Crystal structure of scytalidoglutamic peptidase with its first potent inhibitor provides insights into substrate specificity and catalysis

Scytalidoglutamic peptidase (SGP) from Scytalidium lignicolum is the founding member of the newly discovered\ family of peptidases, G1, so far found exclusively in fungi. The crystal structure of SGP revealed a previously undescribed fold for peptidases and a unique catalytic dyad of residues Gln53 and Glu136. Surprisingly, the beta-sandwich structure of SGP is strikingly similar to members of the carbohydrate-binding concanavalin A-like lectins/glucanases superfamily. By analogy with the active sites of aspartic peptidases, a mechanism employing nucleophillic attack by a water molecule activated by the general base functionality of Glu136 has been proposed. Here, we report the first crystal structures of SGP in complex with two transition state peptide analogs designed to mimic the tetrahedral intermediate of the proteolytic reaction. Of these two analogs, the one containing a central S-hydroxyl group is a potent sub-nanomolar inhibitor of SGP. The inhibitor binds non-covalently to the concave surface of the upper beta-sheet and enables delineation of the S4 to S3' substrate specificity pockets of the enzyme. Structural differences in these pockets account for the unique substrate preferences of SGP among peptidases having an acidic pH optimum. Inhibitor binding is accompanied by a structuring of the region comprising residues Tyr71-Gly80 from being mostly disordered in the apoenzyme and leading to positioning of crucial active site residues for establishing enzyme-inhibitor contacts. In addition, conformational rearrangements are seen in a disulfide bridged surface loop (Cys141-Cys148), which moves inwards, partially closing the open substrate binding cleft of the native enzyme. The non-hydrolysable scissile bond analog of the inhibitor is located in the active site forming close contacts with Gln53 and Glu136. The nucleophilic water molecule is displaced and a unique mode of binding is observed with the S-OH of the inhibitor occupying the oxyanion binding site of the proposed tetrahedral intermediate. Details of the enzyme-inhibitor interactions and mechanistic interpretations are discussed.     

青藏高原栽培青稞淀粉粒蛋白多态性及其与淀粉含量的关系

淀粉粒蛋白(Starch granule proteins, SGPs)是一类与淀粉粒结合的微量蛋白, 其变异可能与淀粉特性相关。实验率先研究了我国青藏高原栽培青稞的SGP组成, 初步探索了所分离的SGP蛋白与淀粉合成的关系。青藏高原青稞的SGP组成存在多态性, 66份青稞材料中分离了10种主要的SGPs, 16种组合带谱。西藏青稞和四川青稞的SGP组成有很大差异, SGP组成具有地域差异性。不同组合带谱材料间淀粉含量差异显著性检验显示, 实验所分离的淀粉粒蛋白可能与淀粉的合成相关。     

A model of the complex between human β-microseminoprotein and CRISP-3 based on NMR data

β-Microseminoprotein (MSP), a 10 kDa seminal plasma protein, forms a tight complex with cysteine-rich secretory protein 3 (CRISP-3) from granulocytes. The 3D structure of human MSP has been determined but there is as yet no 3D structure for CRISP-3. We have now studied the complex between human MSP and CRISP-3 with multidimensional NMR. ¹⁵N-HSQC spectra show substantial differences between free and complexed hMSP. Using several 3D-NMR spectra of triply labeled hMSP in complex with a recombinant N-terminal domain of CRISP-3, most of the backbone of hMSP could be assigned. The data show that only one side of hMSP, comprising β-strands 1, 4, 5, and 8 are affected by the complex formation, indicating that β-strands 1 and 8 form the main binding surface. Based on this we present a tentative structure for the hMSP-CRISP-3 complex using the known crystal structure of triflin as a model of CRISP-3.     

Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline

Background

An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N-acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one.

Methods

We carried out the transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk.

Results

Endo-M-N175Q showed efficient transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield.

Conclusions

Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed transglycosylation.

General significance

Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.     

夏氏疟原虫入侵时小鼠红细胞膜唾液酸糖蛋白的改变

夏氏疟原虫入侵时小鼠红细胞膜唾液酸糖蛋白的改变刘俊凡,卢义钦（湖南医科大学生化教研室,长沙４１Ｏ０７８〕ＲｏｎａｌｄＬ．ＮｏｇｅｌＯｌｇａＯ．Ｂｌｕｍｅｎｆｅｌｄ（爱因斯坦医学院内科血液学研究室和生化学系,美国纽约１０４６１）关键词夏氏疟原虫;红细胞...     

2,3,5-Tris-ethylenimino-1,4-benzoquinone (Trenimon): some chemical and biological properties

The three aziridine rings of 2,3,5-tris-ethylenimino-1,4-benzoquinone (Trenimon) were found to be active alkylating centres in the presence of acid catalyst, but differed in reactivity among themselves. At a given pH Trenimon was readily reduced by a number of agents including cysteine, which probably formed a substitution derivative. The cysteine derivative possessed three alkylating groups which were more reactive than those of pure Trenimon. Trenimon was soluble in fat solvents but the reduction and substitution derivatives were not. Trenimon was bound to hemoglobin and to albumins, probably by a substitution reaction between a sulfhydryl group of the protein and the 6-carbon atom of the drug. Binding to globulin occurred only following reduction of the protein. Trenimon was readily taken up by L5178Y lymphoma cells in suspension culture and was highly toxic but the cysteine derivative was not readily taken up and was less toxic than Trenimon by three orders of magnitude. The implications of the solubility and other physical properties of the alkylating agents are discussed, and evidence is presented that the biologically effective concentration of a drug is that which binds to the cell surface.     

Circulation dynamics of Salmonella enterica subsp. enterica ser. Gallinarum biovar Gallinarum in a poultry farm infested by Dermanyssus gallinae

Dermanyssus gallinae (Mesostigmata: Dermanyssidae, De Geer, 1778) is an ectoparasite of poultry, suspected to play a role as a vector of Salmonella enterica subsp. enterica ser. Gallinarum. Despite an association between them being reported, the actual dynamics in field remain unclear. Therefore, the present study aimed to confirm the interactions among mites, pathogen and chickens. The study was carried out in an industrial poultry farm infested by D. gallinae, during an outbreak of fowl typhoid. The presence of S. Gallinarum in mites was assessed and quantified by a semi‐nested polymerase chain reaction (PCR) and real‐time PCR, respectively, in mites collected during two subsequent productive cycles and the sanitary break. The anti‐group D Salmonella antibodies were quantified by an enzyme‐linked immunosorbent assay. During the outbreak and the sanitary break, S. Gallinarum was constantly present in mites. In the second cycle, scattered positivity was observed, although hens did not exhibit signs of fowl typhoid, as a result of the vaccination with BIO‐VAC SGP695 (Fatro, Ozzano Emilia Bo, Italy). The data strongly suggest that D. gallinae acts as reservoir of S. Gallinarum, thus allowing the pathogen to persist in farms. Furthermore, the present study has highlighted the interactions among D. gallinae, S. Gallinarum and hens with respect to enhancing the mite‐mediated circulation of S. Gallinarum in an infested poultry farm.     

Novel aquatic silk genes from Simulium (Psilozia) vittatum (Zett) Diptera: Simuliidae

The silks of arthropods have an elementary role in the natural history of the organisms that spin them, yet they are coded by rapidly evolving genes leading some authors to speculate that silk proteins are non-homologous proteins co-opted multiple times independently for similar functions. However, some general structural patterns are emerging. In this work we identified three major silk gland proteins using a combined biochemical, proteomic, next-generation sequencing and bioinformatic approach. Biochemical characterization determined that they were phosphorylated with multiple isoforms and potentially differential phosphorylation. Structural characterization showed that their structure was more similar to silk proteins from distantly related aquatic Trichopteran species than more closely related terrestrial or aquatic Diptera. Overall, our approach is easily transferable to any non-model species and if used across a larger number of aquatic species, we will be able to better understand the processes involved in linking the secondary structure of silk proteins with their function between in an organisms and its habitat.     

Disulfide bond assignment of the Ebola virus secreted glycoprotein SGP

The non-structural glycoprotein (SGP) of Ebola virus (EboV) is secreted in large amounts from infected cells as a disulfide-linked homodimer. In this communication, highly purified SGP, derived from Vero E6 cultures infected with the Zaire species of EboV, was used to determine the correct localization of inter- and intrachain disulfide bonds. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis of proteolytic cleavage fragments indicates that all cysteines (six per monomeric unit) form unique disulfide bonds. Monomers of the SGP homodimer are joined in a parallel manner by two intersubunit disulfide bonds formed between paired N-terminal and C-terminal cysteines (C53-C53' and C306-C306'). The remaining cysteines are involved in intrachain disulfide bonding (paired as C108-C135 and C121-C147), which resembles the disulfide bond topology of fibronectin type II domains. The findings presented here provide the foundation for future studies aimed at defining the structural and functional properties of SGP.