Blood group type glycosphingolipids of human kidneys. Structural characterization of extended globo-series compounds

Blood group type glycosphingolipids present in kidneys of blood group A and B human individuals have been isolated and structurally characterized by mass spectrometry, proton NMR spectroscopy, degradation studies and by their reactivity with various monoclonal antibodies andEscherichia coli bacteria. The two major complex glycolipids present in the blood group A and B kidneys were globopentaosylceramide (IV³Gal-Gb₄Cer) and the X pentaglycosylceramide (III³Fuc-nLc₄Cer). The major blood group A glycolipid in the blood group A kidneys was based on the type 4 chain (globo-series). There were also small amounts of the type 2 chain and trace amounts of the type 1 and type 3 chain based A glycolipids. In addition, the blood group H type 4 chain structure was present together with Le^a and Le^b compounds. In the blood group B kidneys, the major B glycolipids were monofucosylated hexa- and octaglycosylceramides, where the former were based on the type 2 carbohydrate chain. The blood group B type 4 chain heptaglycosylceramide was found to be a minor component making up only about 1% of the total blood group B structures. Abbreviations: for blood group glycolipid antigens the short hand designation stands for blood group—number of sugar residues—type of carbohydrate chain. Thus A-7-4 means a type 4 chain blood group A heptaglycosylceramide. The sugar types are abbreviated for mass spectrometry to Hex for hexose, HexNAc forN-acetylhexosamine and dHex for deoxyhexose.     

Syntheses and characterization of novel sterically demanding amido ligand derivatives and their alkali metal ion complexes, crystal structures of [SiH2Mes2], [NH(SiHMes2)2] and [Na{N(SiHMes2)2}(OEt2)]

Three bulky silanes, [SiH₂Mes₂] (1), [SiHMeMes₂] (2), SiHMes₃ (3), two novel amines, [NH(SiHMes₂)₂] (4), NH₂(SiMeMes₂) (5), and three novel alkali metal ion complexes, [Na{N(SiHMes₂)₂}(OEt₂)] (6), Li{N(SiHMes₂)₂} (7), K{N(SiHMes₂)₂} (8), have been synthesized and characterized by multinuclear NMR and mass spectroscopy. The structures of compounds 1, 4 and 6 have been determined by X-ray crystallography. The spectroscopy and structural results are discussed.     

Structural characterization of Lyn-SH3 domain in complex with a herpesviral protein reveals an extended recognition motif that enhances binding affinity

The Src homology 3 (SH3) domain of the Src family kinase Lyn binds to the herpesviral tyrosine kinase interacting protein (Tip) more than one order of magnitude stronger than other closely related members of the Src family. In order to identify the molecular basis for high-affinity binding, the structure of free and Tip-bound Lyn-SH3 was determined by NMR spectroscopy. Tip forms additional contacts outside its classical proline-rich recognition motif and, in particular, a strictly conserved leucine (L186) of the C-terminally adjacent sequence stretch packs into a hydrophobic pocket on the Lyn surface. Although the existence of this pocket is no unique property of Lyn-SH3, Lyn is the only Src family kinase that contains an additional aromatic residue (H41) in the n-Src loop as part of this pocket. H41 covers L186 of Tip by forming tight hydrophobic contacts, and model calculations suggest that the increase in binding affinity compared with other SH3 domains can mainly be attributed to these additional interactions. These findings indicate that this pocket can mediate specificity even between otherwise closely related SH3 domains.     

Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.     

Synthesis, characterization and structure of metallocene dicyanamide complexes

The bis(cyclopentadienyl) complexes [Cp₂Ti(dca)]₂O and Cp₂V(dca)₂ (dca = dicyanamide) have been prepared by reaction of sodium dicyanamide with aqueous solution of titanocene dichloride and vanadocene dichloride, respectively. The X-ray structure analyses of both complexes confirmed monodentate coordination of dicyanamide ligand through the terminal nitrogen atom of cyano group.     

Structural characterization of the self‐association domain of swallow

Swallow, a 62 kDa multidomain protein, is required for the proper localization of several mRNAs involved in the development of Drosophila oocytes. The dimerization of Swallow depends on a 71‐residue self‐association domain in the center of the protein sequence, and is significantly stabilized by a binding interaction with dynein light chain (LC8). Here, we detail the use of solution‐state nuclear magnetic resonance spectroscopy to characterize the structure of this self‐association domain, thereby establishing that this domain forms a parallel coiled‐coil and providing insight into how the stability of the dimerization interaction is regulated.     

Structural basis for the dual U4 and U4atac snRNA-binding specificity of spliceosomal protein hPrp31

Human proteins 15.5K and hPrp31 are components of the major spliceosomal U4 snRNP and of the minor spliceosomal U4atac snRNP. The two proteins bind to related 5'-stem loops (5'SLs) of the U4 and U4atac snRNAs in a strictly sequential fashion. The primary binding 15.5K protein binds at K-turns that exhibit identical sequences in the two snRNAs. However, RNA sequences contacted by the secondary binding hPrp31 differ in U4 and U4atac snRNAs, and the mechanism by which hPrp31 achieves its dual specificity is presently unknown. We show by crystal structure analysis that the capping pentaloops of the U4 and U4atac 5'SLs adopt different structures in the ternary hPrp31-15.5K-snRNA complexes. In U4atac snRNA, a noncanonical base pair forms across the pentaloop, based on which the RNA establishes more intimate interactions with hPrp31 compared with U4 snRNA. Stacking of hPrp31-His270 on the noncanonical base pair at the base of the U4atac pentaloop recapitulates intramolecular stabilizing principles known from the UUCG and GNRA families of RNA tetraloops. Rational mutagenesis corroborated the importance of the noncanonical base pair and the U4atac-specific hPrp31-RNA interactions for complex stability. The more extensive hPrp31-U4atac snRNA interactions are in line with a higher stability of the U4atac compared with the U4-based ternary complex seen in gel-shift assays, which may explain how U4atac snRNA can compete with the more abundant U4 snRNA for the same protein partners in vivo.     

Structural and functional characterization of TgpA,a critical protein for the viability of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen associated with severe diseases, such as cystic fibrosis. During an extensive search for novel essential genes, we identified tgpA (locus PA2873) in P. aeruginosa PAO1, as a gene playing a critical role in bacterial viability. TgpA, the translated protein, is an internal membrane protein with a periplasmic soluble domain, predicted to be endowed with a transglutaminase-like fold, hosting the Cys404, His448, and Asp464 triad. We report here that Cys404 mutation hampers the essential role of TgpA in granting P. aeruginosa viability. Moreover, we present the crystal structure of the TgpA periplasmic domain at 1.6?Å resolution as a first step towards structure–activity analysis of a new potential target for the discovery of antibacterial compounds.     

Structures and target RNA preferences of the RNA-binding protein family of IGF2BPs: An overview

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Structural characterization of an intrinsically unfolded mini-HBX protein from hepatitis B virus

The hepatitis B virus x protein (HBX) is expressed in HBVinfected liver cells and can interact with a wide range of cellular proteins. In order to understand such promiscuous behavior of HBX we expressed a truncated mini-HBX protein (named Tr-HBX) (residues 18-142) with 5 Cys → Ser mutations and characterized its structural features using circular dichroism (CD) spectropolarimetry, NMR spectroscopy as well as bioinformatics tools for predicting disorder in intrinsically unstructured proteins (IUPs). The secondary structural content of Tr-HBX from CD data suggests that Tr-HBX is only partially folded. The protein disorder prediction by IUPred reveals that the unstructured region encompasses its N-terminal ～30 residues of Tr-HBX. A two-dimensional (1)H-(15)N HSQC NMR spectrum exhibits fewer number of resonances than expected, suggesting that Tr-HBX is a hybrid type IUP where its folded C-terminal half coexists with a disordered N-terminal region. Many IUPs are known to be capable of having promiscuous interactions with a multitude of target proteins. Therefore the intrinsically disordered nature of Tr-HBX revealed in this study provides a partial structural basis for the promiscuous structure-function behavior of HBX.     

Structure and functional characterization of the periplasmic N-terminal polypeptide domain of the sugar-specific ion channel protein (ScrY porin)

The structure of the sucrose-specific porin (ScrY) from Salmonella typhimurium has been elucidated by X-ray crystallography to consist of 18 antiparallel beta-strands, associated as a trimer complex similar to ion-transport channels. However, the 71-amino-acid-residue N-terminal periplasmic domain was not determined from the crystal structure due to the absence of sufficient electron density. The N-terminal polypeptide contains a coiled-coil structural motif and has been assumed to play a role in the sugar binding of ScrY porin. In this study the proteolytic stability and a specific proteolytic truncation site at the N-terminal domain were identified by the complete primary structure characterization of ScrY porin, using MALDI mass spectrometry and post-source-decay fragmentation. The secondary structure and supramolecular association of the coiled-coil N-terminal domain were determined by chemical synthesis of the complete N-terminal polypeptide and several partial sequences and their spectroscopic, biophysical, and mass spectrometric characterization. Circular dichroism spectra revealed predominant alpha-helical conformation for the putative coiled-coil domain comprising residues 4-46. Specific association to both dimer and trimer complexes was identified by electrospray ionization mass spectra and was ascertained by dynamic light scattering and electrophoresis data. The role of the N-terminal domain in sugar binding was examined by comparative TR-NOE-NMR spectroscopy of the complete ScrY porin and a recombinant mutant, ScrY(delta1-62), lacking the N-terminal polypeptide. The TR-NOE-NMR data showed a strong influence of ScrY porin on the sugar-binding affinity and suggested a possible function of the periplasmic N terminus for supramolecular stabilization and low-affinity sugar binding.     

Expression, purification, and initial structural characterization of nonstructural protein 2B, an integral membrane protein of Dengue-2 virus, in detergent micelles

Dengue virus causes serious diseases affecting people in tropical and sub-tropical regions. The nonstructural (NS) protein 2B is an integral membrane protein and important for the regulation of viral protease NS3, which is significant for virus replication. The NS2B-NS3 complex is an important drug target for treating dengue fever. However, little is known about the structure of NS2B in its entirety. Herein, we describe the expression and purification of this integral membrane protein from cell membrane and inclusion bodies of Escherichia coli cells. The initial nuclear magnetic resonance (NMR) and circular dichroism (CD) results indicate that the purified protein adopts alpha-helical structures in LMPG and TDPC micelles.     

Synthesis, spectroscopic properties, X-ray single crystal analysis and antimicrobial activities of organotin(IV) 4-(4-methoxyphenyl)piperazine-1-carbodithioates

A series of mononuclear organotin(IV) complexes of the types, R₃SnL {R = C₄H₉ (1), C₆H₁₁ (2), CH₃ (3) and C₆H₅ (4)}, R₂SnClL {R = C₄H₉ (5), C₂H₅ (7) and CH₃ (9)} and R₂SnL₂ {R = C₄H₉ (6), C₂H₅ (8) and CH₃ (10)}, have been synthesized, where L = 4-(4-methoxyphenyl)piperazine-1-carbodithioate. The ligand-salt and the complexes have been characterized by Raman, FT-IR and multinuclear NMR (¹H, ¹³C and ¹¹⁹Sn) spectroscopy and elemental microanalysis (CHNS). The spectroscopic data substantiate coordination of the ligands to the organotin moieties. The structures of complexes 4 and 6 have been determined by single-crystal X-ray diffraction and illustrate the asymmetric bidentate bonding of the ligand. The packing diagrams indicate O···H and π···H intermolecular interactions in complex 4 and intermolecular S₂C···H interactions in complex 6, resulting in layer structures for both complexes. A subsequent antimicrobial study indicates that the compounds are active biologically and may well be the basis for a new class of fungicides.     

Purification, identification and activity of phomodione, a furandione from an endophytic Phoma species

Phomodione, [(4aS(*),9bR(*))-2,6-diacetyl-7-hydroxy-4a,9-dimethoxy-8,9b-dimethyl-4a.9b-dihydrodibenzo[b,d]furan-1,3(2H,4H)-dione], an usnic acid derivative, was isolated from culture broth of a Phoma species, discovered as an endophyte on a Guinea plant (Saurauia scaberrinae). It was identified using NMR, X-ray crystallography, high resolution mass spectrometry, as well as infrared and Raman spectroscopy. In addition to phomodione, usnic acid and cercosporamide, known compounds with antibiotic activity, were also found in the culture medium. Phomodione exhibited a minimum inhibitory concentration of 1.6 microg/mL against Staphylococcus aureus using the disk diffusion assay, and was active against a representative oomycete, ascomycete and basidiomycete at between three and eight micro-grams per mL.     

Isolation,characterization and molecular 3D model of human GDE4, a novel membrane protein containing glycerophosphodiester phosphodiesterase domain

As a transmembrane protein family, glycerophosphodiester phosphodiesterase (GDPD/GDE) catalyzes the hydrolysis of deacylated glycerophospholipids to glycerol phosphate and alcohol. To date, seven mammalian GDEs have been virtually cloned or predicted by bioinformatics analysis, however, GDE4 has not been molecular isolated and characterized in mammal. Here we report molecular cloning of human GDE4 encoding cDNA sequence, which is 945 base pairs long encoding a 314-amino acid protein with 2 transmembrane regions and a GDE motif. The human GDE1 gene is located on chromosome 19q22 and contains ten exons and nine introns. A molecular 3-D model provides the first structural information of human GDE4 and suggests a triose-phosphate-isomerase barrel core as typically found in bacterial GDPDs. Furthermore, a model of the putative catalytic residues highlights that the individual core residues Glu72, Asp74, and His87 are crucial to maintaining GDE4 catalytic activity. Western blotting shows that human GDE4 is a 36 kDa protein. Subcellular localization of GDE4 tagged with enhanced green fluorescence protein is in the cytoplasm, especially accumulated in the perinuclear region and the cell periphery. Moreover, over-expression of GDE4 did not induce neurite formation or change cell morphology. These results indicate GDE4 protein is a member of the GDE family and suggest it may play different roles from other members of GDE family.