Reduced Stability and Increased Dynamics in the Human Proliferating Cell Nuclear Antigen (PCNA) Relative to the Yeast Homolog

Proliferating Cell Nuclear Antigen (PCNA) is an essential factor for DNA replication and repair. PCNA forms a toroidal, ring shaped structure of 90 kDa by the symmetric association of three identical monomers. The ring encircles the DNA and acts as a platform where polymerases and other proteins dock to carry out different DNA metabolic processes. The amino acid sequence of human PCNA is 35% identical to the yeast homolog, and the two proteins have the same 3D crystal structure. In this report, we give evidence that the budding yeast (sc) and human (h) PCNAs have highly similar structures in solution but differ substantially in their stability and dynamics. hPCNA is less resistant to chemical and thermal denaturation and displays lower cooperativity of unfolding as compared to scPCNA. Solvent exchange rates measurements show that the slowest exchanging backbone amides are at the β-sheet, in the structure core, and not at the helices, which line the central channel. However, all the backbone amides of hPCNA exchange fast, becoming undetectable within hours, while the signals from the core amides of scPCNA persist for longer times. The high dynamics of the α-helices, which face the DNA in the PCNA-loaded form, is likely to have functional implications for the sliding of the PCNA ring on the DNA since a large hole with a flexible wall facilitates the establishment of protein-DNA interactions that are transient and easily broken. The increased dynamics of hPCNA relative to scPCNA may allow it to acquire multiple induced conformations upon binding to its substrates enlarging its binding diversity

Modular Architecture and Unique Teichoic Acid Recognition Features of Choline-Binding Protein L (CbpL) Contributing to Pneumococcal Pathogenesis

The human pathogen Streptococcus pneumoniae is decorated with a special class of surface-proteins known as choline-binding proteins (CBPs) attached to phosphorylcholine (PCho) moieties from cell-wall teichoic acids. By a combination of X-ray crystallography, NMR, molecular dynamics techniques and in vivo virulence and phagocytosis studies, we provide structural information of choline-binding protein L (CbpL) and demonstrate its impact on pneumococcal pathogenesis and immune evasion. CbpL is a very elongated three-module protein composed of (i) an Excalibur Ca 2+ -binding domain -reported in this work for the very first time-, (ii) an unprecedented anchorage module showing alternate disposition of canonical and non-canonical choline-binding sites that allows vine-like binding of fully-PCho-substituted teichoic acids (with two choline moieties per unit), and (iii) a Ltp-Lipoprotein domain. Our structural and infection assays indicate an important role of the whole multimodular protein allowing both to locate CbpL at specific places on the cell wall and to interact with host components in order to facilitate pneumococcal lung infection and transmigration from nasopharynx to the lungs and blood. CbpL implication in both resistance against killing by phagocytes and pneumococcal pathogenesis further postulate this surface-protein as relevant among the pathogenic arsenal of the pneumococcus.We gratefully acknowledge Karsta Barnekow and Kristine Sievert-Giermann, for technical assistance and Lothar Petruschka for in silico analysis (all Dept. of Genetics, University of Greifswald). We are further grateful to the staff from SLS synchrotron beamline for help in data collection. This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG GRK 1870 (to SH) and the Spanish Ministry of Economy and Competitiveness (BFU2014-59389-P to JAH, CTQ2014-52633-P to MB and SAF2012-39760-C02-02 to FG) and S2010/BMD- 2457 (Community of Madrid to JAH and FG).Peer Reviewe

Catalytic residues in hydrolases: analysis of methods designed for ligand-binding site prediction

The comparison of eight tools applicable to ligand-binding site prediction is presented. The methods examined cover three types of approaches: the geometrical (CASTp, PASS, Pocket-Finder), the physicochemical (Q-SiteFinder, FOD) and the knowledge-based (ConSurf, SuMo, WebFEATURE). The accuracy of predictions was measured in reference to the catalytic residues documented in the Catalytic Site Atlas. The test was performed on a set comprising selected chains of hydrolases. The results were analysed with regard to size, polarity, secondary structure, accessible solvent area of predicted sites as well as parameters commonly used in machine learning (F-measure, MCC). The relative accuracies of predictions are presented in the ROC space, allowing determination of the optimal methods by means of the ROC convex hull. Additionally the minimum expected cost analysis was performed. Both advantages and disadvantages of the eight methods are presented. Characterization of protein chains in respect to the level of difficulty in the active site prediction is introduced. The main reasons for failures are discussed. Overall, the best performance offers SuMo followed by FOD, while Pocket-Finder is the best method among the geometrical approaches

Cashew nut allergy: clinical relevance and allergen characterisation

Cashew plant (Anacardium occidentale L.) is the most relevant species of the Anacardium genus. It presents high economic value since it is widely used in human nutrition and in several industrial applications. Cashew nut is a well-appreciated food (belongs to the tree nut group), being widely consumed as snacks and in processed foods by the majority of world's population. However, cashew nut is also classified as a potent allergenic food known to be responsible for triggering severe and systemic immune reactions (e.g. anaphylaxis) in sensitised/allergic individuals that often demand epinephrine treatment and hospitalisation. So far, three groups of allergenic proteins have been identified and characterised in cashew nut: Ana o 1 and Ana o 2 (cupin superfamily) and Ana o 3 (prolamin superfamily), which are all classified as major allergens. The prevalence of cashew nut allergy seems to be rising in industrialised countries with the increasing consumption of this nut. There is still no cure for cashew nut allergy, as well as for other food allergies; thus, the allergic patients are advised to eliminate it from their diets. Accordingly, when carefully choosing processed foods that are commercially available, the allergic consumers have to rely on proper food labelling. In this sense, the control of labelling compliance is much needed, which has prompted the development of proficient analytical methods for allergen analysis. In the recent years, significant research advances in cashew nut allergy have been accomplished, which are highlighted and discussed in this review.This work was supported by FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 with grant no. UID/QUI/50006/2013–POCI/01/ 0145/FEDER/007265. Joana Costa is grateful to FCT post-doctoral grant (SFRH/BPD/102404/2014) financed by POPH-QREN (subsidised by FSE and MCTES).info:eu-repo/semantics/publishedVersio

Direct correlation of consecutive C′-N groups in proteins: A method for the assignment of intrinsically disordered proteins

Two novel 3D 13C-detected experiments, hNcocaNCO and hnCOcaNCO, are proposed to facilitate the resonance assignment of intrinsically disordered proteins. The experiments correlate the 15N and 13C′ chemical shifts of two consecutive amide moieties without involving other nuclei, thus taking advantage of the good dispersion shown by the 15N-13C′ correlations, even for proteins that lack a well defined tertiary structure. The new pulse sequences were successfully tested using Nupr1, an intrinsically disordered protein of 93 residues. © 2013 Springer Science+Business Media Dordrecht.Peer Reviewe

New 13C-detected experiments for the assignment of intrinsically disordered proteins

NMR assignment of intrinsically disordered proteins (IDPs) by conventional HN-detected methods is hampered by the small dispersion of the amide protons chemical shifts and exchange broadening of amide proton signals. Therefore several alternative assignment strategies have been proposed in the last years. Attempting to seize that dispersion of 13C′ and 15N chemical shifts holds even in IDPs, we recently proposed two 13C-detected experiments to directly correlate the chemical shifts of two consecutive 13C′-15N groups in proteins, i.e. without mediation of other nuclei. Main drawback of these experiments is the interruption of the connection at prolines. Here we present new 13C-detected experiments to correlate consecutive 13C′-15N groups in IDPs, hacacoNcaNCO and hacaCOncaNCO, that overcome this limitation. Moreover, the experiments provide recognition of glycine residues, thereby facilitating the assignment process. © 2014 Springer Science+Business Media.Peer Reviewe