Structural and functional analysis of the ligand specificity of the HtrA2/Omi PDZ domain

The mitochondrial serine protease HtrA2/Omi helps to maintain mitochondrial function by handling misfolded proteins in the intermembrane space. In addition, HtrA2/Omi has been implicated as a proapoptotic factor upon release into the cytoplasm during the cell death cascade. The protein contains a C-terminal PDZ domain that packs against the protease active site and inhibits proteolytic activity. Engagement of the PDZ domain by peptide ligands has been shown to activate the protease and also has been proposed to mediate substrate recognition. We report a detailed structural and functional analysis of the human HtrA2/Omi PDZ domain using peptide libraries and affinity assays to define specificity, X-ray crystallography to view molecular details of PDZ-ligand interactions, and alanine-scanning mutagenesis to probe the peptide-binding groove. We show that the HtrA2/Omi PDZ domain recognizes both C-terminal and internal stretches of extended, hydrophobic polypeptides. High-affinity ligand recognition requires contacts with up to five hydrophobic side chains by distinct sites on the PDZ domain. However, no particular residue type is absolutely required at any position, and thus, the HtrA2/Omi PDZ domain appears to be a promiscuous module adapted to recognize unstructured, hydrophobic polypeptides. This type of specificity is consistent with the biological role of HtrA2/Omi in mitochondria, which requires the recognition of diverse, exposed stretches of hydrophobic sequences in misfolded proteins. The findings are less consistent with, but do not exclude, a role for the PDZ domain in targeting the protease to specific substrates during apoptosis.     

Paenibacillus curdlanolyticus B-6 xylanase Xyn10C capable of producing a doubly arabinose-substituted xylose, α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp,from rye arabinoxylan

Paenibacillus curdlanolyticus B-6 Xyn10C is a single module xylanase consisting of a glycoside hydrolase family-10 catalytic module. The recombinant enzyme, rXyn10C, was produced by Escherichia coli and characterized. rXyn10C was highly active toward soluble xylans derived from rye, birchwood, and oat spelt, and slightly active toward insoluble wheat arabinoxylan. It hydrolyzed xylooligosaccharides larger than xylotetraose to produce xylotriose, xylobiose, and xylose. When rye arabinoxylan and oat spelt xylan were treated with the enzyme and the hydrolysis products were analyzed by thin layer chromatography (TLC), two unknown hydrolysis products, U1 and U2, were detected in the upper position of xylose on a TLC plate. Electrospray ionization mass spectrometry and enzymatic analysis using Bacillus licheniformis α-l-arabinofuranosidase Axh43A indicated that U1 was α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp and U2 was α-l-Araf-(1 → 2)-d-Xylp, suggesting that rXyn10C had strong activity toward a xylosidic linkage before and after a doubly arabinose-substituted xylose residue and was able to accommodate an α-1,2- and α-1,3-linked arabinose-substituted xylose unit in both the −1 and +1 subsites. A molecular docking study suggested that rXyn10C could accommodate a doubly arabinose-substituted xylose residue in its catalytic site, at subsite −1. This is the first report of a xylanase capable of producing α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp from highly arabinosylated xylan.     

Allostery mediates ligand binding to WWOX tumor suppressor via a conformational switch

While being devoid of the ability to recognize ligands itself, the WW2 domain is believed to aid ligand binding to the WW1 domain in the context of a WW1–WW2 tandem module of WW domain‐containing oxidoreductase (WWOX) tumor suppressor. In an effort to test the generality of this hypothesis, we have undertaken here a detailed biophysical analysis of the binding of WW domains of WWOX alone and in the context of the WW1–WW2 tandem module to an array of putative proline‐proline‐x–tyrosine (PPXY) ligands. Our data show that while the WW1 domain of WWOX binds to all ligands in a physiologically relevant manner, the WW2 domain does not. Moreover, ligand binding to the WW1 domain in the context of the WW1–WW2 tandem module is two‐to‐three‐fold stronger than when treated alone. We also provide evidence that the WW domains within the WW1–WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. Of particular note is the observation that the physical association of the WW2 domain with WW1 blocks access to ligands. Consequently, ligand binding to the WW1 domain not only results in the displacement of the WW2 lid but also disrupts the physical association of WW domains in the liganded conformation. Taken together, our study underscores a key role of allosteric communication in the ability of the WW2 orphan domain to chaperone physiological action of the WW1 domain within the context of the WW1–WW2 tandem module of WWOX. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification and characterization of an ABA‐activated MAP kinase cascade in Arabidopsis thaliana

Abscisic acid (ABA) is a major phytohormone involved in important stress‐related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA‐triggered phosphoproteins as putative mitogen‐activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA‐activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3‐1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA‐dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR‐SnRK2‐PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA‐induced MAPK pathway in plant stress signalling.     

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.