Ye Xiang

Two novel antifungal peptides distinct with a five-disulfide motif from the bark of Eucommia ulmoides Oliv

Two antifungal peptides, named EAFP1 and EAFP2, have been purified from the bark of Eucommia ulmoides Oliv. Each of the sequences consists of 41 residues with a N‐terminal blockage by pyroglutamic acid determined by automated Edman degradation in combination with the tandem mass spectroscopy and the C‐terminal ladder sequencing analysis. The primary structurs all contain 10 cysteines, which are cross‐linked to form five disulfide bridges with a pairing pattern (C1–C5, C2–C9, C3–C6, C4–C7, C8–C10). This is the first finding of a plant antifungal peptide with a five‐disulfide motif. EAFP1 and EAFP2 show characteristics of hevein domain and exhibit chitin‐binding properties similar to the previously identified hevein‐like peptides. They exhibit relatively broad spectra of antifungal activities against eight pathogenic fungi from cotton, wheat, potato, tomato and tobacco. The inhibition activity of EAFP1 and EAFP2 can be effective on both chitin‐containing and chitin‐free fungi. The values of IC50 range from 35 to 155 μg/ml for EAFP1 and 18 to 109 μg/ml for EAFP2. Their antifungal effects are strongly antagonized by calcium ions.

Structural basis for the tumor cell apoptosis-inducing activity of an antitumor lectin from the edible mushroom Agrocybe aegerita

Lectin AAL (Agrocybe aegerita lectin) from the edible mushroom A. aegerita is an antitumor protein that exerts its tumor-suppressing function via apoptosis-inducing activity in cancer cells. The crystal structures of ligand-free AAL and its complex with lactose have been determined. The AAL structure shows a dimeric organization, and each protomer adopts a prototype galectin fold. To identify the structural determinants for antitumor effects arising from the apoptosis-inducing activity of AAL, 11 mutants were prepared and subjected to comprehensive investigations covering oligomerization detection, carbohydrate binding test, apoptosis-inducing activity assay, and X-ray crystallographic analysis. The results show that dimerization of AAL is a prerequisite for its tumor cell apoptosis-inducing activity, and both galactose and glucose are basic moieties of functional carbohydrate ligands for lectin bioactivity. Furthermore, we have identified a hydrophobic pocket that is essential for the proteins apoptosis-inducing activity but independent of its carbohydrate binding and dimer formation. This hydrophobic pocket comprises a hydrophobic cluster including residues Leu33, Leu35, Phe93, and Ile144, and is involved in AALs function mechanism as an integrated structural motif. Single mutants such as F93G or I144G do not disrupt carbohydrate binding and homodimerization capabilities, but abolish the bioactivity of the protein. These findings reveal the structural basis for the antitumor property of AAL, which may lead to de novo designs of antitumor drugs based on AAL as a prototype model.

Optimizing protein crystal growth through dynamic seeding

A dynamic seeding method that is different from the conventional method of seeding drops that have been equilibrated is described. The method basically consists of two steps. Firstly, microseeding was used in association with adjustment of the seeding-drop components, including buffer, additive and concentrations of the precipitants and protein, in order to screen suitable seeding conditions under which microseeds are seeded into a new non-equilibrated drop as the dynamic macroseed drop for the following step. Secondly, after being equilibrated for various times against the reservoir solution, the macroseed drops were used to prepare a dilution series with which the qualified crystals could be harvested using macroseeding. Compared with a conventional seeding technique, this method is distinct with a dynamic situation of macroseed drops before macroseeding and a non-equilibrium serial seeding where all the seeds are seeded into new non-equilibrated drops and the micro/macroseeding are efficiently combined into a whole system. The method simplifies control of the number of microseeds because an excess of microseeds has little effect on the final result. The method also simplifies the manipulation of macroseeds by optimizing the equilibration time and the dilution multiple of the macroseed drops before macroseeding. This dynamic seeding technique has been used in the crystallization of novel protein CutCm, which has a fast crystal-growth rate, and proved that the method is useful for optimizing protein crystallization.

Crystallization and preliminary crystallographic studies of an antitumour lectin from the edible mushroom Agrocybe aegerita.

An antitumour lectin named AAL has been purified from the fruiting body of edible mushroom Agrocybe aegerita. In addition to having a distinct bioactivity, AAL shows strong inhibition effects on human and mouse tumour cells. It has been shown that AAL exerts its antitumour effects via apoptosis-induction. AAL and AAL-lactose complex have been crystallized and their diffraction data were collected with resolution of 2.6 A and 3.0 A, respectively. Both crystals belong to space group P 6122 with unit cell parameters a = 123.98 A, b = 123.98 A, c = 56.86 A, alpha= beta=90 degrees, gamma = 120 degrees and a = 123.69 A, b = 123.69 A, c = 56.64 A, alpha = beta = 90 degrees, gamma = 120 degrees, respectively.

Purification, crystallization and preliminary X-ray diffraction analysis of a novel mannose-binding lectin from Gastrodia elata with antifungal properties.

A plant antifungal protein, Gastrodia antifungal protein (GAFP-1) has been isolated from terminal corms of the orchid Gastrodia elata B1 f. elata, purified to homogeneity and crystallized by means of the hanging-drop vapour-diffusion method. The best quality crystals grew over several months at 277 K. The crystal used for data collection belongs to the space group P2(1)2(1)2, with unit-cell parameters a = 61.087, b = 91.488, c = 81.132 A. Using a synchrotron-radiation source, the resolution limit of the data reached 2.0 A, with an overall R(merge) of 0.097 and a completeness of 99.8%. Four independent molecules were estimated to be present in the asymmetric unit, with a solvent content of 46.3%. This data will help to solve the first structure of a monomeric monocot mannose-binding lectin.

A simple and efficient innovation of the vapor- diffusion method for controlling nucleation and growth of large protein crystals

The rate of water vaporization in the vapor-diffusion method is critical for the protein crystallization process. Present methods, however, allow little or no control of the equilibration rates. This paper presents a relatively simple innovation of the conventional vapor-diffusion method by introducing a capillary barrier (for hanging drop) or a punched film barrier (for both hanging and sitting drop) between drop and reservoir, which can be beneficial in controlling the water vaporization rate, thereby promoting growth of large protein crystals. The crystallization experiments for lysozyme, trichosanthin and a novel neurotoxin BmK Mu9 show that this modified vapor-controlling-diffusion method is very effective for producing large protein crystals. The improved technique can be routinely used as a method for the preparation of other macromolecular and small-molecule crystals whose crystallization involves vaporization of water.

Crystal structure of the copper homeostasis protein (CutCm) from Shigella flexneri at 1.7 Å resolution: The first structure of a new sequence family of TIM barrels

De-Yu Zhu, Yong-Qun Zhu, Ren-Huai Huang, Ye Xiang, Na Yang, Hong-Xia Lu, Gen-Pei Li, Qi Jin, and Da-Cheng Wang* Center for Structural and Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People’s Republic of China State Key Laboratory for Molecular Virology and Genetic Engineering, Beijing, People’s Republic of China Graduate School of the Chinese Academy of Sciences, Beijing, People’s Republic of China

Crystal structure of human SH3BGRL protein: the first structure of the human SH3BGR family representing a novel class of thioredoxin fold proteins

Introduction. The humanSH3BGRL (h-SH3BGRL) gene is a member of the human SH3BGR family. The human SH3BGR (SH3 binding glutamic acid-rich) gene was cloned and characterized in 1997 in an effort to identify new genes located to chromosome 21. After the characterization of SH3BGR, three novel human genes, h-SH3BGRL, h-SH3BGRL2, and h-SH3BGRL3, were identified, showing a high homology to the N-terminal region of the h-SH3BGR protein. They are therefore believed to be a new family of human gene, the h-SH3BGR gene family. The h-SH3BGRL gene located to chromosome Xq13.3 encodes for a small protein of 114 amino acids, which is apparently widely expressed in many tissues including liver and blood. The h-SH3BGRLprotein features a prolinerich sequence (PLPPQIF), which contains both the SH3 binding (PXXP) and the Homer EVH1 binding (PPXXF) motif. This proline-rich region is highly conserved in h-SH3BGR family and was expected to be functionally important. Although it is known that the SH3BGRL gene is missing in a mentally retarded male patient, the exact functional role of h-SH3BGRL is still needed to be defined. Here we report the crystal structure of h-SH3BGRL protein determined by the SIRAS method. The structure indicates that SH3BGRL can not bind to the SH3 or Homer EVH1 domain as previously expected due to our finding that the binding motif (PLPPQIF) is buried in the tertiary structure. As the first structure of the human SH3BGR protein family, the tertiary structure of h-SH3BGRL shows a typical thioredoxin fold at the Nterminal part and a helix-loop-helix motif at the Cterminal lobe. Sequence and structure comparisons show that h-SH3BGRL belongs to the thioredoxin fold protein family but it is distinct from all five classes of the thioredoxin fold proteins identified thus far. According to the unique structural and functional characterizations, h-SH3BGRL represents a novel class of the thioredoxin fold proteins.

Crystallization and preliminary crystallographic studies of a novel antifungal protein with five disulfide bridges from Eucommia ulmoides Oliver

Two antifungal proteins, named Eucommia antifungal peptides 1 and 2 (EAFP1 and EAFP2), have been purified from the bark of the tree E. ulmoides Oliver and show a relatively broad spectrum of antifungal activity against several agriculturally important plant pathogens. One of these small proteins (EAFP2) has been crystallized. The crystal belongs to space group P2(1), with unit-cell parameters a = 19.01, b = 23.16, c = 30.69 A, beta = 98.54 degrees. 1.0 A resolution data were collected from an EAFP2 crystal and have been used to obtain phase information directly by an ab initio method.

Surface morphology and kinetic properties in rapid growth of EAFP protein crystals investigated by atomic force microscopy

The whole process of EAFP protein monoclinic crystal growth with an extremely fast rate has been observed by atomic force microscopy. The results showed that the patterns of the growth images in rapidly growing crystals are complicated. The two-dimensional multi-layered stacks of growth steps are characteristic of higher supersaturation and the growth of steps proceeds in a manner of strong anisotropic spiral dislocations dominantly under lower supersaturation conditions. The complex dislocation sources, including multiple dislocation and multi-interacting single dislocation sources, the constant step-split and the propagation of trooped steps were observed on the {100} surfaces of growing EAFP crystals. The step height of each layer generated either by two-dimensional nucleation at higher supersaturation or by screw dislocation at lower supersaturation is about 2-3 nm, which corresponds to the length of the crystallographic unit cell. Although the rate of advancement for each growth step is similar to that of other protein crystal growth, the unique way of the propagation distinct with the trooped steps, by which a bundle of steps are strapped together, would be responsible for the rapid growth of EAFP crystals. All features show a possible mechanism by which the fast growth of EAFP crystals could be attained. The structural basis of the growth mechanism is also discussed.