New Crystal Form of Human Neuropilin-1 b1 Fragment with Six Electrostatic Mutations Complexed with KDKPPR Peptide Ligand.

Neuropilin 1 (NRP1), a cell-surface co-receptor of a number of growth factors and other signaling molecules, has long been the focus of attention due to its association with the development and the progression of several types of cancer. For example, the KDKPPR peptide has recently been combined with a photosensitizer and a contrast agent to bind NRP1 for the detection and treatment by photodynamic therapy of glioblastoma, an aggressive brain cancer. The main therapeutic target is a pocket of the fragment b1 of NRP1 (NRP1-b1), in which vascular endothelial growth factors (VEGFs) bind. In the crystal packing of native human NRP1-b1, the VEGF-binding site is obstructed by a crystallographic symmetry neighbor protein, which prevents the binding of ligands. Six charged amino acids located at the protein surface were mutated to allow the protein to form a new crystal packing. The structure of the mutated fragment b1 complexed with the KDKPPR peptide was determined by X-ray crystallography. The variant crystallized in a new crystal form with the VEGF-binding cleft exposed to the solvent and, as expected, filled by the C-terminal moiety of the peptide. The atomic interactions were analyzed using new approaches based on a multipolar electron density model. Among other things, these methods indicated the role played by Asp320 and Glu348 in the electrostatic steering of the ligand in its binding site. Molecular dynamics simulations were carried out to further analyze the peptide binding and motion of the wild-type and mutant proteins. The simulations revealed that specific loops interacting with the peptide exhibited mobility in both the unbound and bound forms.

Biochemical and Structural Characterization of Chi-Class Glutathione Transferases: A Snapshot on the Glutathione Transferase Encoded by sll0067 Gene in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Glutathione transferases (GSTs) constitute a widespread superfamily of enzymes notably involved in detoxification processes and/or in specialized metabolism. In the cyanobacterium Synechocsytis sp. PCC 6803, SynGSTC1, a chi-class GST (GSTC), is thought to participate in the detoxification process of methylglyoxal, a toxic by-product of cellular metabolism. A comparative genomic analysis showed that GSTCs were present in all orders of cyanobacteria with the exception of the basal order Gloeobacterales. These enzymes were also detected in some marine and freshwater noncyanobacterial bacteria, probably as a result of horizontal gene transfer events. GSTCs were shorter of about 30 residues compared to most cytosolic GSTs and had a well-conserved SRAS motif in the active site (10SRAS13 in SynGSTC1). The crystal structure of SynGSTC1 in complex with glutathione adopted the canonical GST fold with a very open active site because the α4 and α5 helices were exceptionally short. A transferred multipolar electron-density analysis allowed a fine description of the solved structure. Unexpectedly, Ser10 did not have an electrostatic influence on glutathione as usually observed in serinyl-GSTs. The S10A variant was only slightly less efficient than the wild-type and molecular dynamics simulations suggested that S10 was a stabilizer of the protein backbone rather than an anchor site for glutathione.

Biochemical and Structural Insights on the Poplar Tau Glutathione Transferase GSTU19 and 20 Paralogs Binding Flavonoids.

Glutathione transferases (GSTs) constitute a widespread superfamily of enzymes notably involved in xenobiotic detoxification and/or in specialized metabolism. Populus trichocarpa genome (V4.1 assembly, Phytozome 13) consists of 74 genes coding for full-length GSTs and ten likely pseudogenes. These GSTs are divided into 11 classes, in which the tau class (GSTU) is the most abundant with 54 isoforms. PtGSTU19 and 20, two paralogs sharing more than 91% sequence identity (95% of sequence similarity), would have diverged from a common ancestor of P. trichocarpa and P. yatungensis species. These enzymes display the distinctive glutathione (GSH)-conjugation and peroxidase activities against model substrates. The resolution of the crystal structures of these proteins revealed significant structural differences despite their high sequence identity. PtGSTU20 has a well-defined deep pocket in the active site whereas the bottom of this pocket is disordered in PtGSTU19. In a screen of potential ligands, we were able to identify an interaction with flavonoids. Some of them, previously identified in poplar (chrysin, galangin, and pinocembrin), inhibited GSH-conjugation activity of both enzymes with a more pronounced effect on PtGSTU20. The crystal structures of PtGSTU20 complexed with these molecules provide evidence for their potential involvement in flavonoid transport in P. trichocarpa.

Structural and Biochemical Analysis of OrfG: The VirB8-like Component of the Conjugative Type IV Secretion System of ICESt3 From Streptococcus thermophilus.

Conjugative transfer is a major threat to global health since it contributes to the spread of antibiotic resistance genes and virulence factors among commensal and pathogenic bacteria. To allow their transfer, mobile genetic elements including Integrative and Conjugative Elements (ICEs) use a specialized conjugative apparatus related to Type IV secretion systems (Conj-T4SS). Therefore, Conj-T4SSs are excellent targets for strategies that aim to limit the spread of antibiotic resistance. In this study, we combined structural, biochemical and biophysical approaches to study OrfG, a protein that belongs to Conj-T4SS of ICESt3 from Streptococcus thermophilus. Structural analysis of OrfG by X-ray crystallography revealed that OrfG central domain is similar to VirB8-like proteins but displays a different quaternary structure in the crystal. To understand, at a structural level, the common and the diverse features between VirB8-like proteins from both Gram-negative and -positive bacteria, we used an in silico structural alignment method that allowed us to identify different structural classes of VirB8-like proteins. Biochemical and biophysical characterizations of purified OrfG soluble domain and its central and C-terminal subdomains indicated that they are mainly monomeric in solution but able to form an unprecedented 6-mer oligomers. Our study provides new insights into the structural analysis of VirB8-like proteins and discusses the interplay between tertiary and quaternary structures of these proteins as an essential component of the conjugative transfer.

Structural plasticity among glutathione transferase Phi members: natural combination of catalytic residues confers dual biochemical activities.

The glutathione transferase (GST) gene family is divided into 14 classes in photosynthetic organisms. Among them, the Phi class (GSTF) is composed of a large number of genes that are often induced in response to environmental constraints due to their ability to detoxify xenobiotics, to their peroxidase activity and to their involvement in the biosynthesis and/or transport of secondary metabolites. However, the exact functions of GSTFs from many plants including Populus trichocarpa are unknown. Here, following GSTF1 characterization, we have performed a comparative analysis of the seven other GSTFs found in poplar by systematically evaluating the biochemical and enzymatic properties of the corresponding recombinant proteins and of variants mutated for active site residues and by determining the three-dimensional structures of several representatives. Owing to the presence of a cysteine with a pKa value around 5 in their active site, GSTF3, F7, and F8 displayed a thiol transferase activity in addition to the usual glutathione transferase and peroxidase activities. From structural analyses, it appeared that these dual biochemical properties originate from the existence of a certain variability in the ß1-α1 loop. This allows positioning of several active site residues at proximity of the glutathione molecule, which itself remains unchanged in GSTF three-dimensional structures. These results highlight the promiscuity of some GSTFs and that changes of active site residues in some isoforms during evolution generated functional diversity by modifying their activity profile. DATABASE: Structural data are available in the PDB under the accession numbers 5EY6, 5F05, 5F06, and 5F07.

The poplar Phi class glutathione transferase: expression, activity and structure of GSTF1.

Glutathione transferases (GSTs) constitute a superfamily of enzymes with essential roles in cellular detoxification and secondary metabolism in plants as in other organisms. Several plant GSTs, including those of the Phi class (GSTFs), require a conserved catalytic serine residue to perform glutathione (GSH)-conjugation reactions. Genomic analyses revealed that terrestrial plants have around ten GSTFs, eight in the Populus trichocarpa genome, but their physiological functions and substrates are mostly unknown. Transcript expression analyses showed a predominant expression of all genes both in reproductive (female flowers, fruits, floral buds) and vegetative organs (leaves, petioles). Here, we show that the recombinant poplar GSTF1 (PttGSTF1) possesses peroxidase activity toward cumene hydroperoxide and GSH-conjugation activity toward model substrates such as 2,4-dinitrochlorobenzene, benzyl and phenetyl isothiocyanate, 4-nitrophenyl butyrate and 4-hydroxy-2-nonenal but interestingly not on previously identified GSTF-class substrates. In accordance with analytical gel filtration data, crystal structure of PttGSTF1 showed a canonical dimeric organization with bound GSH or 2-(N-morpholino)ethanesulfonic acid molecules. The structure of these protein-substrate complexes allowed delineating the residues contributing to both the G and H sites that form the active site cavity. In sum, the presence of GSTF1 transcripts and proteins in most poplar organs especially those rich in secondary metabolites such as flowers and fruits, together with its GSH-conjugation activity and its documented stress-responsive expression suggest that its function is associated with the catalytic transformation of metabolites and/or peroxide removal rather than with ligandin properties as previously reported for other GSTFs.

Characterization of a Phanerochaete chrysosporium glutathione transferase reveals a novel structural and functional class with ligandin properties.

Glutathione S-transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes. A new fungal specific class of GST has been highlighted by genomic approaches. The biochemical and structural characterization of one isoform of this class in Phanerochaete chrysosporium revealed original properties. The three-dimensional structure showed a new dimerization mode and specific features by comparison with the canonical GST structure. An additional ß-hairpin motif in the N-terminal domain prevents the formation of the regular GST dimer and acts as a lid, which closes upon glutathione binding. Moreover, this isoform is the first described GST that contains all secondary structural elements, including helix α4' in the C-terminal domain, of the presumed common ancestor of cytosolic GSTs (i.e. glutaredoxin 2). A sulfate binding site has been identified close to the glutathione binding site and allows the binding of 8-anilino-1-naphtalene sulfonic acid. Competition experiments between 8-anilino-1-naphtalene sulfonic acid, which has fluorescent properties, and various molecules showed that this GST binds glutathionylated and sulfated compounds but also wood extractive molecules, such as vanillin, chloronitrobenzoic acid, hydroxyacetophenone, catechins, and aldehydes, in the glutathione pocket. This enzyme could thus function as a classical GST through the addition of glutathione mainly to phenethyl isothiocyanate, but alternatively and in a competitive way, it could also act as a ligandin of wood extractive compounds. These new structural and functional properties lead us to propose that this GST belongs to a new class that we name GSTFuA, for fungal specific GST class A.