Sequence and phylogenetic analysis of viper venom serine proteases

Snakebites are a major neglected tropical disease responsible for as many as 95000 deaths every year worldwide. Viper venom serine proteases disrupt haemostasis of prey and victims by affecting various stages of the blood coagulation system. A better understanding of their sequence, structure, function and phylogenetic relationships will improve the knowledge on the pathological conditions and aid in the development of novel therapeutics for treating snakebites. A large dataset for all available viper venom serine proteases was developed and analysed to study various features of these enzymes. Despite the large number of venom serine protease sequences available, only a small proportion of these have been functionally characterised. Although, they share some of the common features such as a C-terminal extension, GWG motif and disulphide linkages, they vary widely between each other in features such as isoelectric points, potential N-glycosylation sites and functional characteristics. Some of the serine proteases contain substitutions for one or more of the critical residues in catalytic triad or primary specificity pockets. Phylogenetic analysis clustered all the sequences in three major groups. The sequences with substitutions in catalytic triad or specificity pocket clustered together in separate groups. Our study provides the most complete information on viper venom serine proteases to date and improves the current knowledge on the sequence, structure, function and phylogenetic relationships of these enzymes. This collective analysis of venom serine proteases will help in understanding the complexity of envenomation and potential therapeutic avenues

Charcot-Leyden crystal protein/galectin-10 interacts with cationic ribonucleases and is required for eosinophil granulogenesis

BACKGROUND: The human eosinophil Charcot-Leyden Crystal (CLC) protein is a member of the Galectin superfamily and is also known as Galectin-10 (Gal-10). CLC/Gal-10 forms the distinctive hexagonal bipyramidal crystals considered hallmarks of eosinophil participation in allergic responses and related inflammatory reactions; however, the glycan-containing ligands of CLC/Gal-10, its cellular function(s), and its role(s) in allergic diseases are unknown. OBJECTIVE: We sought to determine the binding partners of CLC/Gal-10 and elucidate its role in eosinophil biology. METHODS: Intracellular binding partners were determined by ligand blotting with CLC/Gal-10, followed by co-immunoprecipitation and co-affinity purifications. The role of CLC/Gal-10 in eosinophil function was determined by employing enzyme activity assays, confocal microscopy, and shRNA knock-out of CLC/Gal-10 expression in human CD34(+) cord blood hematopoietic progenitors differentiated to eosinophils. RESULTS: CLC/Gal-10 interacts with both human eosinophil granule cationic ribonucleases, eosinophil-derived neurotoxin (EDN, RNS2) and eosinophil cationic protein (ECP, RNS3), and with murine eosinophil-associated ribonucleases. The interaction is independent of glycosylation and is not inhibitory toward endoribonuclease activity. Activation of eosinophils with INF-γ induces the rapid co-localization of CLC/Gal-10 with EDN/RNS2 and CD63. ShRNA knock-down of CLC/Gal-10 in human cord blood-derived CD34(+) progenitor cells impairs eosinophil granulogenesis. CONCLUSIONS: CLC/Gal-10 functions as a carrier for the sequestration and vesicular transport of the potent eosinophil granule cationic ribonucleases during both differentiation and degranulation, enabling their intracellular packaging and extracellular functions in allergic inflammation

Expression, purification, crystallization and preliminary crystallographic analysis of a putative Clostridium difficile surface protein Cwp19

Cwp19 is a putatively surface-located protein from Clostridium difficile. A recombinant N-terminal protein (residues 27–401) lacking the signal peptide and the C-terminal cell-wall-binding repeats (PFam04122) was crystallized using the sitting-drop vapour-diffusion method and diffracted to 2 Å resolution

Disease Variants of FGFR3 Reveal Molecular Basis for the Recognition and Additional Roles for Cdc37 in Hsp90 Chaperone System

Receptor tyrosine kinase FGFR3 is involved in many signaling networks and is frequently mutated in developmental disorders and cancer. The Hsp90/ Cdc37 chaperone system is essential for function of normal and neoplastic cells. Here we uncover the mechanistic inter-relationships between these pro- teins by combining approaches including NMR, HDX-MS, and SAXS. We show that several disease- linked mutations convert FGFR3 to a stronger client, where the determinant underpinning client strength involves an allosteric network through the N-lobe and at the lobe interface. We determine the architec- ture of the client kinase/Cdc37 complex and dem on- strate, together with site-speci?c information, that binding of Cdc37 to unrelated kinases induces a common, extensive conformational remodeling of the kinase N-lobe, beyond localized changes and in- teractions within the binary complex. As further shown for FGFR3, this processing by Cdc37 deacti- vates the kinase and presents it, in a speci ?c orienta- tion established in the complex, for direct recognition by Hsp90

Crystal structure of a-1,3-galactosyltransferase (a3GT) in a complex with p-nitrophenyl-ß-galactoside (pNPßGal)

The specificities of glycosyltransferases make them useful for the synthesis of biologically active oligosaccharides, but also restrict their range of products. In substrate engineering, substrate promiscuity is enhanced by attaching removable interactive groups to weak substrates. Thus, the attachment of ß p-nitrophenyl converts galactose from a poor into a good substrate of a-1,3-galactosyltransferase. The crystallographic structure of a complex of a3GT containing p-nitrophenyl-ß-galactoside shows that the p-nitrophenyl binds similarly to the N-acetylglucosamine of the substrate, N-acetyllactosamine, interacting with the indole of Trp249. p-Nitrophenyl, unlike N-acetylglucosamine, makes no H-bonds but has more non-polar interactions, making it an effective monosaccharide mimetic

Chimeric microtubule disruptors

A chimeric approach is used to discover microtubule disruptors with excellent in vitro activity and oral bioavailability; a ligand-protein interaction with carbonic anhydrase that enhances bioavailability is characterised by protein X-ray crystallography. Dosing of a representative chimera in a tumour xenograft model confirms the excellent therapeutic potential of the class