence of the Tyr-9 hydroxyl (ΔΔC(p)=-0.73 kJ mol(-1) K(-1)), but less negative for H-site binding to the Y9F mutant (ΔΔC(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.     

Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-A resolution: insight into the range of P450 conformations and the coordination of redox partner binding

A 1.9-A molecular structure of the microsomal cytochrome P450 2B4 with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI) in the active site was determined by x-ray crystallography. In contrast to the previous experimentally determined 2B4 structure, this complex adopted a closed conformation similar to that observed for the mammalian 2C enzymes. The differences between the open and closed structures of 2B4 were primarily limited to the lid domain of helices F through G, helices B' and C, the N terminus of helix I, and the beta(4) region. These large-scale conformational changes were generally due to the relocation of conserved structural elements toward each other with remarkably little remodeling at the secondary structure level. For example, the F' and G' helices were maintained with a sharp turn between them but are placed to form the exterior ceiling of the active site in the CPI complex. CPI was closely surrounded by residues from substrate recognition sites 1, 4, 5, and 6 to form a small, isolated hydrophobic cavity. The switch from open to closed conformation dramatically relocated helix C to a more proximal position. As a result, heme binding interactions were altered, and the putative NADPH-cytochrome P450 reductase binding site was reformed. This suggests a structural mechanism whereby ligand-induced conformational changes may coordinate catalytic activity. Comparison of the 2B4/CPI complex with the open 2B4 structure yields insights into the dynamics involved in substrate access, tight inhibitor binding, and coordination of substrate and redox partner binding.     

Tetanus Toxin and Botulinum A and C Neurotoxins Inhibit Noradrenaline Release from Cultured Mouse Brain

The specific binding protein for substance P (SP) was solubilized in an active form from the crude mitochondrial (P2) fraction of bovine brainstem. After incubation with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and 0.1 M NaCl at 0 degrees C for 30 min, the SP binding to the supernatant fraction (100,000 g, 60 min) was determined by the glass fiber filtration method reported by Bruns et al. (1983). The specific [3H]SP binding to the solubilized fraction was highly specific for SP and was displaced by nanomolar concentrations of SP and physalaemin, but only by micromolar concentrations of eledoisin. In addition, the binding was inhibited by GTP (approximately 40% of the specific binding decreased by 10 microM GTP) in both preparations. These results were virtually identical to those of P2 membrane preparations and suggested that this high-affinity SP binding site belongs to the SP-P type. Scatchard analyses of SP binding to the solubilized fraction revealed a single saturable component with a Bmax of 22.0 +/- 5.10 fmol/mg protein and a KD of 0.79 nM, and these values are almost the same as those obtained in the P2 fraction (Bmax = 31.3 +/- 3.56 fmol/mg protein, KD = 0.82 nM). Gel filtration analysis showed that the detergent-SP binding protein complex has two calculated molecular weights of greater than 1,000,000 and 55,000-60,000 (a corresponding Stokes radius of 35.5 nm).     

Interaction of hemin with placental glutathione transferase

To verify a possible involvement of glutathione transferase pi in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with Kd values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase pi was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The Ki calculated was 4 microM and the inhibition appeared to be non-competitive with respect to 1-chloro-2,4-dinitrobenzene. CD spectra of the bilirubin-glutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest the involvement of glutathione transferase pi in the hemin transport.