No abstract

In green species, sucrose can help antagonize abiotic stress. Sucrose phosphate synthase (SPS) is a well-known rate-limiting enzyme in the synthesis of sucrose. To date, however, there is no known crystal structure of SPS from plant or cyanobacteria. In this study, we report the first co-crystal structure of SPS from Thermosynechococcus elongatus with UDP and sucrose-6-phosphate (S6P). Within the catalytic site, the side chains of His158 and Glu331, along with two phosphate groups from UDP, form hydrogen bonds with the four hydroxyl groups of the glucose moiety in S6P. This association causes these four hydroxyl groups to become partially negatively charged, thus promoting formation of the C1 oxocarbenium ion. Breakage of the hydrogen bond between His158 and one of the hydroxyl groups may trigger covalent bond formation between the C1 oxocarbenium ion and the C2 hydroxyl of fructose-6-phosphate. Consistent with our structural model, we observed that two SPS mutants, H158A and E331A, lost all catalytic activity. Moreover, electron density of residues from two loops (loop1 and loop2) in the SPS A-domain was not observed, suggest their dynamic nature. B-factor analysis and molecular dynamics stimulations of the full-length enzyme and A-domain indicate that both loops are crucial for binding and release of substrate and product. In addition, temperature gradient analysis shows that SPS exhibits its highest activity at 70 • C, suggesting that this enzyme has the potential of being used in industrial production of S6P.

The expression of prototype galectin‐14 (Gal‐14) in human placenta is higher than any other galectin, suggesting that it may play a role in fetal development and regulation of immune tolerance during pregnancy. Here, we solved the crystal structure of dimeric Gal‐14 and found that its global fold is significantly different from that of other galectins with two β‐strands (S5 and S6) extending from one monomer and contributing to the carbohydrate‐binding domain of the other. The hemagglutination assay showed that this lectin could induce agglutination of chicken erythrocytes, even though lactose could not inhibit Gal‐14‐induced agglutination activity. Calorimetry indicates that lactose does not interact with this lectin. Compared to galectin‐1, galectin‐3, and galectin‐8, Gal‐14 has two key amino acids (a histidine and an arginine) in the normally conserved, canonical sugar‐binding site, which are substituted by glutamine (Gln53) and histidine (His57), thus likely explaining why lactose binding to this lectin is very weak. Lactose was observed in the ligand‐binding site of one Gal‐14 structure, most likely because ligand binding is weak and crystals were allowed to grow over a long period of time in the presence of lactose. We also found that EGFP‐tagged Gal‐14 is primarily localized within the nucleus of different cell types. In addition, Gal‐14 colocalized with c‐Rel (a member of NF‐κB family) in HeLa cells. These findings indicate that Gal‐14 might regulate signal transduction pathways through NF‐κB hubs. Overall, the present study provides impetus for further research into the function of Gal‐14 in embryology.

Galectin-13 (Gal-13) plays numerous roles in regulating the relationship between maternal and fetal tissues. Low expression levels or mutations of the lectin can result in pre-eclampsia. The previous crystal structure and gel filtration data show that Gal-13 dimerizes via formation of two disulfide bonds formed by Cys136 and Cys138. In the present study, we mutated them to serine (C136S, C138S and C136S/C138S), crystalized the variants and solved their crystal structures. All variants crystallized as monomers. In the C136S structure, Cys138 formed a disulfide bond with Cys19, indicating that Cys19 is important for regulation of reversible disulfide bond formation in this lectin. Hemagglutination assays demonstrated that all variants are inactive at inducing erythrocyte agglutination, even though gel filtration profiles indicate that C136S and C138S could still form dimers, suggesting that these dimers do not exhibit the same activity as wild-type (WT) Gal-13. In HeLa cells, the three variants were found to be distributed the same as with WT Gal-13. However, a Gal-13 variant (delT221) truncated at T221 could not be transported into the nucleus, possibly explaining why women having this variant get pre-eclampsia. Considering the normally high concentration of glutathione in cells, WT Gal-13 should exist mostly as a monomer in cytoplasm, consistent with the monomeric variant C136S/C138S, which has a similar ability to interact with HOXA1 as WT Gal-13.

The gene for galectin-13 (Gal-13, placental protein 13) is only present in primates, and its low expression level in maternal serum may promote pre-eclampsia. In the present study, we used pull-down experiments and biolayer interferometry to assess the interaction between Gal-13 and actin. These studies uncovered that human Gal-13 (hGal-13) and Saimiri boliviensis boliviensis (sGal-13) strongly bind to α- and β/γ-actin, with Ca2+ and ATP significantly enhancing interactions. This in turn suggests that h/sGal-13 may inhibit myosin-induced contraction when vascular smooth muscle cells undergo polarization. Here, we solved the crystal structure of sGal-13 bound to lactose and found that it exists as a monomer in contrast to hGal-13 that is a dimer. The distribution of sGal-13 in HeLa cells is similar to that of hGal-13, indicating that monomeric Gal-13 is the primary form in cells. Even though sGal-13 also binds to actin, hGal-13 ligand binding site mutants do not influence hGal-13/actin binding, whereas the monomeric mutant C136S/C138S binds to actin more strongly than wild type hGal-13. Overall, our study demonstrates that monomeric Gal-13 binds to actin, an interaction that is independent of the galectin canonical ligand binding site.