Structure and membrane-targeting of a Bordetella pertussis effector N-terminal domain

BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS) effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Like other cytotoxicity-mediating effectors, BteA uses its multifunctional N-terminal domain to target phosphatidylinositol (PI)-rich microdomains in the host membrane. Despite their structural similarity, T3SS effectors exhibit a variable range of membrane interaction modes, and currently only limited structural information is available for the BteA membrane-targeting domain and the molecular mechanisms underlying its function. Employing a synergistic combination of structural methods, here we determine the structure of this functional domain and uncover key molecular determinants mediating its interaction with membranes. Residues 29–121 of BteA form an elongated four-helix bundle packed against two shorter perpendicular helices, the second of which caps the domain in a critical ‘tip motif’. A flexible region preceding the BteA helical bundle contains the characteristic β-motif required for binding its cognate chaperone BtcA. We show that BteA targets PI(4,5)P₂-containing lipoprotein nanodiscs and binds a soluble PI(4,5)P₂ analog via an extensive positively charged surface spanning its first two helices, and that this interaction is weaker for PI(3,5)P₂ and abolished for PI(4)P. We confirmed this model of membrane-targeting by observation of BteA-induced changes in the structure of PI(4,5)P₂-containing phospholipid bilayers using small-angle X-ray scattering (SAXS). We also extended these results to a larger BteA domain (residues 1–287), confirming its interaction with bilayers using calorimetry, fluorescence and SAXS methods. This novel view of the structural underpinnings of membrane targeting by BteA is an important step towards a comprehensive understanding of cytotoxicity in Bordetella, as well as interactions of a broad range of pathogens with their respective hosts.     

Uterine peristalsis-induced stresses within the uterine wall may sprout adenomyosis

Adenomyosis is a disease in which ectopic endometrial glands and stromal cells appear in the uterine myometrium. This pathology is common among women of reproductive age, and in addition to chronic pelvic pain and heavy periods it may also cause infertility. The ‘tissue injury and repair’ mechanism in response to increased intrauterine pressures was proposed as the etiology for migration of fragments of basal endometrium into the myometrial wall. In order to investigate this mechanism, a conceptual two-dimensional model of the uterine wall subjected to intrauterine pressures was implemented using ADINA commercial software. The stress field within the uterine wall was examined for a variety of intrauterine sinusoidal pressure waves with varying frequencies. The results revealed that: (1) as the wavelength of the subjected pressure wave decreased, high concentration of stresses developed near the inner uterine cavity; (2) as the pressure wave frequency increased, high gradients of the stresses were obtained; (3) at menstrual phase, the highest stresses obtained at the endometrial–myometrial interface. Therefore, increased uterine activity results in high stresses which may lead to tissue lesions and detachment of endometrial cells.     

Ion binding by X-537A. Equilibrium and rate of complexation of Co2+ in methanol

The reaction of X-537A (XH) with Co²⁺ in methanol to form the complex CoX⁺ has been studied fluorometrically to determine the equilibrium constant as a function of temperature. The effect of complexation on the proton NMR spectrum of the X-537A was studied to determine the kinetics of complex formation. Comparing the data for the reaction XM⁺→X^? + M²⁺ in methanol at 25° for several M²⁺ we find that the equilibrium constants increase in the order CoX⁺ < MnX⁺ < NiX⁺ and span only a factor of 5 while the rate constants increase in the order NiX⁺ < CoX⁺ < MnX⁺ and span a factor of more than 100.     

Exposing pepper plants to high day temperatures prevents the adverse low night temperature symptoms

Exposing pepper ( Capsicum annuum ) plants to extremely high day temperatures (HDT) (day/night temperatures of 36 ± 2/10 ± 2°C), obtained by keeping the greenhouse closed during the day to exploit solar heating, prevented the development of low night temperature (LNT) symptoms. Plants of cultivars Fiesta and Selica grown under LNTs (10 ± 2°C) and moderate day temperatures (25 ± 2°C) during winter exhibited retarded growth, reduced leaf numbers, and deformed fruits with few or no seeds. LNT caused a reduction in the number and quality of pollen grains: the reduction in pollen quality was associated with reduced starch accumulation in pollen grains at 3 days before anthesis (DBA) and a decrease of more than two-fold in total soluble sugars in the mature pollen grains. This inhibitory effect was associated with more than 50% reduction in the enzymatic activities of the cell wall-bound and soluble acid invertases that catalyze the hydrolysis of incoming sucrose molecules. All these symptoms were prevented by HDT treatment which matched the vegetative and reproductive performance of the plants to those of plants grown under optimal night temperature (ONT) conditions (day/night temperatures of 23 ± 2/18 ± 2°C). HDT also prevented the inhibitory effect of LNT on enzymatic activities of both invertases in pollen at 5 DBA and brought about the accumulation of high levels of starch in pollen at 3 DBA. The results presented could support the development of a novel procedure for producing greenhouse crops with minimum or even with no fuel consumption for heating during the winter nights in regions with bright and sunny days.     

IMPACT World+: a globally regionalized life cycle impact assessment method

The International Journal of Life Cycle Assessment - This paper addresses the need for a globally regionalized method for life cycle impact assessment (LCIA), integrating multiple state-of-the-art...     

Atrophic thyroid follicles and inner ear defects reminiscent of cochlear hypothyroidism in Slc26a4-related deafness

Thyroid hormone is essential for inner ear development and is required for auditory system maturation. Human mutations in SLC26A4 lead to a syndromic form of deafness with enlargement of the thyroid gland (Pendred syndrome) and non-syndromic deafness (DFNB4). We describe mice with an Slc26a4 mutation, Slc26a4 ^loop/loop , which are profoundly deaf but show a normal sized thyroid gland, mimicking non-syndromic clinical signs. Histological analysis of the thyroid gland revealed defective morphology, with a majority of atrophic microfollicles, while measurable thyroid hormone in blood serum was within the normal range. Characterization of the inner ear showed a spectrum of morphological and molecular defects consistent with inner ear pathology, as seen in hypothyroidism or disrupted thyroid hormone action. The pathological inner ear hallmarks included thicker tectorial membrane with reduced β-tectorin protein expression, the absence of BK channel expression of inner hair cells, and reduced inner ear bone calcification. Our study demonstrates that deafness in Slc26a4 ^loop/loop mice correlates with thyroid pathology, postulating that sub-clinical thyroid morphological defects may be present in some DFNB4 individuals with a normal sized thyroid gland. We propose that insufficient availability of thyroid hormone during inner ear development plays an important role in the mechanism underlying deafness as a result of SLC26A4 mutations.     

Architecture of the hepatitis C virus E1 glycoprotein transmembrane domain studied by NMR

Oligomerization of hepatitis C viral envelope proteins E1 and E2 is essential to virus fusion and assembly. Although interactions within the transmembrane (TM) domains of these glycoproteins have proven contributions to the E1/E2 heterodimerization process and consequent infectivity, there is little structural information on this entry mechanism. Here, as a first step towards our long-term goal of understanding the interaction between E1 and E2 TM-domains, we have expressed, purified and characterized E1-TM using structural biomolecular NMR methods. An MBP-fusion expression system yielded sufficient quantities of pure E1-TM, which was solubilized in two membrane-mimicking environments, SDS- and LPPG-micelles, affording samples amenable to NMR studies. Triple resonance assignment experiments and relaxation measurements provided information on the secondary structure and global fold of E1-TM in these environments. In SDS micelles E1-TM adopts a helical conformation, with helical stretches at residues 354–363 and 371–379 separated by a more flexible segment of residues 364–370. In LPPG micelles a helical conformation was observed for residues 354–377 with greater flexibility in the 366–367 dyad, suggesting LPPG provides a more native environment for the peptide. Replacement of key positively charged residue K370 with an alanine did not affect the secondary structure of E1-TM but did change the relative positioning within the micelle of the two helices. These results lay the foundation for structure determination of E1-TM and a molecular understanding of how E1-TM flexibility enhances its interaction with E2-TM during heterodimerization and membrane fusion.     

Global fold and backbone dynamics of the hepatitis C virus E2 glycoprotein transmembrane domain determined by NMR

E1 and E2 are two hepatitis C viral envelope glycoproteins that assemble into a heterodimer that is essential for membrane fusion and penetration into the target cell. Both extracellular and transmembrane (TM) glycoprotein domains contribute to this interaction, but study of TM–TM interactions has been limited because synthesis and structural characterization of these highly hydrophobic segments present significant challenges. In this NMR study, by successful expression and purification of the E2 transmembrane domain as a fusion construct we have determined the global fold and characterized backbone motions for this peptide incorporated in phospholipid micelles. Backbone resonance frequencies, relaxation rates and solvent exposure measurements concur in showing this domain to adopt a helical conformation, with two helical segments spanning residues 717–726 and 732–746 connected by an unstructured linker containing the charged residues D728 and R730 involved in E1 binding. Although this linker exhibits increased local motions on the ps timescale, the dominating contribution to its relaxation is the global tumbling motion with an estimated correlation time of 12.3 ns. The positioning of the helix–linker–helix architecture within the mixed micelle was established by paramagnetic NMR spectroscopy and phospholipid-peptide cross relaxation measurements. These indicate that while the helices traverse the hydrophobic interior of the micelle, the linker lies closer to the micelle perimeter to accommodate its charged residues. These results lay the groundwork for structure determination of the E1/E2 complex and a molecular understanding of glycoprotein heterodimerization.