Catalytic loop motion in human glutathione synthetase: A molecular modeling approach

Conformational changes of three flexible loops (G, A, and S) in human glutathione synthetase (hGS) arise to accommodate the substrates inside the active site. The crystal structure of hGS, a member of the ATP-grasp superfamily, has been reported only for the product-enzyme complex. To study the function of the hGS loops, molecular dynamics simulations are performed on three different conformational models: unbound enzyme, reactant-enzyme, and product-enzyme complex of hGS. The conformational changes among the three models are analyzed and the roles of the loops during the catalytic process are described. The modeled structures of hGS show that the central portions of the G- and A-loop have a double role in the reactant complex conformation: they bind the substrates and simultaneously interact with each other through an extensive network of hydrogen bonds. The present study proposes that these favorable loop-ligand and loop-loop interactions are required for opening and closing of the active site of hGS. Additionally, this research identifies important amino acid residues and explains their function within the catalytic loops of hGS.     

Aspartate 458 of human glutathione synthetase is important for cooperativity and active site structure

Human glutathione synthetase (hGS) catalyzes the second ATP-dependent step in the biosynthesis of glutathione (GSH) and is negatively cooperative to the γ-glutamyl substrate. The hGS active site is composed of three highly conserved catalytic loops, notably the alanine rich A-loop. Experimental and computational investigations of the impact of mutation of Asp458 are reported, and thus the role of this A-loop residue on hGS structure, activity, negativity cooperativity and stability is defined. Several Asp458 hGS mutants (D458A, D458N and D458R) were constructed using site-directed mutagenesis and their activities determined (10%, 15% and 7% of wild-type hGS, respectively). The Michaelis–Menten constant (K_m) was determined for all three substrates (glycine, GAB and ATP): glycine K_m increased by 30–115-fold, GAB K_m decreased by 8–17-fold, and the ATP K_m was unchanged. All Asp458 mutants display a change in cooperativity from negative cooperativity to non-cooperative. All mutants show similar stability as compared to wild-type hGS, as determined by differential scanning calorimetry. The findings indicate that Asp458 is essential for hGS catalysis and that it impacts the allostery of hGS.     

The effects of amino acid replacements of glycine 20 on conformational stability and catalysis of staphylococcal nuclease

Staphylococcal nuclease (SNase) is a well-established model for protein folding studies. Its three-dimensional structure has been determined. The enzyme, Ca2+, and DNA or RNA substrate form a ternary complex. Glycine 20 is the second position of the first beta-turn of SNase, which may serve as the folding initiation site for the SNase polypeptide. To study the role of Gly20 in the conformational stability and catalysis of SNase, three mutants, in which Gly20 was replaced by alanine, valine, or isoleucine, were constructed and studied by using circular dichroism spectra, intrinsic and ANS-binding fluorescence spectra, stability and activity assays. The mutations have little effect on the conformational integrity of the mutants. However, the catalytic activity is reduced drastically by the mutations, and the stability of the protein is progressively decreased in the order G20A相似文献   

Structural Basis for Evolution of Product Diversity in Soybean Glutathione Biosynthesis

The redox active peptide glutathione is ubiquitous in nature, but some plants also synthesize glutathione analogs in response to environmental stresses. To understand the evolution of chemical diversity in the closely related enzymes homoglutathione synthetase (hGS) and glutathione synthetase (GS), we determined the structures of soybean (Glycine max) hGS in three states: apoenzyme, bound to γ-glutamylcysteine (γEC), and with hGSH, ADP, and a sulfate ion bound in the active site. Domain movements and rearrangement of active site loops change the structure from an open active site form (apoenzyme and γEC complex) to a closed active site form (hGSH•ADP•SO₄²⁻ complex). The structure of hGS shows that two amino acid differences in an active site loop provide extra space to accommodate the longer β-Ala moiety of hGSH in comparison to the glycinyl group of glutathione. Mutation of either Leu-487 or Pro-488 to an Ala improves catalytic efficiency using Gly, but a double mutation (L487A/P488A) is required to convert the substrate preference of hGS from β-Ala to Gly. These structures, combined with site-directed mutagenesis, reveal the molecular changes that define the substrate preference of hGS, explain the product diversity within evolutionarily related GS-like enzymes, and reinforce the critical role of active site loops in the adaptation and diversification of enzyme function.     

Valine 44 and valine 45 of human glutathione synthetase are key for subunit stability and negative cooperativity

It was hypothesized that residues Val44 and Val45 serve as important residues for human glutathione synthetase (hGS) function and stability given their location at the dimer interface of this enzyme. Computational studies suggest that mutation at Val45 has more impact on the structure and stability of hGS than does mutation at Val44. Experimentally, enzymes with mutations at the 44 and or 45 positions of hGS were prepared, purified and assayed for initial activity. Val45 position mutations (either to alanine or tryptophan) have a greater impact on enzyme activity than do mutations at Val44. Differential scanning calorimetry experiments reveal a loss of stability in all mutant enzymes, with V45 mutations being less stable than the corresponding Val44 mutations. The γ-GluABA substrate affinity remains unaltered in V44A and V45A mutant enzymes, but increases when tryptophan is introduced at either of these positions. Hill coefficients trend towards less negative cooperativity with the exception of V45W mutant hGS. These results imply that residues V44 and V45 are located along the allosteric pathway of this negatively cooperative dimeric enzyme, that their mutation impacts the allosteric pathway more than it does the active site of hGS, and that these residues (and by extension the dimer interface in which they are located) are integral to the stability of human glutathione synthetase.     

The utility of molecular dynamics simulations for understanding site-directed mutagenesis of glycine residues in biotin carboxylase

Biotin carboxylase from Escherichia coli catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. Comparison of the crystal structures of biotin carboxylase in the absence and presence of ATP showed a central B-domain closure when ATP was bound. Peptidic NH groups from two active site glycine residues (Gly165 and Gly166) that form hydrogen bonds to the phosphate oxygens of ATP were postulated to act as a "trigger" for movement of the B-domain. The function of these two glycine residues in the catalytic mechanism was studied by disruption of the hydrogen bonds using site-directed mutagenesis. Both single (G165V) and (G166V) and double mutants (G165V-G166V) were constructed. The mutations did not affect the maximal velocity of a partial reaction, the bicarbonate-dependent ATPase activity. This suggests that the peptidic NH groups of Gly165 and Gly166 are not triggers for domain movement. However, the K(m) values for ATP for each of the mutants was increased over 40-fold when compared with wild-type indicating the peptidic NH groups of Gly165 and Gly166 play a role in binding ATP. Consistent with ATP binding, the maximal velocity for the biotin-dependent ATPase activity (i.e. the complete reaction) was decreased over 100-fold suggesting the mutations have misaligned the reactants for optimal catalysis. Molecular dynamics studies confirm perturbation of the hydrogen bonds from the mutated residues to ATP, whereas the double mutant exhibits antagonistic effects such that hydrogen bonding from residues 165 and 166 to ATP is similar to that in the wild-type. Consistent with the site-directed mutagenesis results the molecular dynamics studies show that ATP is misaligned in the mutants.     

Interhelical packing modulates conformational flexibility in the lactose permease of Escherichia coli

A key to obtaining an X-ray structure of the lactose permease of Escherichia coli (LacY) (Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H. R., and Iwata, S. (2003) Science 301, 549-716) was the use of a mutant in which Cys154 (helix V) is replaced with Gly. LacY containing this mutation strongly favors an inward-facing conformation, which binds ligand with high affinity, but catalyzes little transport and exhibits few if any of the ligand-dependent conformational changes observed with wild-type LacY. The X-ray structure demonstrates that helix V crosses helix I in the approximate middle of the membrane in such a manner that Cys154 lies close to Gly24 (helix I). Therefore, it seems likely that replacing Cys154 with Gly may lead to tighter packing between helices I and V, thereby resulting in the phenotype observed. Consistently, replacement of Gly24 with Cys in the C154G mutant rescues significant transport activity, and the mutant exhibits properties similar to wild-type LacY with respect to substrate binding and thermostability. However, the only other replacements that rescue transport to any extent whatsoever are Val and Asp, both of which are much less effective than Cys. The results suggest that, although helix packing probably plays an important role with respect to the properties of the C154G mutant, the ability of Cys at position 24 to rescue transport activity of C154G is more complicated than simple replacement of bulk between positions 24 and 154. Rather, activity is dependent on more subtle interactions between the helices, and mutations that disrupt interactions between helix IV and loop 6-7 or between helices II and IV also rescue transport in the C154G mutant.     

Single amino acid changes in domain II of Bacillus thuringiensis CryIAb delta-endotoxin affect irreversible binding to Manduca sexta midgut membrane vesicles.

Deletion of amino acid residues 370 to 375 (D2) and single alanine substitutions between residues 371 and 375 (FNIGI) of lepidopteran-active Bacillus thuringiensis CryIAb delta-endotoxin were constructed by site-directed mutagenesis techniques. All mutants, except that with the I-to-A change at position 373 (I373A), produced delta-endotoxin as CryIAb and were stable upon activation either by Manduca sexta gut enzymes or by trypsin. Mutants D2, F371A, and G374A lost most of the toxicity (400 times less) for M. sexta larvae, whereas N372A and I375A were only 2 times less toxic than CryIAb. The results of homologous and heterologous competition binding assays to M. sexta midgut brush border membrane vesicles (BBMV) revealed that the binding curves for all mutant toxins were similar to those for the wild-type toxin. However, a significant difference in irreversible binding was observed between the toxic (CryIAb, N372A, and I375A) and less-toxic (D2, F371A, and G374A) proteins. Only 20 to 25% of bound, radiolabeled CryIAb, N372A, and I375A toxins was dissociated from BBMV, whereas about 50 to 55% of the less-toxic mutants, D2, F371A, and G374A, was dissociated from their binding sites by the addition of excess nonlabeled ligand. Voltage clamping experiments provided further evidence that the insecticidal property (inhibition of short-circuit current across the M. sexta midgut) was directly correlated to irreversible interaction of the toxin with the BBMV. We have also shown that CryIAb and mutant toxins recognize 210- and 120-kDa peptides in ligand blotting. Our results imply that mutations in residues 370 to 375 of domain II of CrylAb do not affect overall binding but do affect the irreversible association of the toxin to the midgut columnar epithelial cells of M. sexta.     

In vitro characterization of four novel non-functional variants of the thiopurine S-methyltransferase

Human thiopurine S-methyltransferase (TPMT) is an enzyme responsible for the detoxification of widely used thiopurine drugs such as azathioprine (Aza). Its activity is inversely related to the risk of developing severe hematopoietic toxicity in certain patients treated with standard doses of thiopurines. DNA samples from four leucopenic patients treated with Aza were screened by PCR-SSCP analysis for mutations in the 10 exons of the TPMT gene. Four missense mutations comprising two novel mutations, A83T (TPMT*13, Glu(28)Val) and C374T (TPMT*12, Ser(125)Leu), and two previously described mutations, G430C (TPMT*10, Gly(144)Arg) and T681G (TPMT*7, His(227)Gln) were identified. Using a recombinant yeast expression system, kinetic parameters (K(m) and V(max)) of 6-thioguanine S-methylation of the four TPMT variants were determined and compared to those obtained with wild-type TPMT. This functional analysis suggests that these rare allelic variants are defective TPMT alleles. The His(227)Gln variant retained only 10% of the intrinsic clearance value (V(max)/K(m) ratio) of the wild-type enzyme. The Ser(125)Leu and Gly(144)Arg variants were associated with a significant decrease in intrinsic clearance values, retaining about 30% of the wild-type enzyme, whereas the Glu(28)Val variant produced a more modest decrease (57% of the wild-type enzyme). The data suggest that the sporadic contribution of the rare Glu(28)Val, Ser(125)Leu, Gly(144)Arg, and His(227)Gln variants may account for the occurrence of altered metabolism of TPMT substrates. These findings improve our knowledge of the genetic basis of interindividual variability in TPMT activity and would enhance the efficiency of genotyping methods to predict patients at risk of inadequate responses to thiopurine therapy.     

Effects of mutations at Gly114 on the stability and refolding of haloarchaeal nucleoside diphosphate kinase in low salt solution

We have shown before that mutation of Gly114 to Arg enhances folding of hexameric nucleoside diphosphate kinase (HsNDK) from Halobacterium salinarum. In this study, we constructed three mutant forms, Gly114Lys (G114K), Gly114Ser (G114S) and Gly114Asp (G114D), to further clarify the role residue 114 plays in the stability and folding of HsNDK. While expression of G114D mutant resulted in inactive enzyme, other mutant HsNDKs were successfully expressed in active form. The G114K mutant, similar to Gly114Arg (G114R) mutant, refolded in 1 M NaCl after heat-denaturation, under which the wild-type HsNDK and G114S proteins showed no refolding.     

Structural basis for decreased affinity of Emodin binding to Val66-mutated human CK2α as determined by molecular dynamics

Protein kinase CK2 (casein kinase 2) is a multifunctional serine/threonine kinase that is involved in a broad range of physiological events. The decreased affinity of Emodin binding to human CK2α resulting from single-point mutation of Val66 to Ala (V66A) has been demonstrated by experimental mutagenesis. Molecular dynamics (MD) simulations and energy analysis were performed on wild type (WT) and V66A mutant CK2α-Emodin complexes to investigate the subtle influences of amino acid replacement on the structure of the complex. The structure of CK2α and the orientation of Emodin undergo changes to different degrees in V66A mutant. The affected positions in CK2α are mainly distributed over the glycine-rich loop (G-loop), the α-helix and the loop located at the portion between G-loop and α-helix (C-loop). Based on the coupling among these segments, an allosteric mechanism among the C-loop, the G-loop and the deviated Emodin is proposed. Additionally, an estimated energy calculation and residue-based energy decomposition also indicate the lower instability of V66A mutant in contrast to WT, as well as the unfavorable energetic influences on critical residue contributions.     

Screening for and Verification of Novel Mutations Associated with Drug Resistance in the HIV Type 1subtype B′ in China

Objective

Mutations associated with HIV drug resistance have been extensively characterized at the HIV-1 polymerase domain, but more studies have verified that mutations outside of the polymerase domain also results in resistance to antiviral drugs. In this study, mutations were identified in 354 patients experiencing antiretroviral therapy (ART) failure and in 97 naïve-therapy patients. Mutations whose impact on antiviral drugs was unknown were verified by phenotypic testing.

Methods

Pol sequences of HIV subtype B^′ obtained from patients experiencing ART failure and from naïve-therapy patients were analyzed for mutations distinct between two groups. Mutations that occurred at a significantly higher frequency in the ART failure than the naïve-therapy group were submitted to the Stanford HIV Drug Resistance Database (SHDB) to analyze the correlation between HIV mutations and drug resistance. For mutations whose impact on the antiviral drug response is unknown, the site-directed mutagenesis approach was applied to construct plasmids containing the screened mutations. 50% inhibitory concentration (IC₅₀) to AZT, EFV and NVP was measured to determine the response of the genetically constructed viruses to antiviral drugs.

Results

7 mutations at 6 positions of the RT region, D123E, V292I, K366R, T369A, T369V, A371V and I375V, occurred more frequently in the ART failure group than the naïve-therapy group. Phenotypic characterization of these HIV mutants revealed that constructed viruses with mutations A371V and T369V exhibited dual resistance to AZT and EFV respectively, whereas the other 5 mutations showed weak resistance. Although the impact of the other six mutations on response to NVP was minimal, mutation T369V could enhance resistance to NVP.

Conclusions

This study demonstrated that mutations at the RT C-terminal in subtype B′ could result in resistance to RT inhibitors if the mutations occurred alone, but that some mutations could promote susceptibility to antiviral drugs.     

Membrane structure of the human immunodeficiency virus gp41 fusion peptide by molecular dynamics simulation. II. The glycine mutants

In this work, molecular dynamics (MD) simulation of the interaction of three mutants, G3V, G5V and G10V, of the human immunodeficiency virus (HIV) gp41 16-residue fusion peptide (FP) with an explicit palmitoyloleoylphosphatidyl-ethanolamine (POPE) lipid bilayer was performed. The goals of this work are to study the correlation of the fusogenic activity of the FPs with the mode of their interaction with the bilayer and to examine the roles of the many glycine residues in the FP in the fusion process. The results of this work corroborate the main conclusion of our earlier MD work of the WT FP and several mutants with polar substitution. These two studies provide correlation between the mode of insertion and the fusogenic activity of these peptides and support the hypothesis that an oblique insertion of the fusion domain of the viral protein is required for fusogenic activity. Inactive mutants interact with the bilayer by a surface-binding mode. The results of this work, combined with the results of our earlier work, show that, while the secondary structures of the wild-type FP and its mutants do not affect the fusogenic activities, the conformational flexibility appears to be an important factor. The active WT FP and its partially active mutants, G3V and G5V, all have significant conformational transitions at one of the glycine sites. They occur at Gly(5) in FP-wt, at Gly(10) in FP-G5V and at Gly(13) in FP-G3V. Thus, a glycine site in each of these active (or partially active) FPs provides conformational flexibility. On the other hand, the inactive mutants FP-G10V, FP-L9R and FP-V2E do not have any conformational transitions except at either terminus and thus possess no conformational flexibility. Thus, the results of this work support the suggestion that the role of glycine residues in the fusion domain is to provide the necessary conformational flexibility for fusion activity.The glycines also form a "glycine strip" in the FP that locates on one (the less hydrophobic) face of the helix (the "sided helix"). However, whether this "glycine strip" is disrupted or not does not seem to correlate with the retention of fusogenic activities. Finally, although the FLGFL (8-12) motif is absolutely conserved in the HIV fusion domain, a well-structured motif stabilized by hydrogen bonding does not appear to be required for activity. In fact, hydrogen bonding in this motif was found to be missing in FP-G3V and FP-G5V. Both of these mutants are partially active.     

The role of glutathione biosynthesis in heavy metal resistance in the fission yeast Schizosaccharomyces pombe

Abstract: Plants and the fission yeast Schizosaccharomyces pombe synthesize small cadmium-binding peptides, called phytochelatins, in response to cadmium. Derived from glutathione (GSH: λ-Glu-Cys-Gly), they have the general structure (λ-Glu-Cys) n Gly, where n is 2–11. In order to study the biosynthesis of phytochelatins, we used the mutagen N -methyl- N '-nitro- N nitrosoguanidine (MNNG) to select mutants with a lowered GSH content. GSH-deficient mutants show a Cd-sensitive phenotype, whereas resistance to Cu is only slightly influenced. These Cd-sensitive mutants contain 2–15% of the wild-type GSH level. For three mutants a lowered activity of λ-glutamylcysteine synthetase was measured. One of the mutants was transformed to Cd-resistance and the complementing fragment was analyzed further. The complementing fragment hybridized with chromosome III. In the transformants, GSH content was restored up to wild-type levels, whereas the activity of λ-glutamylcysteine synthetase was significantly increased compared with the wild-type. Possible mechanisms for Cd-resistance in the transformants are discussed.     

Discovery of a novel and potent class of anti-HIV-1 maturation inhibitors with improved virology profile against gag polymorphisms

A new class of betulin-derived α-keto amides was identified as HIV-1 maturation inhibitors. Through lead optimization, GSK8999 was identified with IC₅₀ values of 17 nM, 23 nM, 25 nM, and 8 nM for wild type, Q369H, V370A, and T371A respectively. When tested in a panel of 62 HIV-1 isolates covering a diversity of CA-SP1 genotypes including A, AE, B, C, and G using a PBMC based assay, GSK8999 was potent against 57 of 62 isolates demonstrating an improvement over the first generation maturation inhibitor BVM. The data disclosed here also demonstrated that the new α-keto amide GSK8999 has a mechanism of action consistent with inhibition of the proteolytic cleavage of CA-SP1.     

An andersen-Tawil syndrome mutation in Kir2.1 (V302M) alters the G-loop cytoplasmic K+ conduction pathway

Loss-of-function mutations in the inward rectifier potassium channel, Kir2.1, cause Andersen-Tawil syndrome (ATS-1), an inherited disorder of periodic paralysis and ventricular arrhythmias. Here, we explore the mechanism by which a specific ATS-1 mutation (V302M) alters channel function. Val-302 is located in the G-loop, a structure that is believed to form a flexible barrier for potassium permeation at the apex of the cytoplasmic pore. Consistent with a role in stabilizing the G-loop in an open conformation, we found the V302M mutation specifically renders the channel unable to conduct potassium without altering subunit assembly or attenuating cell surface expression. As predicted by the position of the Val-302 side chain in the crystal structure, amino acid substitution analysis revealed that channel activity and phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity are profoundly sensitive to alterations in the size, shape, and hydrophobicity of side chains at the Val-302 position. The observations establish that the Val-302 side chain is a critical determinant of potassium conduction through the G-loop. Based on our functional studies and the cytoplasmic domain crystal structure, we suggest that Val-302 may influence PIP2 gating indirectly by translating PIP2 binding to conformational changes in the G-loop pore.     

Site-directed mutagenesis of the phosphate-binding consensus sequence in Escherichia coli adenylosuccinate synthetase.

Adenylosuccinate synthetases from different sources contain an N-terminal glycine-rich sequence GDEGKGK, which is homologous to the conserved sequence GXXXXGK found in many other guanine nucleotide-binding proteins or enzymes. To determine the role of this sequence in the structure and function of Escherichia coli adenylosuccinate synthetase, site-directed mutagenesis was performed to generate five mutant enzymes: G12V (Gly12----Val), G15V (Gly15----Val), G17V (Gly17----Val), K18R (Lys18----Arg), and I19T (Ile19----Thr). Comparison of the kinetic properties of the wild-type enzyme and those of the mutant enzymes revealed that the sequence is critical for enzyme activity. Replacement of Gly12, Gly15, or Gly17 with Val, or replacement of Lys18 with Arg, resulted in significant decreases in the kcat/Km values of the enzyme. Because the consensus sequence GXXXXGK(T/S) has been found in many GTP-binding proteins, isoleucine at position 19 in the E. coli adenylosuccinate synthetase was changed to threonine to produce the sequence GDEGKGKT. This mutation, which more closely resembles the consensus sequence, resulted in a 160-fold increase in the Km value for substrate GTP; however, there were no great changes for the other two substrates, IMP and aspartate. Based on these data, we suggest that the N-terminal glycinerich sequence in E. coli adenylosuccinate synthetase plays a more important role in enzyme catalysis than in substrate binding. In addition, a hydrophobic amino acid residue such as isoleucine, leucine, or valine, rather than threonine, may play a critical role in GTP binding in adenosuccinate synthetase. These findings suggest that the glycine-rich sequence in adenylosuccinate synthetase functions differently relative to those in other GTP binding proteins or enzymes.     

Palmitoylation of the vasopressin V1a receptor reveals different conformational requirements for signaling, agonist-induced receptor phosphorylation, and sequestration

In this study, we establish that the V1a vasopressin receptor (V1aR) is palmitoylated, and we show that this modification has an important functional role. Palmitoylation of the V1aR occurs within the Cys371/Cys372 couplet located in the proximal C-terminal tail domain. Substitution of these residues in a [C371G/C372G]V1aR construct effectively disrupted receptor palmitoylation. Our data also indicate an additional palmitoylation site at another locus in the receptor, as yet undefined. [3H]Palmitate incorporation was agonist-sensitive and increased following exposure to [Arg8]vasopressin (AVP). Given the hydrophobic nature of the acyl chain, palmitoylation of the C terminus of G-protein-coupled receptors has been proposed to form an additional intracellular loop. Consequently, palmitoylation/depalmitoylation will have a profound effect on the local conformation of this domain. The V1aR palmitoylation status regulated both phosphorylation and sequestration of the receptor, and furthermore, palmitoylation, phosphorylation, and sequestration were all regulated by AVP. The palmitoylation-defective construct [C371G/C372G]V1aR exhibited decreased phosphorylation compared to wild-type V1aR, under both basal and AVP-stimulated conditions, and was sequestered at a faster rate. In contrast, the binding of four different classes of ligand and intracellular signaling were not affected by palmitoylation. This study therefore establishes that there are different conformational requirements for signaling, agonist-induced phosphorylation, and sequestration of the V1aR.