Structural basis of the cystein protease inhibitor Clonorchis sinensis Stefin-1.

Cystein protease plays a critical role as a virulence factor in the development and progression of various diseases. Cystatin is a superfamily of cysteine protease inhibitors that participates in various physiological and pathological processes. The cysteine protease inhibitor CsStein-1 isolated from Clonorchis sinensis belongs to the type 1 stefin of cystatins. This inhibitor regulates the activity and processing of CsCF (Cathepsin F of Clonorchis sienesis), which plays an important role in parasite nutrition and host-parasite interaction. CsStefin-1 has also been proposed as a host immune modulator and a participant in the mechanism associated with anti-inflammatory ability. Here, we report the first crystal structure of CsStefin-1 determined by the multi-wavelength anomalous diffraction (MAD) method to 2.3 Å. There are six molecules of CsStefin-1 per asymmetric unit, with a solvent content of 36.5%. The structure of CsStefin-1 is composed of twisted four-stranded antiparallel ß-sheets, a central α-helix, and a short α-helix. We also demonstrate that CsStefin-1 binds to CsCF-8 cysteine protease and inhibits its activity. In addition, a molecular docking model of CsStefin-1 and CsCF-8 was developed using homology modeling based on their structures. The structural information regarding CsStefin-1 and molecular insight into its interaction with CsCF-8 are important to understanding their biological function and to design of inhibitors that modulate cysteine protease activity.

Structure and function of vascular endothelial growth factor and its receptor system.

Vascular endothelial growth factor and its receptor (VEGF-VEGFR) system play a critical role in the regulation of angiogenesis and lymphangiogenesis in vertebrates. Each of the VEGF has specific receptors, which it activates by binding to the extracellular domain of the receptors, and, thus, regulates the angiogenic balance in the early embryonic and adult stages. However, de-regulation of the VEGF-VEGFR implicates directly in various diseases, particularly cancer. Moreover, tumor growth needs a dedicated blood supply to provide oxygen and other essential nutrients. Tumor metastasis requires blood vessels to carry tumors to distant sites, where they can implant and begin the growth of secondary tumors. Thus, investigation of signaling systems related to the human disease, such as VEGF-VEGFR, will facilitate the development of treatments for such illnesses. [BMB Reports 2018; 51(2): 73-78].

Crystal structure of the C-terminal domain of Bacillus subtilis GabR reveals a closed conformation by γ-aminobutyric acid binding, inducing transcriptional activation.

Bacillus subtilis GabR (BsGabR) is involved in the γ-aminobutyric acid (GABA) catabolism as a transcriptional regulator, consisting of an N-terminal helix-turn-helix DNA-binding domain and a C-terminal aminotransferase-like (AT-like) domain. Research on the C-terminal AT-like domain of BsGabR (BsGabR-CTD) has focused on the interaction with GABA as an effector, but most its functional details remain unclear. To understand the underlying mechanism, we report the crystal structure of BsGabR-CTD in complex with pyridoxal 5'-phosphate (PLP) and GABA at 2.0 Å resolution. The structure of ligand-bound BsGabR-CTD revealed two distinct monomeric states in a homodimer. One subunit is a closed-form containing the PLP-GABA adduct, and the other subunit is a PLP-bound open-form. Our structural studies provide a detailed mechanism indicating that the open-to-closed transition by the binding of GABA induces the conformational rearrangement of BsGabR-CTD, which may trigger the activation of transcription.

Structure-based functional studies for the cellular recognition and cytolytic mechanism of pneumolysin from Streptococcus pneumoniae.

Cholesterol-dependent cytolysins (CDCs) contribute to various pathogenesis by Gram-positive bacterial pathogens. Among them, pneumolysin (PLY) produced by Streptococcus pneumoniae is a major contributor to pneumococcal infections. Despite numerous studies of the cytolytic mechanism of PLY, little structural information on its interactions with a specific receptor of the cell membrane is available. We report here the first crystal structures of PLY in an apo-form and in a ternary complex with two mannoses at 2.8Å and 2.5Å resolutions, respectively. Both structures contained one monomer in an asymmetric unit and were comprised of four discontinuous domains, similar to CDC structures reported previously. The ternary complex structure showed that loop 3 and the undecapeptide region in domain 4 might contribute to cellular recognition by binding to mannose, as a component of a specific cell-surface receptor. Moreover, mutational studies and docking simulations for four residues (Leu431, Trp433, Thr459, and Leu460) in domain 4 indicated that Leu431 and Trp433 in the undecapeptide might be involved in the binding of cholesterol, together with the Thr459-Leu460 pair in loop 1. Our results provide structure-based molecular insights into the interaction of PLY with the target cell membrane, including the binding of mannose and cholesterol.

Structural insight into the substrate inhibition mechanism of NADP(+)-dependent succinic semialdehyde dehydrogenase from Streptococcus pyogenes.

Succinic semialdehyde dehydrogenases (SSADHs) are ubiquitous enzymes that catalyze the oxidation of succinic semialdehyde (SSA) to succinic acid in the presence of NAD(P)(+), and play an important role in the cellular mechanisms including the detoxification of accumulated SSA or the survival in conditions of limited nutrients. Here, we report the inhibitory properties and two crystal structures of SSADH from Streptococcus pyogenes (SpSSADH) in a binary (ES) complex with SSA as the substrate and a ternary (ESS) complex with the substrate SSA and the inhibitory SSA, at 2.4 Å resolution for both structures. Analysis of the kinetic inhibitory parameters revealed significant substrate inhibition in the presence of NADP(+) at concentrations of SSA higher than 0.02 mM, which exhibited complete uncompetitive substrate inhibition with the inhibition constant (Ki) value of 0.10 ± 0.02 mM. In ES-complex of SpSSADH, the SSA showed a tightly bound bent form nearby the catalytic residues, which may be caused by reduction of the cavity volume for substrate binding, compared with other SSADHs. Moreover, structural comparison of ESS-complex with a binary complex with NADP(+) of SpSSADH indicated that the substrate inhibition was induced by the binding of inhibitory SSA in the cofactor-binding site, instead of NADP(+). Our results provide first structure-based molecular insights into the substrate inhibition mechanism of SpSSADH as the Gram-positive bacterial SSADH.

Kinetic and structural characterization for cofactor preference of succinic semialdehyde dehydrogenase from Streptococcus pyogenes.

The γ-Aminobutyric acid (GABA) that is found in prokaryotic and eukaryotic organisms has been used in various ways as a signaling molecule or a significant component generating metabolic energy under conditions of nutrient limitation or stress, through GABA catabolism. Succinic semialdehyde dehydrogenase (SSADH) catalyzes the oxidation of succinic semialdehyde to succinic acid in the final step of GABA catabolism. Here, we report the catalytic properties and two crystal structures of SSADH from Streptococcus pyogenes (SpSSADH) regarding its cofactor preference. Kinetic analysis showed that SpSSADH prefers NADP(+) over NAD(+) as a hydride acceptor. Moreover, the structures of SpSSADH were determined in an apo-form and in a binary complex with NADP(+) at 1.6 Šand 2.1 Šresolutions, respectively. Both structures of SpSSADH showed dimeric conformation, containing a single cysteine residue in the catalytic loop of each subunit. Further structural analysis and sequence comparison of SpSSADH with other SSADHs revealed that Ser158 and Tyr188 in SpSSADH participate in the stabilization of the 2'-phosphate group of adenine-side ribose in NADP(+). Our results provide structural insights into the cofactor preference of SpSSADH as the gram-positive bacterial SSADH.

Kinetic characterization and molecular modeling of NAD(P)(+)-dependent succinic semialdehyde dehydrogenase from Bacillus subtilis as an ortholog YneI.

Succinic semialdehyde dehydrogenase (SSADH) catalyzes the oxidation of succinic semialdehyde (SSA) into succinic acid in the final step of γ-aminobutyric acid degradation. Here, we characterized Bacillus subtilis SSADH (BsSSADH) regarding its cofactor discrimination and substrate inhibition. BsSSADH showed similar values of the catalytic efficiency (kcat/Km) in both NAD(+) and NADP(+) as cofactors, and exhibited complete uncompetitive substrate inhibition at higher SSA concentrations. Further analyses of the sequence alignment and homology modeling indicated that the residues of catalytic and cofactor-binding sites in other SSADHs were highly conserved in BsSSADH.

Characterization of pneumolysin from Streptococcus pneumoniae, interacting with carbohydrate moiety and cholesterol as a component of cell membrane.

The cytolytic mechanism of cholesterol-dependent cytolysins (CDCs) requires the presence of cholesterol in the target cell membrane. Membrane cholesterol was thought to serve as the common receptor for these toxins, but not all CDCs require cholesterol for binding. One member of this toxin family, pneumolysin (PLY) is a major virulence factor of Streptococcus pneumoniae, and the mechanism via which PLY binds to its putative receptor or cholesterol on the cell membrane is still poorly understood. Here, we demonstrated that PLY interacted with carbohydrate moiety and cholesterol as a component of the cell membrane, using the inhibitory effect of hemolytic activity. The hemolytic activity of PLY was inhibited by cholesterol-MßCD, which is in a 3ß configuration at the C3-hydroxy group, but is not in a 3α-configuration. In the interaction between PLY and carbohydrate moiety, the mannose showed a dose-dependent increase in the inhibition of PLY hemolytic activity. The binding ability of mannose with truncated PLYs, as determined by the pull-down assay, showed that mannose might favor binding to domain 4 rather than domains 1-3. These studies provide a new model for the mechanism of cellular recognition by PLY, as well as a foundation for future investigations into whether non-sterol molecules can serve as receptors for other members of the CDC family of toxins.