eIF4G stimulates the activity of the DEAD box protein eIF4A by a conformational guidance mechanism

The activity of eIF4A, a key player in translation initiation, is regulated by other translation factors through currently unknown mechanisms. Here, we provide the necessary framework to understand the mechanism of eIF4A's regulation by eIF4G. In solution, eIF4A adopts a defined conformation that is different from the crystal structure. Binding of eIF4G induces a ‘half-open' conformation by interactions with both domains, such that the helicase motifs are pre-aligned for activation. A primary interface acts as an anchor for complex formation. We show here that formation of the secondary interface is essential for imposing the ‘half-open' conformation on eIF4A, and it is critical for the functional interaction of eIF4G with eIF4A. Via this bipartite interaction, eIF4G guides the transition of eIF4A between the ‘half-open' and closed conformations, and stimulates its activity by accelerating the rate-limiting step of phosphate release. Subtle changes induced by eIF4G may be amplified by input signals from other translation factors, leading to an efficient regulation of translation initiatio

Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled (2H/15N/13C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1β were assessed by surface plasmon resonance yielding KD values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineerin

Komplexität als Determinante der Qualitätswahrnehmung von Dienstleistungen

Viel ist in den letzten Jahren über die Wahrnehmung von Dienstleistungsqualität geschrieben worden. Ein Großteil der Forschungsarbeiten hat sich dabei auf die Dimensionalität des Qualitätsbegriffs konzentriert; Determinanten der Qualitätswahrnehmung wurden nur am Rande thematisiert. Der vorliegende Beitrag untersucht als eine wichtige Determinante der Qualitätswahrnehmung die wahrgenommene Komplexität der Dienstleistung. Auf der Basis theoretischer Überlegungen wird ein Kausalmodell zur Untersuchung dieser Zusammenhänge entwickelt. Im Ergebnis zeigt sich ein deutlicher Einfluß der wahrgenommenen Komplexität auf die Qualitätswahrnehmung, die ihrerseits die Kundenbindung nachhaltig beeinflusst

Screening prisoners for intellectual disabilities in three English prisons

Background Prisoners with intellectual disabilities are known to be disadvantaged in prisons and to be more susceptible to bullying, segregation, depression and anxiety than other prisoners. Method In this study, nearly 3000 new prisoners entering three English prisons were offered screening for intellectual disabilities, using the LDSQ. Results On average, 75% of all new prisoners entering prison were offered screening, and only 14% refused screening. Overall, just less than 7% were screened positive on the LDSQ and prisons made some reasonable adjustments as a result. Conclusions It is argued that it is feasible to screen for intellectual disabilities in prisons and, given the inequalities to which prisoners with intellectual disabilities are subject in prison, it is time for such screening to be rolled out to all prisons

eIF4G stimulates the activity of the DEAD box protein eIF4A by a conformational guidance mechanism

The activity of eIF4A, a key player in translation initiation, is regulated by other translation factors through currently unknown mechanisms. Here, we provide the necessary framework to understand the mechanism of eIF4A’s regulation by eIF4G. In solution, eIF4A adopts a defined conformation that is different from the crystal structure. Binding of eIF4G induces a ‘half-open’ conformation by interactions with both domains, such that the helicase motifs are pre-aligned for activation. A primary interface acts as an anchor for complex formation. We show here that formation of the secondary interface is essential for imposing the ‘half-open’ conformation on eIF4A, and it is critical for the functional interaction of eIF4G with eIF4A. Via this bipartite interaction, eIF4G guides the transition of eIF4A between the ‘half-open’ and closed conformations, and stimulates its activity by accelerating the rate-limiting step of phosphate release. Subtle changes induced by eIF4G may be amplified by input signals from other translation factors, leading to an efficient regulation of translation initiation

Membrane transporters in the bioproduction of organic acids: state of the art and future perspectives for industrial applications

Organic acids such as monocarboxylic acids, dicarboxylic acids or even more complex molecules such as sugar acids, have displayed great applicability in the industry as these compounds are used as platform chemicals for polymer, food, agricultural and pharmaceutical sectors. Chemical synthesis of these compounds from petroleum derivatives is currently their major source of production. However, increasing environmental concerns have prompted the production of organic acids by microorganisms. The current trend is the exploitation of industrial biowastes to sustain microbial cell growth and valorize biomass conversion into organic acids. One of the major bottlenecks for the efficient and cost-effective bioproduction is the export of organic acids through the microbial plasma membrane. Membrane transporter proteins are crucial elements for the optimization of substrate import and final product export. Several transporters have been expressed in organic acid-producing species, resulting in increased final product titers in the extracellular medium and higher productivity levels. In this review, the state of the art of plasma membrane transport of organic acids is presented, along with the implications for industrial biotechnology.This work was supported by the strategic programme UID/BIA/04050/2019 funded by Portuguese fundsthrough the FCT I.P., and the projects: PTDC/BIAMIC/5184/2014, funded by national funds through the Fundacao para a Ciencia e Tecnologia (FCT) I.P. and by the European Regional Development Fund (ERDF) through the COMPETE 2020-Programa Operacional Competitividade e Internacionalizacao (POCI), and EcoAgriFood: Innovative green products and processes to promote AgriFood BioEconomy (operacao NORTE-01-0145-FEDER-000009), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). DR acknowledges FCT for the SFRH/BD/96166/2013 PhD grant. MSS acknowledges the Norte2020 for the UMINHO/BD/25/2016 PhD grant with the reference NORTE-08-5369-FSE-000060. TR acknowledges Yeastdoc European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 764927

Structural and functional characterization of the integral membrane proteins CitS and CCR5 by electron microscopy

Secondary transport proteins are integral membrane proteins found in every cell. They facilitate the transport of versatile substrates (e.g. nutrients, ions and drugs) across the hydrophobic membrane barrier. Independent on their mode of transport (symport/antiport) the uphill transport of the main substrate is driven by the coupled flux of a co-substrate downhill its electrochemical gradient. Malfunction of secondary transporter can cause severe physiological disorders like depression and obesity and therefore these transport proteins constitute attractive drug targets. The main part of this PhD thesis is the structural and functional characterization of the secondary citrate/sodium symporter CitS from Klebsiella pneumonia, mainly by transmission electron microscopy (TEM). CitS is the best characterized member of the bacterial 2-hydroxycarboxylate transporter (2-HCT) family. It facilitates the secondary transport of bivalent citrate ions driven by a coupled flux of Na+ across the inner membrane of the host. Hydropathy profiling and extensive biochemical experimentation prior to this study predicted CitS to represent a new structural fold as paradigm for numerous related proteins, so that it constitutes a highly attractive target for structural studies. As a first step, two-dimensional (2D) crystals of recombinant CitS were produced by dialysis assisted reconstitution of pure detergent solubilized protein into bilayer forming phospholipids. Extensive screening of crystallization conditions led to highly ordered tubular 2D crystals suitable for structure determination by cryo-electron crystallography. Therefore, numerous sample preparation methods were evaluated, while plunge-freezing provided significantly better results compared to commonly used sugar embedding methods. As described in chapter 2, image processing of electron micrographs from plunge-frozen 2D crystals provided the projection structure of CitS at 6 Å resolution. The transporter appears as oval shaped dimer measuring 5*9 nm in the membrane plane. The dimer reveals three distinct structural domains being formed by two dense clusters of α-helices at each molecule’s tip and a third, less dense domain in the center of the dimer. The domains are separated by solvent areas. Surprisingly, this architecture highly resembles that of the unrelated Na+/H+ antiporter NhaP1. In projection, each CitS monomer reveals eleven TMS that well match previous membrane topology predictions. Finally, we developed several models describing possible monomer-monomer interfaces and domain organizations. In chapter 3, we describe the 3D structure of CitS at 6/15 Å resolution obtained by electron micrographs of tilted 2D crystal samples. Based on the 3D volume, we developed a molecular model that reveals eleven α-helices and two additional helical reentrant loops. The central dimerization domain is formed by seven partially tilted helices, while the distal cluster reveals 4 transmembrane segments surrounding the two reentrant loops. We also find internal structural symmetry for the strongly intertwined N- and C-terminal domains as prerequisite for substrate translocation by the ‘alternating access’ mechanism. Additional projection structures of CitS in various substrate environments (Na+, K+, acetate and citrate) allowed us to map the conformational space. The binding of citrate as main substrate induces a defined movement of α-helices spatially limited to the helix cluster in each monomer. This primarily occurs in the presence of Na+, and much less with K+ and highlights the high co-ion specificity. These findings also enable us to assign the dense helix cluster as substrate binding and translocation site. In a second project, various biophysical techniques were used to characterize the recombinant G protein-coupled receptor (GPCR) CCR5. Besides its important role in immune responses, CCR5 also acts as co-receptor during HIV-1 target cell entry. In chapter 4, an innovative E. coli based expression platform is presented that enables the production of 10 mg purified protein from 1L cell culture. We could demonstrate ligand binding, structural integrity, homogeneity and stability of triply isotope labeled CCR5. This provides a promising starting point for ongoing structural studies, especially by nuclear magnetic resonance (NMR) spectroscopy