Streptococcus pneumoniae NanC: STRUCTURAL INSIGHTS INTO THE SPECIFICITY AND MECHANISM OF A SIALIDASE THAT PRODUCES A SIALIDASE INHIBITOR*

Streptococcus pneumoniae is an important human pathogen that causes a range of disease states. Sialidases are important bacterial virulence factors. There are three pneumococcal sialidases: NanA, NanB, and NanC. NanC is an unusual sialidase in that its primary reaction product is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, also known as DANA), a nonspecific hydrolytic sialidase inhibitor. The production of Neu5Ac2en from α2–3-linked sialosides by the catalytic domain is confirmed within a crystal structure. A covalent complex with 3-fluoro-β-N-acetylneuraminic acid is also presented, suggesting a common mechanism with other sialidases up to the final step of product formation. A conformation change in an active site hydrophobic loop on ligand binding constricts the entrance to the active site. In addition, the distance between the catalytic acid/base (Asp-315) and the ligand anomeric carbon is unusually short. These features facilitate a novel sialidase reaction in which the final step of product formation is direct abstraction of the C3 proton by the active site aspartic acid, forming Neu5Ac2en. NanC also possesses a carbohydrate-binding module, which is shown to bind α2–3- and α2–6-linked sialosides, as well as N-acetylneuraminic acid, which is captured in the crystal structure following hydration of Neu5Ac2en by NanC. Overall, the pneumococcal sialidases show remarkable mechanistic diversity while maintaining a common structural scaffold.     

Notch signaling and Notch signaling modifiers

Originally discovered nearly a century ago, the Notch signaling pathway is critical for virtually all developmental programs and modulates an astounding variety of pathogenic processes. The DSL (Delta, Serrate, LAG-2 family) proteins have long been considered canonical activators of the core Notch pathway. More recently, a wide and expanding network of non-canonical extracellular factors has also been shown to modulate Notch signaling, conferring newly appreciated complexity to this evolutionarily conserved signal transduction system. Here, I review current concepts in Notch signaling, with a focus on work from the last decade elucidating novel extracellular proteins that up- or down-regulate signal potency.     

Junker: An Intergenic Explorer for Bacterial Genomes

In the past few decades, scientists from all over the world have taken a keen interest in novel functional units such as small regulatory RNAs, small open reading frames, pseudogenes, transposons, integrase binding attB/attP sites, repeat elements within the bacterial intergenic regions (IGRs) and in the analysis of those junk regions for ge- nomic complexity. Here we have developed a web server, named Junker, to facilitate the in-depth analysis of IGRs for examining their length distribution, four-quadrant...     

Impaired nuclear translocation of CAR in hepatic preneoplastic lesions: association with an attenuated CYP2B induction by phenobarbital

Phenobarbital (PB) induction of CYP2B, a representative target gene of constitutive androstane receptor (CAR), has been observed to be attenuated in preneoplastic lesions of rat liver; however, molecular basis for this attenuation is poorly understood. In this report, we provide evidence indicating that the CAR expressed in the hepatic preneoplastic lesions of rats and mice was resistant to nuclear translocation and transactivation of the PB-responsive enhancer module upon PB treatment. These observations suggest that the attenuation of the induction of CYP2B by PB in hepatic preneoplastic lesions is evidently a consequence of impaired nuclear translocation of CAR.     

A third sea urchin sperm receptor for egg jelly module protein, suREJ2, concentrates in the plasma membrane over the sperm mitochondrion

Sea urchin spermatozoa are model cells for studying signal transduction events underlying flagellar motility and the acrosome reaction. We previously described the sea urchin sperm receptor for egg jelly 1 (suREJ1) which consists of 1450 amino acids, has one transmembrane segment and binds to the fucose sulfate polymer of egg jelly to induce the sperm acrosome reaction. We also cloned suREJ3 which consists of 2681 amino acids and has 11 putative transmembrane segments. Both these proteins localize to the plasma membrane over the acrosomal vesicle. While cloning suREJ1, we found suREJ2, which consists of 1472 amino acids, has two transmembrane segments and is present in the entire sperm plasma membrane, but is concentrated over the sperm mitochondrion. Experimental evidence suggests that, unlike suREJ1 and suREJ3, suREJ2 does not project extracellularly from the plasma membrane, but is an intracellular plasma membrane protein. All three sea urchin sperm REJ proteins possess a protein module of > 900 amino acids, termed 'the REJ module', that is shared by the human autosomal dominant polycystic kidney disease protein, polycystin-1, and PKDREJ, a testis-specific protein in mammals whose function is unknown. In the present study, we describe the sequence, domain structure and localization of suREJ2 and speculate on its possible function.     

Heparin binds to the laminin alpha4 chain LG4 domain at a site different from that found for other laminins

We previously reported that the LG4 domain of the laminin alpha4 chain is responsible for high-affinity heparin binding. To specify the amino acid residues involved in this activity, we produced a series of alpha4 LG4-fusion proteins in which each of the 27 basic residues (arginine, R; histidine; lysine, K) were replaced one by one with alanine (A). When the effective residues R1520A, K1531A, K1533A, and K1539A are mapped on a structural model, they form a track on the concave surface of the beta-sandwich, suggesting that they interact with adjacent sulfate groups along the heparin chain. Whereas low-affinity heparin-binding sites of other LG domains have been located at the top of the beta-sheet sandwich opposite the N and C termini, the residues for high-affinity heparin binding of alpha4 LG4 reveal a new topological area of the LG module.     

Binding sub-site dissection of a carbohydrate-binding module reveals the contribution of entropy to oligosaccharide recognition at "non-primary" binding subsites

The optimal ligands for many carbohydrate-binding proteins are often oligosaccharides comprising two, three, or more monosaccharide units. The binding affinity for these sugars is increased incrementally by contributions from binding subsites on the protein that accommodate the individual monosaccharide residues of the oligosaccharide. Here, we use CsCBM6-1, a xylan-specific type B carbohydrate-binding module (CBM) from Clostridium stercorarium falling into amino acid sequence family CBM6, as a model system to investigate the structural and thermodynamic contributions of binding subsites in this protein to carbohydrate recognition. The three-dimensional structures of uncomplexed CsCBM6-1 (at 1.8 A resolution) and bound to the oligosaccharides xylobiose, xylotriose, and xylotetraose (at 1.70 A, 1.89 A, and 1.69 A resolution, respectively) revealed the sequential occupation of four subsites within the binding site in the order of subsites 2, 3, 4 then 1. Overall, binding to all of the xylooligosaccharides tested was enthalpically favourable and entropically unfavourable, like most protein-carbohydrate interactions, with the primary subsites 2 and 3 providing the bulk of the free energy and enthalpy of binding. In contrast, the contributions to the changes in entropy of the non-primary subsites 1 and 4 to xylotriose and xylotetraose binding, respectively, were positive. This observation is remarkable, in that it shows that the 10-20-fold improvement in association constants for oligosaccharides longer than a disaccharide is facilitated by favourable entropic contributions from the non-primary binding subsites.     

钻形紫菀开花期种群构件的生物量分配

在野外用样方法,选取60株钻形紫菀(Aster subulatus Michx.)开花植株,进行根、茎、叶及花等构件的生物量及其物质分配关系的研究.结果表明:钻形紫菀开花期构件生物量为茎>花>根>叶,其变异系数分别为57.15%、64.66%、57.65%和55.2%,具有较大表型可塑性;在各构件物质分配变异系数中,花生物量分配的变异系数相对较大,说明其调节生殖分配的能力较强;植株高度与各构件生物量呈显著的正相关性,随着各构件生物量的增加均呈幂函数形式增加;花生物量分配与总生物量呈显著的正相关性,其余构件生物量分配均与总生物量及花生物量分配呈负相关性,物质分配由营养构件、支持构件、光合构件向生殖构件转移.反映出钻形紫菀具有自我调节生长力的分配策略,对异质环境具有较强适应能力.     

Structure of a polyisoprenoid binding domain from Saccharophagus degradans implicated in plant cell wall breakdown

Saccharophagus degradans belongs to a recently discovered group of marine bacteria equipped with an arsenal of sugar cleaving enzymes coupled to carbohydrate-binding domains to degrade various insoluble complex polysaccharides. The modular Sde-1182 protein consists of a family 2 carbohydrate binding module linked to a X158 domain of unknown function. The 1.9 Å and 1.55 Å resolution crystal structures of the isolated X158 domain bound to the two related polyisoprenoid molecules, ubiquinone and octaprenyl pyrophosphate, unveil a β-barrel architecture reminiscent of the YceI-like superfamily that resembles the architecture of the lipocalin fold. This unprecedented association coupling oxidoreduction and carbohydrate recognition events may have implications for effective nutrient uptake in the marine environment.