Maikun Teng

Crystal Structure of the Cysteine-rich Secretory Protein Stecrisp Reveals That the Cysteine-rich Domain Has a K+ Channel Inhibitor-like Fold

Stecrisp from Trimeresurus stejnegeri snake venom belongs to a family of cysteine-rich secretory proteins (CRISP) that have various functions related to sperm-egg fusion, innate host defense, and the blockage of ion channels. Here we present the crystal structure of stecrisp refined to 1.6-Å resolution. It shows that stecrisp contains three regions, namely a PR-1 (pathogenesis-related proteins of group1) domain, a hinge, and a cysteine-rich domain (CRD). A conformation of solvent-exposed and -conserved residues (His60, Glu75, Glu96, and His115) in the PR-1 domain similar to that of their counterparts in homologous structures suggests they may share some molecular mechanism. Three flexible loops of hypervariable sequence surrounding the possible substrate binding site in the PR-1 domain show an evident difference in homologous structures, implying that a great diversity of species- and substrate-specific interactions may be involved in recognition and catalysis. The hinge is fixed by two crossed disulfide bonds formed by four of ten characteristic cysteines in the carboxyl-terminal region and is important for stabilizing the N-terminal PR-1 domain. Spatially separated from the PR-1 domain, CRD possesses a similar fold with two K+ channel inhibitors (Bgk and Shk). Several candidates for the possible functional sites of ion channel blocking are located in a solvent-exposed loop in the CRD. The structure of stecrisp will provide a prototypic architecture for a structural and functional exploration of the diverse members of the CRISP family.

Core Structure of the Yeast Spt4-Spt5 Complex: A Conserved Module for Regulation of Transcription Elongation

The Spt4-Spt5 complex is an essential RNA polymerase II elongation factor found in all eukaryotes and important for gene regulation. We report here the crystal structure of Saccharomyces cerevisiae Spt4 bound to the NGN domain of Spt5. This structure reveals that Spt4-Spt5 binding is governed by an acid-dipole interaction between Spt5 and Spt4. Mutations that disrupt this interaction disrupt the complex. Residues forming this pivotal interaction are conserved in the archaeal homologs of Spt4 and Spt5, which we show also form a complex. Even though bacteria lack a Spt4 homolog, the NGN domains of Spt5 and its bacterial homologs are structurally similar. Spt4 is located at a position that may help to maintain the functional conformation of the following KOW domains in Spt5. This structural and evolutionary perspective of the Spt4-Spt5 complex and its homologs suggest that it is an ancient, core component of the transcription elongation machinery.

Crystal structure of human vacuolar protein sorting protein 29 reveals a phosphodiesterase/nuclease-like fold and two protein-protein interaction sites

Vacuolar protein sorting protein 29 (Vps29p), which is involved in retrograde trafficking from prevacuolar endosomes to the trans-Golgi network, performs its biological functions by participating in the formation of a “retromer complex.” In human cells, this complex comprises four conserved proteins: hVps35p, hVps29p, hVps26p, and sorting nexin 1 protein (SNX1). Here, we report the crystal structure of hVps29p at 2.1 Å resolution, the first three-dimensional structure of the retromer subunits. This novel structure adopts a four-layered α-β-β-α sandwich fold. hVps29p contains a metal-binding site that is very similar to the active sites of some proteins of the phosphodiesterase/nuclease protein family, indicating that hVps29p may carry out chemically similar functions. Structure and sequence conservation analysis suggests that hVps29p contains two protein-protein interaction sites. One site, which potentially serves as the interface between hVps29p and hVps35p, comprises 5 conserved hydrophobic and 8 hydrophilic residues. The other site is relatively more hydrophilic and may serve as a binding interface with hVps26p, SNX1, or other target proteins.

Crystal Structures and Amidolytic Activities of Two Glycosylated Snake Venom Serine Proteinases

We deduced that Agkistrodon actus venom serine proteinases I and II, previously isolated from the venom of A. acutus (Zhu, Z., Gong, P., Teng, M., and Niu, L. (2003) Acta Crystallogr. Sect. D Biol. Crystallogr. 59, 547–550), are encoded by two almost identical genes, with only the single substitution Asp for Asn at residue 62. Amidolytic assays indicated that they possess slightly different enzymatic properties. Crystal structures of A. actus venom serine proteinases I and II were determined at resolution of 2.0 and 2.1 Å with the identification of trisaccharide (NAG301-FUC302-NAG303) and monosaccharide (NAG301) residues in them, respectively. The substrate binding sites S3 of the two proteinases appear much shallower than that of Trimeresurus stejnegeri venom plasminogen activator despite the overall structural similarity. Based on structural analysis, we showed that these Asn35-linked oligosaccharides collide spatially with some inhibitors, such as soybean trypsin inhibitor, and would therefore hinder their inhibitory binding. Difference of the carbohydrates in both the proteinases might also lead to their altered catalytic activities.

Purification, partial characterization, crystallization and structural determination of AHP-LAAO, a novel L-amino-acid oxidase with cell apoptosis-inducing activity from Agkistrodon halys pallas venom

A snake-venom protein named AHP-LAAO has been purified from Agkistrodon halys pallas venom using four-stage chromatography. AHP-LAAO is a novel member of the snake-venom L-amino-acid oxidase family. Its amino-acid sequence shows high homology to other members of this family. For L-leucine, the values of k(cat) and K(M) are 31.1 s(-1) and 0.25 mM, respectively. The molecular weight of AHP-LAAO is about 60.7 kDa as determined by MALDI-TOF mass spectrometry. AHP-LAAO can also induce apoptosis of cultured Hela cells. Two sets of diffraction data with similar resolution limits (about 2.5 A) were collected independently at MacCHESS (Cornell High Energy Synchrotron Source, USA) and IHEP (Institute of High Energy Physics, Beijing, China). The crystals belong to space group I2(1)3, with unit-cell parameter a = 169.31 A, corresponding to one molecule in the asymmetric unit and a volume-to-weight ratio of 3.33 A(3) Da(-1). The final structural model is similar to that of L-amino-acid oxidase from Calloselasma rhodostoma venom.

NetAlign: a web-based tool for comparison of protein interaction networks

UNLABELLED NetAlign is a web-based tool designed to enable comparative analysis of protein interaction networks (PINs). NetAlign compares a query PIN with a target PIN by combining interaction topology and sequence similarity to identify conserved network substructures (CoNSs), which may derive from a common ancestor and disclose conserved topological organization of interactions in evolution. To exemplify the application of NetAlign, we perform two genome-scale comparisons with (1) the Escherichia coli PIN against the Helicobacter pylori PIN and (2) the Saccharomyces cerevisiae PIN against the Caenorrhabditis elegans PIN. Many of the identified CoNSs correspond to known complexes; therefore, cross-species PIN comparison provides a way for discovery of conserved modules. In addition, based on the species-to-species differences in CoNSs, we reformulate the problems of protein-protein interaction (PPI) prediction and species divergence from a network perspective. AVAILABILITY http://www1.ustc.edu.cn/lab/pcrystal/NetAlign.

Comparison of protein interaction networks reveals species conservation and divergence

BackgroundRecent progresses in high-throughput proteomics have provided us with a first chance to characterize protein interaction networks (PINs), but also raised new challenges in interpreting the accumulating data.ResultsMotivated by the need of analyzing and interpreting the fast-growing data in the field of proteomics, we propose a comparative strategy to carry out global analysis of PINs. We compare two PINs by combining interaction topology and sequence similarity to identify conserved network substructures (CoNSs). Using this approach we perform twenty-one pairwise comparisons among the seven recently available PINs of E.coli, H.pylori, S.cerevisiae, C.elegans, D.melanogaster, M.musculus and H.sapiens. In spite of the incompleteness of data, PIN comparison discloses species conservation at the network level and the identified CoNSs are also functionally conserved and involve in basic cellular functions. We investigate the yeast CoNSs and find that many of them correspond to known complexes. We also find that different species harbor many conserved interaction regions that are topologically identical and these regions can constitute larger interaction regions that are topologically different but similar in framework. Based on the species-to-species difference in CoNSs, we infer potential species divergence. It seems that different species organize orthologs in similar but not necessarily the same topology to achieve similar or the same function. This attributes much to duplication and divergence of genes and their associated interactions. Finally, as the application of CoNSs, we predict 101 protein-protein interactions (PPIs), annotate 339 new protein functions and deduce 170 pairs of orthologs.ConclusionOur result demonstrates that the cross-species comparison strategy we adopt is powerful for the exploration of biological problems from the perspective of networks.

Hepatitis B virus and hepatocellular carcinoma at the miRNA level

AIM To study Hepatitis B virus (HBV) infection and its association with hepatocellular carcinoma (HCC) at the miRNA level. METHODS Three cellular models were used to investigate miRNA expression changes during HBV infection: human HepG2 hepatoblastoma cell line as a model without HBV infection; HepG2 cell line transfected with a 1.3-fold full-length HBV genome as an acute infection model; and HepG2.2.15 cell line, which is derived from HepG2 and stably transfected with a complete HBV genome, as a chronic infection model. The miRNA levels were examined using microarray technology. To explore the relationship between HBV infection and HCC genesis at the miRNA level, we downloaded from national center for biotechnology information Gene Expression Omnibus an miRNA expression dataset derived from HCC patients, most of whom are HBV carriers. We compared the miRNA expression alterations during HBV infection with those in HCC patients, by analyzing miRNA expression change profiles statistically. RESULTS Seventy-seven and 48 miRNAs were differentially expressed during acute and chronic HBV infection, respectively. Among these miRNAs, 25 were in common, the intersection of which was significant under the hypergeometric test (P = 1.3 × 10⁻¹¹). Fourteen miRNAs were observed to change coherently in the acute and chronic infections, with one upregulated and 13 downregulated. Eleven showed inverse changes during the two phases of infection; downregulated in the acute infection and upregulated in the chronic infection. The results imply that common and specific mechanisms exist at the miRNA level during acute and chronic HBV infection. Besides, comparative analysis of the miRNA expression changes during HBV infection with those in HCC indicates that, although miRNA expression changes during HBV infection are distinct from those in HCC patients (P < 2.2 × 10⁻¹⁶), they exhibited significant correlations (P = 0.0229 for acute infection; P = 0.0084 for chronic infection). Perturbation of miRNA expression during chronic HBV infection was closer to that in HCC patients than that during acute HBV infection. This observation implies the contribution of miRNAs to HCC genesis from HBV infection. According to their patterns of differential expression in acute and chronic HBV infection, as well as in HCC, miRNAs of potential research interest could be identified, such as miR-18a/miR-18b, miR-106a, miR-221 and miR-101. For instance, the gradient expression alteration of miR-221 in the above three phases, which is downregulated in acute HBV infection, normally expressed in chronic HBV infection, and upregulated in HCC, indicates that it may be a key effector for progression of the disease. CONCLUSION Our analysis provides insights into HBV infection and related HCC in relation to miRNAs, and reveals some candidate miRNAs for future studies.

cDNA cloning, functional expression and antifungal activities of a dimeric plant defensin SPE10 from Pachyrrhizus erosus seeds

SPE10 is an antifungal protein isolated from the seeds of Pachyrrhizus erosus. cDNA encoding a 47 amino acid peptide was cloned by RT-PCR and the gene sequence proved SPE10 to be a new member of plant defensin family. The synthetic cDNA with codons preferred in yeast was cloned into the pPIC9 plasmid directly in-frame with the secretion signal α-mating factor, and highly expressed in methylotrophic Pichia pastoris. Activity assays showed the recombinant SPE10 inhibited specifically the growth of several pathogenic fungi as native SPE10. Circular dichroism and fluorescence spectroscopy analysis indicated that the native and recombinant protein should have same folding, though there are eight cystein residues in the sequence. Several evidence suggested SPE10 should be the first dimeric plant defensin reported so far.

PALS1 specifies the localization of ezrin to the apical membrane of gastric parietal cells

The ERM (ezrin/radixin/moesin) proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to regulated HCl secretion in gastric parietal cells. Here, we show that the integrity of ezrin is essential for parietal cell activation and provide the first evidence that ezrin interacts with PALS1, an evolutionarily conserved PDZ and SH3 domain-containing protein. Our biochemical study verifies that ezrin binds to PALS1 via its N terminus and is co-localized with PALS1 to the apical membrane of gastric parietal cells. Furthermore, our study shows that PALS1 is essential for the apical localization of ezrin, as either suppression of PALS1 protein accumulation or deletion of the PALS1-binding domain of ezrin eliminated the apical localization of ezrin. Finally, our study demonstrates the essential role of ezrin-PALS1 interaction in the apical membrane remodeling associated with parietal cell secretion. Taken together, these results define a novel molecular mechanism linking ezrin to the conserved apical polarity complexes and their roles in polarized epithelial secretion of gastric parietal cells.