Structural basis for the carbohydrate specificities of artocarpin: variation in the length of a loop as a strategy for generating ligand specificity

Artocarpin, a tetrameric lectin of molecular mass 65 kDa, is one of the two lectins extracted from the seeds of jackfruit. The structures of the complexes of artocarpin with mannotriose and mannopentose reported here, together with the structures of artocarpin and its complex with Me-α-mannose reported earlier, show that the lectin possesses a deep-seated binding site formed by three loops. The binding site can be considered as composed of two subsites; the primary site and the secondary site. Interactions at the primary site composed of two of the loops involve mainly hydrogen bonds, while those at the secondary site comprising the third loop are primarily van der Waals in nature. Mannotriose in its complex with the lectin interacts through all the three mannopyranosyl residues; mannopentose interacts with the protein using at least three of the five mannose residues. The complexes provide a structural explanation for the carbohydrate specificities of artocarpin. A detailed comparison with the sugar complexes of heltuba, the only other mannose-specific jacalin-like lectin with known three-dimensional structure in sugar-bound form, establishes the role of the sugar-binding loop constituting the secondary site, in conferring different specificities at the oligosaccharide level. This loop is four residues longer in artocarpin than in heltuba, providing an instance where variation in loop length is used as a strategy for generating carbohydrate specificity

Structural basis of the carbohydrate specificities of Jacalin: an X-ray and modeling study

The structures of the complexes of tetrameric jacalin with Gal, Me-α-GalNAc, Me-α-T-antigen, GalNAcβ1-3Gal-α-O-Me and Galα1-6Glc (mellibiose) show that the sugar-binding site of jacalin has three components: the primary site, secondary site A, and secondary site B. In these structures and in the two structures reported earlier, Gal or GalNAc occupy the primary site with the anomeric carbon pointing towards secondary site A. The α-substituents, when present, interact, primarily hydrophobically, with secondary site A which has variable geometry. O-H···π and C-H···π hydrogen bonds involving this site also exist. On the other hand, β-substitution leads to severe steric clashes. Therefore, in complexes involving β-linked disaccharides, the reducing sugar binds at the primary site with the non-reducing end located at secondary site B. The interactions at secondary site B are primarily through water bridges. Thus, the nature of the linkage determines the mode of the association of the sugar with jacalin. The interactions observed in the crystal structures and modeling based on them provide a satisfactory qualitative explanation of the available thermodynamic data on jacalin-carbohydrate interactions. They also lead to fresh insights into the nature of the binding of glycoproteins by jacalin

Stable kinetochore–microtubule interactions depend on the Ska complex and its new component Ska3/C13Orf3

Ska1 and Ska2 form a complex at the kinetochore–microtubule (KT–MT) interface and are required for timely progression from metaphase to anaphase. Here, we use mass spectrometry to search for additional components of the Ska complex. We identify C13Orf3 (now termed Ska3) as a novel member of this complex and map the interaction domains among the three known components. Ska3 displays similar characteristics as Ska1 and Ska2: it localizes to the spindle and KT throughout mitosis and its depletion markedly delays anaphase transition. Interestingly, a more complete removal of the Ska complex by concomitant depletion of Ska1 and Ska3 results in a chromosome congression failure followed by cell death. This severe phenotype reflects a destabilization of KT–MT interactions, as demonstrated by reduced cold stability of KT fibres. Yet, the depletion of the Ska complex only marginally impairs KT localization of the KMN network responsible for MT attachment. We propose that the Ska complex functionally complements the KMN, providing an additional layer of stability to KT–MT attachment and possibly signalling completion of attachment to the spindle checkpoint

Crystal structure of the jacalin-T-antigen complex and a comparative study of lectin-T-antigen complexes

Thomsen-Friedenreich antigen (Galβ1-3GalNAc), generally known as T-antigen, is expressed in more than 85% of human carcinomas. Therefore, proteins which specifically bind T-antigen have potential diagnostic value. Jacalin, a lectin from jack fruit (Artocarpus integrifolia) seeds, is a tetramer of molecular mass 66 kDa. It is one of the very few proteins which are known to bind T-antigen. The crystal structure of the jacalin-T-antigen complex has been determined at 1.62 Å resolution. The interactions of the disaccharide at the binding site are predominantly through the GalNAc moiety, with Gal interacting only through water molecules. They include a hydrogen bond between the anomeric oxygen of GalNAc and the π electrons of an aromatic side-chain. Several intermolecular interactions involving the bound carbohydrate contribute to the stability of the crystal structure. The present structure, along with that of the Me-α-Gal complex, provides a reasonable qualitative explanation for the known affinities of jacalin to different carbohydrate ligands and a plausible model of the binding of the lectin to T-antigen O-linked to seryl or threonyl residues. Including the present one, the structures of five lectin-T-antigen complexes are available. GalNAc occupies the primary binding site in three of them, while Gal occupies the site in two. The choice appears to be related to the ability of the lectin to bind sialylated sugars. In either case, most of the lectin-disaccharide interactions are at the primary binding site. The conformation of T-antigen in the five complexes is nearly the same

The SPARC complex defines RNAPII promoters in Trypanosoma brucei

Kinetoplastids are a highly divergent lineage of eukaryotes with unusual mechanisms for regulating gene expression. We previously surveyed 65 putative chromatin factors in the kinetoplastid Trypanosoma brucei. Our analyses revealed that the predicted histone methyltransferase SET27 and the Chromodomain protein CRD1 are tightly concentrated at RNAPII transcription start regions (TSRs). Here, we report that SET27 and CRD1, together with four previously uncharacterized constituents, form the SET27 promoter-associated regulatory complex (SPARC), which is specifically enriched at TSRs. SET27 loss leads to aberrant RNAPII recruitment to promoter sites, accumulation of polyadenylated transcripts upstream of normal transcription start sites, and conversion of some normally unidirectional promoters to bidirectional promoters. Transcriptome analysis in the absence of SET27 revealed upregulated mRNA expression in the vicinity of SPARC peaks within the main body of chromosomes in addition to derepression of genes encoding variant surface glycoproteins (VSGs) located in subtelomeric regions. These analyses uncover a novel chromatin-associated complex required to establish accurate promoter position and directionality

Reduction in Nuclear Size by DHRS7 in Prostate Cancer Cells and by Estradiol Propionate in DHRS7-Depleted Cells

Increased nuclear size correlates with lower survival rates and higher grades for prostate cancer. The short-chain dehydrogenase/reductase (SDR) family member DHRS7 was suggested as a biomarker for use in prostate cancer grading because it is largely lost in higher-grade tumors. Here, we found that reduction in DHRS7 from the LNCaP prostate cancer cell line with normally high levels of DHRS7 increases nuclear size, potentially explaining the nuclear size increase observed in higher-grade prostate tumors where it is lost. An exogenous expression of DHRS7 in the PC3 prostate cancer cell line with normally low DHRS7 levels correspondingly decreases nuclear size. We separately tested 80 compounds from the Microsource Spectrum library for their ability to restore normal smaller nuclear size to PC3 cells, finding that estradiol propionate had the same effect as the re-expression of DHRS7 in PC3 cells. However, the drug had no effect on LNCaP cells or PC3 cells re-expressing DHRS7. We speculate that separately reported beneficial effects of estrogens in androgen-independent prostate cancer may only occur with the loss of DHRS7/ increased nuclear size, and thus propose DHRS7 levels and nuclear size as potential biomarkers for the likely effectiveness of estrogen-based treatments