Modulating catalytic activity by unnatural amino acid residues in a GSH-binding loop of GST P1-1

The loop following helix α2 in glutathione transferase P1-1 has two conserved residues, Cys48 and Tyr50, important for glutathione (GSH) binding and catalytic activity. Chemical modification of Cys48 thwarts the catalytic activity of the enzyme, and mutation of Tyr50 generally decreases the k_cat value and the affinity for GSH in a differential manner. Cys48 and Tyr50 were targeted by site-specific mutations and chemical modifications in order to investigate how the α2 loop modulates GSH binding and catalysis. Mutation of Cys48 into Ala increased K_M^GSH 24-fold and decreased the binding energy of GSH by 1.5 kcal/mol. Furthermore, the protein stability against thermal inactivation and chemical denaturation decreased. The crystal structure of the Cys-free variant was determined, and its similarity to the wild-type structure suggests that the mutation of Cys48 increases the flexibility of the α2 loop rather than dislocating the GSH-interacting residues. On the other hand, replacement of Tyr50 with Cys, producing mutant Y50C, increased the Gibbs free energy of the catalyzed reaction by 4.8 kcal/mol, lowered the affinity for S-hexyl glutathione by 2.2 kcal/mol, and decreased the thermal stability. The targeted alkylation of Cys50 in Y50C increased the affinity for GSH and protein stability. Characterization of the most active alkylated variants, S-n-butyl-, S-n-pentyl-, and S-cyclobutylmethyl-Y50C, indicated that the affinity for GSH is restored by stabilizing the α2 loop through positioning of the key residue into the lock structure of the neighboring subunit. In addition, k_cat can be further modulated by varying the structure of the key residue side chain, which impinges on the rate-limiting step of catalysis.     

Penicillin-binding protein 2x of Streptococcus pneumoniae: three new mutational pathways for remodelling an essential enzyme into a resistance determinant

Mutations in the transpeptidase domain of penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae that reduce the affinity to beta-lactams are important determinants of resistance to these antibiotics. We have now analyzed in vitro and in vivo properties of PBP2x variants from cefotaxime-resistant laboratory mutants and a clinical isolate. The patterns of two to four resistance-specific mutations present in each of the proteins, all of which are placed between 6.6 and 24 Å around the active site, fall into three categories according to their positions in the three-dimensional structure. The first PBP2x group is characterized by mutations at the end of helix α11 and carries the well-known T550A change and/or one mutation on the surface of the penicillin-binding domain in close contact with the C-terminal domain. All group I proteins display very low acylation efficiencies, ≤ 1700 M^− 1 s^− 1, for cefotaxime. The second class represented by PBP2x of the mutant C505 shows acylation efficiencies below 100 M^− 1 s^− 1 for both cefotaxime and benzylpenicillin and contains the mutation L403F at a critical site close to the active serine. PBP2x of the clinical isolate 669 reveals a third mutational pathway where at least the two mutations Q552E and S389L are important for resistance, and acylation efficiency is reduced for both beta-lactams to around 10,000 M^− 1 s^− 1. In each group, at least one mutation is located in close vicinity to the active site and mediates a resistance phenotype in vivo alone, whereas other mutations might exhibit secondary effects only in context with other alterations.     

Solution Conformation and Dynamics of the HIV-1 Integrase Core Domain

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a critical enzyme involved in infection. It catalyzes two reactions to integrate the viral cDNA into the host genome, 3′ processing and strand transfer, but the dynamic behavior of the active site during catalysis of these two processes remains poorly characterized. NMR spectroscopy can reveal important structural details about enzyme mechanisms, but to date the IN catalytic core domain has proven resistant to such an analysis. Here, we present the first NMR studies of a soluble variant of the catalytic core domain. The NMR chemical shifts are found to corroborate structures observed in crystals, and confirm prior studies suggesting that the α4 helix extends toward the active site. We also observe a dramatic improvement in NMR spectra with increasing MgCl₂ concentration. This improvement suggests a structural transition not only near the active site residues but also throughout the entire molecule as IN binds Mg²⁺. In particular, the stability of the core domain is linked to the conformation of its C-terminal helix, which has implications for relative domain orientation in the full-length enzyme. ¹⁵N relaxation experiments further show that, although conformationally flexible, the catalytic loop of IN is not fully disordered in the absence of DNA. Indeed, automated chemical shift-based modeling of the active site loop reveals several stable clusters that show striking similarity to a recent crystal structure of prototype foamy virus IN bound to DNA.     

Dynamic conformational equilibria in the physiological function of the Bombyx mori pheromone-binding protein

The Bombyx mori pheromone-binding protein (BmorPBP) undergoes a pH-dependent conformational transition from a form at basic pH, which contains an open cavity suitable for ligand binding (BmorPBP^B), to a form at pH 4.5, where this cavity is occupied by an additional helix (BmorPBP^A). This helix α7 is formed by the C-terminal dodecapeptide 131-142, which is flexibly disordered on the protein surface in BmorPBP^B and in its complex with the pheromone bombykol. Previous work showed that the ligand-binding cavity cannot accommodate both bombykol and helix α7. Here we further investigated mechanistic aspects of the physiologically crucial ejection of the ligand at lower pH values by solution NMR studies of the variant protein BmorPBP(1-128), where the C-terminal helix-forming tetradecapeptide is removed. The NMR structure of the truncated protein at pH 6.5 corresponds closely to BmorPBP^B. At pH 4.5, BmorPBP(1-128) maintains a B-type structure that is in a slow equilibrium, on the NMR chemical shift timescale, with a low-pH conformation for which a discrete set of ¹⁵N-¹H correlation peaks is NMR unobservable. The full NMR spectrum was recovered upon readjusting the pH of the protein solution to 6.5. These data reveal dual roles for the C-terminal tetradecapeptide of BmorPBP in the mechanism of reversible pheromone binding and transport, where it governs dynamic equilibria between two locally different protein conformations at acidic pH and competes with the ligand for binding to the interior cavity.     

Developmental studies on the Sigma and Delta-1 glutathione transferases of Lucilia cuprina

The glutathione transferases (GSTs) are a large group of enzymes having both detoxication roles and specialist metabolic functions. The present work represents an initial approach to identifying some of these roles by examining the variation of specific members of the family under differing conditions. The GSTs from Lucilia cuprina have been partially purified, members of two families being isolated, by the use of glutathione immobilised on epichlorhydrin-activated Sepharose 6B. The GSTs were separated by 2D SDS-PAGE and characterised by MALDI-TOF analysis of tryptic peptides. The mass fragments were then matched against the corresponding Drosophila melanogaster and Musca domestica sequences. GSTs were identified as coming from only the Sigma and Delta classes. The multiple Delta zones appear all to be derived from the Lucilia GSTD1 isoform. The distribution of these GST proteins has been studied during different developmental stages of the insect. Delta isoforms were present in all developmental stages of L. cuprina. The Sigma GST was not detectable in the egg, was just detectable in the larval and pupal stages and was the major GST isolated in the adult. Sigma and Delta isoforms were both found in all body segments of the insect. Both isoforms appear to undergo extensive post-translational modification. Activities of the two types of protein with model substrates have been determined.     

Structure of a Drosophila sigma class glutathione S-transferase reveals a novel active site topography suited for lipid peroxidation products

Insect glutathione-S-transferases (GSTs) are grouped in three classes, I, II and recently III; class I (Delta class) enzymes together with class III members are implicated in conferring resistance to insecticides. Class II (Sigma class) GSTs, however, are poorly characterized and their exact biological function remains elusive. Drosophila glutathione S-transferase-2 (GST-2) (DmGSTS1-1) is a class II enzyme previously found associated specifically with the insect indirect flight muscle. It was recently shown that GST-2 exhibits considerable conjugation activity for 4-hydroxynonenal (4-HNE), a lipid peroxidation product, raising the possibility that it has a major anti-oxidant role in the flight muscle. Here, we report the crystal structure of GST-2 at 1.75A resolution. The GST-2 dimer shows the canonical GST fold with glutathione (GSH) ordered in only one of the two binding sites. While the GSH-binding mode is similar to other GST structures, a distinct orientation of helix alpha6 creates a novel electrophilic substrate-binding site (H-site) topography, largely flat and without a prominent hydrophobic-binding pocket, which characterizes the H-sites of other GSTs. The H-site displays directionality in the distribution of charged/polar and hydrophobic residues creating a binding surface that explains the selectivity for amphipolar peroxidation products, with the polar-binding region formed by residues Y208, Y153 and R145 and the hydrophobic-binding region by residues V57, A59, Y211 and the C-terminal V249. A structure-based model of 4-HNE binding is presented. The model suggest that residues Y208, R145 and possibly Y153 may be key residues involved in catalysis.     

Structural and Functional Analysis of Human SIRT1

SIRT1 is a NAD⁺-dependent deacetylase that plays important roles in many cellular processes. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an open apo form and a closed conformation in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a β hairpin structure that complements the β sheet of the NAD⁺-binding domain, covering an essentially invariant hydrophobic surface. The apo form adopts a distinct open conformation, in which the smaller subdomain of SIRT1 undergoes a rotation with respect to the larger NAD⁺-binding subdomain. A biochemical analysis identifies key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain.     

Structure of the extracellular glutathione S-transferase OvGST1 from the human pathogenic parasite Onchocerca volvulus

Onchocerciasis or river blindness, caused by the filarial worm Onchocerca volvulus, is the world’s second leading infectious cause of blindness. In order to chronically infect the host, O. volvulus has evolved molecular strategies that influence and direct immune responses away from the modes most damaging to it. The O. volvulus GST1 (OvGST1) is a unique glutathione S-transferase (GST) in that it is a glycoprotein and possesses a signal peptide that is cleaved off in the process of maturation. The mature protein starts with a 25-amino-acid extension not present in other GSTs. In all life stages of the filarial worm, it is located directly at the parasite-host interface. Here, the OvGST1 functions as a highly specific glutathione-dependent prostaglandin D synthase (PGDS). The enzyme therefore has the potential to participate in the modulation of immune responses by contributing to the production of parasite-derived prostanoids and restraining the host’s effector responses, making it a tempting target for chemotherapy and vaccine development. Here, we report the crystal structure of the OvGST1 bound to its cofactor glutathione at 2.0 Å resolution. The structure reveals an overall structural homology to the haematopoietic PGDS from vertebrates but, surprisingly, also a large conformational change in the prostaglandin binding pocket. The observed differences reveal a different vicinity of the prostaglandin H₂ binding pocket that demands another prostaglandin H₂ binding mode to that proposed for the vertebrate PGDS. Finally, a putative substrate binding mode for prostaglandin H₂ is postulated based on the observed structural insights.     

Saturation transfer difference NMR studies on substrates and inhibitors of succinic semialdehyde dehydrogenases

Saturation transfer difference (STD) NMR experiments on Escherichia coli and Drosophila melanogaster succinic semialdehyde dehydrogenase (SSADH, EC1.2.1.24) suggest that only the aldehyde forms and not the gem-diol forms of the specific substrate succinic semialdehyde (SSA), of selected aldehyde substrates, and of the inhibitor 3-tolualdehyde bind to these enzymes. Site-directed mutagenesis of the active site cysteine311 to alanine in D. melanogaster SSADH leads to an inactive product binding both SSA aldehyde and gem-diol. Thus, the residue cysteine311 is crucial for their discrimination. STD experiments on SSADH and NAD⁺/NADP⁺ indicate differential affinity in agreement with the respective cosubstrate properties. Epitope mapping by STD points to a strong interaction of the NAD⁺/NADP⁺ adenine H2 proton with SSADH. Adenine H8, nicotinamide H2, H4, and H6 also show STD signals. Saturation transfer to the ribose moieties is limited to the anomeric protons of E. coli SSADH suggesting that the NAD⁺/NADP⁺ adenine and nicotinamide, but not the ribose moieties are important for the binding of the coenzymes.     

Accord insertion in the 5′ flanking region of CYP6G1 confers nicotine resistance in Drosophila melanogaster

What has driven the sweep of the Accord retrotransposon insertion allele of CYP6G1 in the natural populations of Drosophila melanogaster is unknown. Previous studies on the DDT selection hypothesis produced conflicting data. To reexamine the DDT selection hypothesis and search for alternative explanations, we conducted a series of correlation and genetic linkage experiments with eight D. melanogaster natural populations collected from California (CM1, CM2, CM3, and CM7) and Africa (AM2, AM3, AM4, AM7). Diagnostic PCR showed that CM1, CM2, CM7, and AM3 have the Accord insertion in the CYP6G1 locus, whereas the other four strains do not. RT-PCR analysis exhibits a 100% correlation between Accord insertion and CYP6G1 overexpression. However, among the four strains with Accord-mediated CYP6G1 overexpression only CM1 and CM7 are resistant to DDT, and the other two strains (CM2 and AM3), like the four Accord-free strains, are susceptible to DDT. By contrast, all the four strains with Accord-mediated CYP6G1 overexpression are resistant to nicotine, a plant allelochemical. Genetic crosses between DDT resistant and susceptible Accord-insertion strains, as well as crosses between Accord-insertion and Accord-free strains demonstrated that Accord insertion and CYP6G1 overexpression are genetically linked to nicotine resistance rather than DDT resistance. These results suggest that naturally-occurring allelochemicals such as nicotine are the initial driving force for the worldwide prevalence of the Accord insertion allele of CYP6G1 in D. melanogaster natural populations.     

Structural Basis for Featuring of Steroid Isomerase Activity in Alpha Class Glutathione Transferases

Glutathione transferases (GSTs) are abundant enzymes catalyzing the conjugation of hydrophobic toxic substrates with glutathione. In addition to detoxication, human GST A3-3 displays prominent steroid double-bond isomerase activity; e.g. transforming Δ⁵-androstene-3-17-dione into Δ⁴-androstene-3-17-dione (AD). This chemical transformation is a crucial step in the biosynthesis of steroids, such as testosterone and progesterone. In contrast to GST A3-3, the homologous GST A2-2 does not show significant steroid isomerase activity. We have solved the 3D structures of human GSTs A2-2 and A3-3 in complex with AD. In the GST A3-3 crystal structure, AD was bound in an orientation suitable for the glutathione (GSH)-mediated catalysis to occur. In GST A2-2, however, AD was bound in a completely different orientation with its reactive double bond distant from the GSH-binding site. The structures illustrate how a few amino acid substitutions in the active site spectacularly alter the binding mode of the steroid substrate in relation to the conserved catalytic groups and an essentially fixed polypeptide chain conformation. Furthermore, AD did not bind to the GST A2-2-GSH complex. Altogether, these results provide a first-time structural insight into the steroid isomerase activity of any GST and explain the 5000-fold difference in catalytic efficiency between GSTs A2-2 and A3-3. More generally, the structures illustrate how dramatic diversification of functional properties can arise via minimal structural alterations. We suggest a novel structure-based mechanism of the steroid isomerization reaction.     

Crystallographic survey of active sites of an unclassified glutathione transferase from Bombyx mori

Background

Glutathione transferase (GST) catalyzes a major step in the xenobiotic detoxification pathway. We previously identified a novel, unclassified GST that is upregulated in an insecticide-resistant silkworm (Bombyx mori) upon insecticide exposure. Here, we sought to further characterize this GST, bmGSTu, by solving and refining its crystal structure and identifying its catalytic residues.

Methods

The structure of wild-type bmGSTu was determined with a resolution of 2.1 Å by synchrotron radiation and molecular modeling. Potential catalytic residues were mutated to alanine by means of site-directed mutagenesis, and kinetic data determined for wild-type and mutated bmGSTu.

Results

We found that bmGSTu occurred as a dimer, and that, like other GSTs, each subunit displayed a G-site and an H-site in the active center. Bound glutathione could be localized at the G-site. Kinetic data of the mutated forms of bmGSTu show that Val55, Glu67, and Ser68 in the G-site are important for catalysis. Furthermore, the H-site showed some unique features.

Conclusions

This is the first study to our knowledge to elucidate the molecular conformation of this B. mori GST. Our results indicate that residues Val55, Glu67, and Ser68, as well as Tyr7 and Ser12, in the glutathione-binding region of bmGSTu are critical for catalytic function.

General Significance

Our results, together with our previous finding that bmGSTu was preferentially induced in an insecticide-resistant strain, support the idea that bmGSTu functions in the transformation of exogenous chemical agents. Furthermore, the unique features observed in bmGSTu may shed light on mechanisms of insecticide resistance.     

High-resolution structure of shikimate dehydrogenase from Thermotoga maritima reveals a tightly closed conformation

Shikimate dehydrogenase (SDH), which catalyses the NADPH-dependent reduction of 3-dehydroshikimate to shikimate in the shikimate pathway, is an attractive target for the development of herbicides and antimicrobial agents. Structural analysis of a SDH from Thermotoga maritima encoded by the Tm0346 gene was performed to facilitate further structural comparisons between the various shikimate dehydrogenases. The crystal structure of SDH from T. maritima was determined at 1.45 Å by molecular replacement. SDH from T. maritima showed a monomeric architecture. The overall structure of SDH from T. maritima comprises the N-terminal α/β sandwich domain for substrate binding and the C-terminal domain for NADP binding. When the T. maritima SDH structure was compared with those of the SDHs from other species, the SDH from T. maritima was in a tightly closed conformation, which should be open for catalysis. Notably, α7 moves toward the active site (∼5 Å), which forces the SDH of T. maritima in a more closed form. Four ammonium sulfate (AMS) ions were identified in the structure. They were located in the active site and appeared to mimic the role of the substrate in terms of the enzyme activity and stability. The new high resolution structural information reported in this study, including the AMS binding sites as a potent inhibitor binding site of SDHs, is expected to supplement the existing structural data and will be useful for structure-based antibacterial discovery against SDHs.     

Characterising the Binding Specificities of the Subunits Associated with the KMT2/Set1 Histone Lysine Methyltransferase

KMT2/Set1 is the catalytic subunit of the complex of proteins associated with Set1 (COMPASS) that is responsible for the methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. Whereas monomethylated H3K4 (H3K4me1) is found throughout the genome, di- (H3K4me2) and tri- (H3K4me3) methylated H3K4 are enriched at specific loci, which correlates with the promoter and 5′-ends of actively transcribed genes in the case of H3K4me3. The COMPASS subunits contain a number of domains that are conserved in homologous complexes in higher eukaryotes and are reported to interact with modified histones. However, the exact organization of these subunits and their role within the complex have not been elucidated. In this study we showed that: (1) subunits Swd1 and Swd3 form a stable heterodimer that dissociates upon binding to a modified H3K4me2 tail peptide, suggesting a regulatory role in COMPASS; (2) the affinity of the subunit Spp1 for modified histone H3 substrates is much higher than that of Swd1 and Swd3; (3) Spp1 has a preference for H3K4me2/3 methylation state; and (4) Spp1 contains a high-affinity DNA-binding domain in the previously uncharacterised C-terminal region. These data allow us to suggest a mechanism for the regulation of COMPASS activity at an actively transcribed gene.     

Conformational Changes in a Plant Ketol-Acid Reductoisomerase upon Mg and NADPH Binding as Revealed by Two Crystal Structures

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) is an enzyme in the branched-chain amino acid biosynthesis pathway where it catalyzes the conversion of 2-acetolactate into (2R)-2,3-dihydroxy-3-isovalerate or the conversion of 2-aceto-2-hydroxybutyrate into (2R,3R)-2,3-dihydroxy-3-methylvalerate. KARI catalyzes two reactions—alkyl migration and reduction—and requires Mg²⁺ and NADPH for activity. To date, the only reported structures for a plant KARI are those of the spinach enzyme-Mn²⁺-(phospho)ADP ribose-(2R,3R)-2,3-dihydroxy-3-methylvalerate complex and the spinach KARI-Mg²⁺-NADPH-N-hydroxy-N-isopropyloxamate complex, where N-hydroxy-N-isopropyloxamate is a predicted transition-state analog. These studies demonstrated that the enzyme consists of two domains, N-domain and C-domain, with the active site at the interface of these domains. Here, we have determined the structures of the rice KARI-Mg²⁺ and rice KARI-Mg²⁺-NADPH complexes to 1.55 Å and 2.80 Å resolutions, respectively. In comparing the structures of all the complexes, several differences are observed. Firstly, the N-domain is rotated up to 15° relative to the C-domain, expanding the active site by up to 4 Å. Secondly, an α-helix in the C-domain that includes residues V510-T519 and forms part of the active site moves by ∼ 3.9 Å upon binding of NADPH. Thirdly, the 15 C-terminal amino acid residues in the rice KARI-Mg²⁺ complex are disordered. In the rice KARI-Mg²⁺-NADPH complex and the spinach KARI structures, many of the 15 residues bind to NADPH and the N-domain and cover the active site. Fourthly, the location of the metal ions within the active site can vary by up to 2.7 Å. The new structures allow us to propose that an induced-fit mechanism operates to (i) allow substrate to enter the active site, (ii) close over the active site during catalysis, and (iii) open the active site to facilitate product release.     

Statistical coupling analysis of aspartic proteinases based on crystal structures of the Trichoderma reesei enzyme and its complex with pepstatin A

The crystal structures of an aspartic proteinase from Trichoderma reesei (TrAsP) and of its complex with a competitive inhibitor, pepstatin A, were solved and refined to crystallographic R-factors of 17.9% (R_free = 21.2%) at 1.70 Å resolution and 15.8% (R_free = 19.2%) at 1.85 Å resolution, respectively. The three-dimensional structure of TrAsP is similar to structures of other members of the pepsin-like family of aspartic proteinases. Each molecule is folded in a predominantly β-sheet bilobal structure with the N-terminal and C-terminal domains of about the same size. Structural comparison of the native structure and the TrAsP-pepstatin complex reveals that the enzyme undergoes an induced-fit, rigid-body movement upon inhibitor binding, with the N-terminal and C-terminal lobes tightly enclosing the inhibitor. Upon recognition and binding of pepstatin A, amino acid residues of the enzyme active site form a number of short hydrogen bonds to the inhibitor that may play an important role in the mechanism of catalysis and inhibition. The structures of TrAsP were used as a template for performing statistical coupling analysis of the aspartic protease family. This approach permitted, for the first time, the identification of a network of structurally linked residues putatively mediating conformational changes relevant to the function of this family of enzymes. Statistical coupling analysis reveals coevolved continuous clusters of amino acid residues that extend from the active site into the hydrophobic cores of each of the two domains and include amino acid residues from the flap regions, highlighting the importance of these parts of the protein for its enzymatic activity.     

On the benefit of bivalency in peptide ligand/pin1 interactions

The human peptidyl prolyl cis/trans isomerase (PPIase) Pin1 has a key role in developmental processes and cell proliferation. Pin1 consists of an N-terminal WW domain and a C-terminal catalytic PPIase domain both targeted specifically to Ser(PO₃H₂)/Thr(PO₃H₂)-Pro sequences. Here, we report the enhanced affinity originating from bivalent binding of ligands toward Pin1 compared to monovalent binding. We developed composite peptides where an N-terminal segment represents a catalytic site-directed motif and a C-terminal segment exhibits a predominant affinity to the WW domain of Pin1 tethered by polyproline linkers of different chain length. We used NMR shift perturbation experiments to obtain information on the specific interaction of a bivalent ligand to both targeted sites of Pin1. The bivalent ligands allowed a considerable range of thermodynamic investigations using isothermal titration calorimetry and PPIase activity assays. They expressed up to 350-fold improved affinity toward Pin1 in the nanomolar range in comparison to the monovalent peptides. The distance between the two binding motifs was highly relevant for affinity. The optimum in affinity manifested by a linker length of five prolyl residues between active site- and WW domain-directed peptide fragments suggests that the corresponding domains in Pin1 are allowed to adopt preferred spatial arrangement upon ligand binding.