Functions, structures and Triton X-100 effect for the catalytic subunits of heterodimeric phospholipases A2 from Vipera nikolskii venom.

Phospholipases A(2) (PLA(2)s) from snake venoms have diverse pharmacological functions including neurotoxicity, and more studies are necessary to understand relevant mechanisms. Here we report the different crystal structures for two enzymatically active basic subunits (HDP-1P and HDP-2P) of heterodimeric neurotoxic PLA(2)s isolated from Vipera nikolskii venom. Structural comparisons with similar PLA(2)s clearly show some flexible regions which might be important for the catalytic function and neurotoxicity. Unexpectedly, Triton X-100 molecule bound in the hydrophobic channel of HDP-1P and HDP-2P was observed, and its binding induced conformational changes in the Ca(2+) binding loop. Enzymatic activity measurements indicated that Triton X-100 decreased the activity of PLA(2), although with comparatively low inhibitory activity. For the first time exocytosis experiments in pancreatic beta cells were used to confirm the presynaptic neurotoxicity of relevant snake PLA(2). These experiments also indicated that Triton X-100 inhibited the influence of HDP-1P on exocytosis, but the inhibition was smaller than that of MJ33, a phospholipid-analogue inhibitor of PLA(2). Our studies performed at a cellular level are in good agreement with earlier findings that enzymatic activity of the snake presynaptic PLA(2) neurotoxins is essential for effective block of nerve terminals.

Octameric structure of the human bifunctional enzyme PAICS in purine biosynthesis.

Phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is an important bifunctional enzyme in de novo purine biosynthesis in vertebrate with both 5-aminoimidazole ribonucleotide carboxylase (AIRc) and 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide synthetase (SAICARs) activities. It becomes an attractive target for rational anticancer drug design, since rapidly dividing cancer cells rely heavily on the purine de novo pathway for synthesis of adenine and guanine, whereas normal cells favor the salvage pathway. Here, we report the crystal structure of human PAICS, the first in the entire PAICS family, at 2.8 A resolution. It revealed that eight PAICS subunits, each composed of distinct AIRc and SAICARs domains, assemble a compact homo-octamer with an octameric-carboxylase core and four symmetric periphery dimers formed by synthetase domains. Based on structural comparison and functional complementation analyses, the active sites of SAICARs and AIRc were identified, including a putative substrate CO(2)-binding site. Furthermore, four symmetry-related, separate tunnel systems in the PAICS octamer were found that connect the active sites of AIRc and SAICARs. This study illustrated the octameric nature of the bifunctional enzyme. Each carboxylase active site is formed by structural elements from three AIRc domains, demonstrating that the octamer structure is essential for the carboxylation activity. Furthermore, the existence of the tunnel system implies a mechanism of intermediate channeling and suggests that the quaternary structure arrangement is crucial for effectively executing the sequential reactions. In addition, this study provides essential structural information for designing PAICS-specific inhibitors for use in cancer chemotherapy.

Crystallization and preliminary crystallographic studies of human septin 1 with site-directed mutations.

Septin 1 is a member of an evolutionarily conserved family of GTP-binding and filament-forming proteins named septins, which function in diverse processes including cytokinasis, vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration and neoplasia. Human septin 1 has been expressed and purified, but suffers from severe aggregation. Studies have shown that septin 1 with site-directed mutations of five serine residues (Ser19, Ser206, Ser307, Ser312 and Ser315) has a much lower degree of aggregation and better structural homogeneity and that the mutations cause only slight perturbations in the secondary structure of septin 1. This septin 1 mutant was crystallized and diffraction data were collected to 2.5 A resolution. The space group is P422, with unit-cell parameters a = b = 106.028, c = 137.852 A.

An attempt to increase the efficiency of protein crystal screening: a simplified screen and experiments.

Macromolecular crystallization remains a bottleneck in structure determination by X-ray diffraction. Based on the data reflecting success rates of crystallization conditions in different screens and the information derived from the BMCD and other related studies, a simplified screen has been designed to increase the success rate of traditional screening and to save samples, time and cost. The screen has been tested with six protein samples which had been crystallized before and its comparison with Crystal Screen (Hampton Research) was also performed with lysozyme crystallization. The experimental results show that for obtaining crystal leads, the success rate of the simplified screen is reasonably higher. In addition, it has been successful in crystallizing two new proteins from snake venom using the simplified screen that had failed with Crystal Screen. These results indicate that the simplified screen, which assembles and optimizes the efficient crystallization conditions from distinct screens, extends the region of crystallization and improves the success rate of screening. Based on information of newly published efficient crystallization conditions, the simplified screen could be developed and optimized continuously in the future.

Isolation and preliminary crystallographic studies of two new phospholipases A2 from Vipera nikolskii venom.

Snake-venom phospholipases A2 (PLA2s) represent a good model for studies of structure-function relationships, mainly because of their small size and diverse pharmacological and toxicological activities. To obtain new members of the abundant PLA2 family, the venom of the viper Vipera nikolskii was fractionated for the first time and two new proteins, VN5-3 and VN4-3, were isolated. Both proteins show phospholipase A2 activity and may possess neurotoxic activity. Based on the determined partial amino-acid sequences, the new proteins can be classified as basic Asp49 phospholipases A2. They were crystallized using the hanging-drop vapour-diffusion method and crystals of both proteins belong to space group R32, with similar unit-cell parameters: a = b = 76.29, c = 303.35 A for protein VN5-3 and a = b = 76.28, c = 304.39 A for protein VN4-3. Diffraction data sets to 3.0 and 2.2 A resolution were collected and processed for the VN5-3 and VN4-3 crystals, respectively. Preliminary analysis indicates that there are two molecules in the asymmetric unit for both crystals. Further crystallographic studies will help in understanding the structural basis for the multiple functions of snake-venom PLA2s.

Crystallization and preliminary X-ray crystallographic studies of human cyclophilin J.

Human cyclophilin J, a new member of the cyclophilin family, has been expressed and crystallized. Diffraction data have been collected to 2.0 A resolution and preliminary crystallographic studies have been completed. The space group of the crystals is P3(1)21, with unit-cell parameters a = b = 40.597, c = 170.732 A, alpha = beta = 90, gamma = 120 degrees.

2.6 A resolution crystal structure of the bacterioferritin from Azotobacter vinelandii.

The crystal structure of the bacterioferritin from Azotobacter vinelandii has been determined at 2.6 A resolution. Both the low occupancy of one iron ion in the dinuclear iron center and the deviation of its adjacent residue His130 from the center suggest migration of the iron ion from the dinuclear iron site to the inner nucleation site. The concerted movement of His130 and Glu47 may admit a dynamic gating mechanism for shift of the oxidized iron ion. Ba2+ binding to the fourfold channel implicates that the channel bears Fe2+ conductivity and selectivity to provide a route for iron access to the inner cavity during core formation.

Purification and refolding of human alpha5-subunit (PSMA5) of the 20S proteasome, expressed as inclusion bodies in Escherichia coli.

The 20S proteasome is the central enzyme of nonlysosomal protein degradation in both the cytosol and nucleus. It is composed of 28 protein subunits which are arranged into four staggered heptameric rings. The outer rings consist of alpha-subunits which are responsible for binding of proteasome activators, inhibitors, and regulators. To better characterize human alpha5-subunit (PSMA5) of the 20S proteasome, we have established a high-efficiency Escherichia coli expression system. The DNA-coding sequence for the human PSMA5, which was subcloned into the vector pET-22b (+), has been expressed as inclusion bodies in E. coli BL21 (DE3). To produce the native PSMA5, straightforward protocols have been developed for refolding the human PSMA5 in the presence of surfactants using dilution refolding and size-exclusion chromatography matrix refolding methods. After refolding, recovery yields of about 20% were obtained, respectively, with purity above 95%. The human PSMA5 was detected by dynamic light scattering in refolding process, and the molecular weight of the final refolded product was measured using gel filtration chromatography, which indicates that the human PSMA5 exists mainly as tetramer.

The space experiment of protein crystallization aboard the Chinese spacecraft SZ-3.

Using new flight hardware, a Chinese mission of space protein crystallization has been performed aboard the Chinese spacecraft SZ-3. Preliminary analyses of the experimental results have shown that a few proteins produced better crystals in space. At least, the crystals of cytochrome b5 mutant could diffract X-ray beyond the highest resolution reported so far for the same kind of crystals. In addition, some rules derived from our numerical studies of the liquid/liquid diffusion protein crystallization were proved by the crystallization of lysozyme as model protein in this space experiment, which also clearly showed the advantages and disadvantages of the gelation of the protein solution used in microgravity growth of protein crystals.