Insights into the structures and dynamics of the pathogen secreted effectors AVR3A11 and tarp through the application of NMR spectroscopy

The lifecycles of obligate pathogenic and parasitic microorganisms depend on a myriad of interactions with their hosts at the molecular level. The class of bacterial proteins directly responsible for these inter-organism interactions have been termed e�ector proteins and can function in either an extracellular or, once secreted into the host cell, an intracellular environment. Primarily through the use of nuclear magnetic resonance spectroscopy (NMR), I have investigated the biophysical properties of two such bacterial e�ector proteins. TARP (translocated actin recruiting protein) is a largely disordered 100 kDa e�ector, common to all chlamydial species, which functions to remodel the host actin cytoskeleton to facilitate the internalisation of the chlamydial cell. Using constructs of TARP comprising an expected actin binding domain, I have shown through NMR chemical shift indexing and 15N relaxation that although the unbound domain is intrinsically disordered a short region, which aligns to other helical actin binding domains, maintains some helical propensity. Furthermore, these residues map to chemical shift variations in the bound state and the Kd for the interaction has also been determined using isothermal titration calorimetry. AVR3a11 is an 8 kDa e�ector from the pepper pathogen Phytophthora capsici that has been shown to inhibit plant programmed cell death. Using a combination of 2D and 3D NMR experiments I have assigned the majority of the backbone and side-chain resonances from the structured regions of AVR3a11. Through the acquisition and analysis of 13C and 15N edited HSQCNOESY spectra I have also calculated a water re�ned, structural ensemble for AVR3a11. Additionally, analysis of the slow (H:D exchange) and fast (T1, T2 and heteronuclear NOE) dynamic regimes, describes AVR3a11 as a relatively tightly folded helical bundle which also exhibits a signi�cant degree of conformational exchange

Somatostatin, an in vivo binder to Aβ oligomers, Binds to βPFOAβ(1−42) Tetramers

Somatostatin (SST14) is strongly related to Alzheimer's disease (AD), as its levels decline during aging, it regulates the proteolytic degradation of the amyloid beta peptide (Aβ), and it binds to Aβ oligomers in vivo. Recently, the 3D structure of a membrane-associated β-sheet pore-forming tetramer (βPFOAβ(1−42) tetramer) has been reported. Here, we show that SST14 binds selectively to the βPFOAβ(1−42) tetramer with a KD value of ∼40 μM without binding to monomeric Aβ(1−42). Specific NMR chemical shift perturbations, observed during titration of SST14, define a binding site in the βPFOAβ(1−42) tetramer and are in agreement with a 2:1 stoichiometry determined by both native mass spectroscopy and isothermal titration calorimetry. These results enabled us to perform driven docking and model the binding mode for the interaction. The present study provides additional evidence on the relation between SST14 and the amyloid cascade and positions the βPFOAβ(1−42) tetramer as a relevant aggregation form of Aβ and as a potential target for AD

Improving theatre turnaround time

The NHS Institute for Innovation and Improvement has determined that a £7 million saving can be achieved per trust by improving theatre efficiency. The aim of this quality improvement project was to improve orthopaedic theatre turnaround without compromising the patient safety. We process mapped all the stages from application of dressing to knife to skin on the next patient in order to identify potential areas for improvement. Several suggestions arose which were tested in multiple PDSA cycles in a single theatre. These changes were either adopted, adapted or rejected on the basis of run chart data and theatre team feedback. Successful ideas which were adopted included, the operating department practitioner (ODP) seeing and completing check-in paperwork during the previous case rather than during turnaround, a 15 minute telephone warning to ensure the next patient was fully ready, a dedicated cleaning team mobilised during wound closure, sending for the next patient as theatre cleaning begins. Run charts demonstrate that as a result of these interventions the mean turnaround time almost halved from 66.5 minutes in July to 36.8 minutes over all PDSA cycles. This improvement has been sustained and rolled out into another theatre. As these improvements become more established we hope that additional cases will be booked, improving theatre output. The PDSA cycle continues as we believe that further gains may yet be made, and our improvements may be rolled out across other surgical specialities

The intrinsically disordered Tarp protein from chlamydia binds actin with a partially preformed helix

Tarp (translocated actin recruiting phosphoprotein) is an effector protein common to all chlamydial species that functions to remodel the host-actin cytoskeleton during the initial stage of infection. In C. trachomatis, direct binding to actin monomers has been broadly mapped to a 100-residue region (726-825) which is predicted to be predominantly disordered, with the exception of a ~10-residue α helical patch homologous to other WH2 actin-binding motifs. Biophysical investigations demonstrate that a Tarp726-825 construct behaves as a typical intrinsically disordered protein; within it, NMR relaxation measurements and chemical shift analysis identify the ten residue WH2-homologous region to exhibit partial α-helix formation. Isothermal titration calorimetry experiments on the same construct in the presence of monomeric G-actin show a well defined binding event with a 1:1 stoichiometry and Kd of 102 nM, whilst synchrotron radiation circular dichroism spectroscopy suggests the binding is concomitant with an increase in helical secondary structure. Furthermore, NMR experiments in the presence of G-actin indicate this interaction affects the proposed WH2-like α-helical region, supporting results from in silico docking calculations which suggest that, when folded, this α helix binds within the actin hydrophobic cleft as seen for other actin-associated proteins

Smad7 Binds Differently to Individual and Tandem WW3 and WW4 Domains of WWP2 Ubiquitin Ligase Isoforms

WWP2 is an E3 ubiquitin ligase that differentially regulates the contextual tumour suppressor/progressor TGFβ signalling pathway by alternate isoform expression. WWP2 isoforms select signal transducer Smad2/3 or inhibitor Smad7 substrates for degradation through different compositions of protein–protein interaction WW domains. The WW4 domain containing WWP2-C induces Smad7 turnover in vivo and positively regulates the metastatic epithelial–mesenchymal transition programme. This activity and the overexpression of these isoforms in human cancers make them candidates for therapeutic intervention. Here, we use NMR spectroscopy to solve the solution structure of the WWP2 WW4 domain and observe the binding characteristics of Smad7 substrate peptide. We also reveal that WW4 has an enhanced affinity for a Smad7 peptide phosphorylated at serine 206 adjacent to the PPxY motif. Using the same approach, we show that the WW3 domain also binds Smad7 and has significantly enhanced Smad7 binding affinity when expressed in tandem with the WW4 domain. Furthermore, and relevant to these biophysical findings, we present evidence for a novel WWP2 isoform (WWP2C-ΔHECT) comprising WW3–WW4 tandem domains and a truncated HECT domain that can inhibit TGFβ signalling pathway activity, providing a further layer of complexity and feedback to the WWP2 regulatory apparatus. Collectively, our data reveal a structural platform for Smad substrate selection by WWP2 isoform WW domains that may be significant in the context of WWP2 isoform switching linked to tumorigenesis

PDBe: improved findability of macromolecularstructure data in the PDB

© 2019 The Authors. Published by OUP. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1093/nar/gkz990The Protein Data Bank in Europe (PDBe), a founding member of the Worldwide Protein Data Bank (wwPDB), actively participates in the deposition, curation, validation, archiving and dissemination of macromolecular structure data. PDBe supports diverse research communities in their use of macromolecular structures by enriching the PDB data and by providing advanced tools and services for effective data access, visualization and analysis. This paper details the enrichment of data at PDBe, including mapping of RNA structures to Rfam, and identification of molecules that act as cofactors. PDBe has developed an advanced search facility with ∼100 data categories and sequence searches. New features have been included in the LiteMol viewer at PDBe, with updated visualization of carbohydrates and nucleic acids. Small molecules are now mapped more extensively to external databases and their visual representation has been enhanced. These advances help users to more easily find and interpret macromolecular structure data in order to solve scientific problems.The Protein Data Bank in Europe is supported by European Molecular Biology Laboratory-European Bioinformatics Institute; Wellcome Trust [104948]; Biotechnology and Biological Sciences Research Council [BB/N019172/1, BB/G022577/1, BB/J007471/1, BB/K016970/1, BB/K020013/1, BB/M013146/1, BB/M011674/1, BB/M020347/1, BB/M020428/1, BB/P024351/1]; European Union [284209]; ELIXIR and Open Targets. Funding for open access charge: EMB

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly

J. Agric. Food Chem.

(+)-2,3-trans-3,4-cis-Leucocyanidin was produced by acidic epimerization of (+)-2,3-trans-3,4-trans-leucocyanidin synthesized by reduction of (+)-dihydroquercetin with NaBH4, and structures of the two stereoisomers purified by C18- and phenyl-reverse-phase high-performance liquid chromatography (HPLC) were confirmed by NMR spectroscopy. We confirm that only 3,4-cis-leucocyanidin is used by leucoanthocyanidin reductase as substrate. The two stereoisomers are quite stable in aqueous solution at -20 degrees C. Characterization of the two stereoisomers was also performed using electrospray ionization tandem mass spectrometry (ESI-MS/MS), and we discuss here for the first time the corresponding MS/MS fragmentation pathways, which are clearly distinct. The main difference is that of the mode of dehydration of the 3,4-diol in positive ionization mode, which involves a loss of hydroxyl group at either C-3 or C-4 for the 3,4-cis isomer but only at C-3 for the 3,4-trans isomer. Tandem mass spectrometry therefore proves useful as a complementary methodology to NMR to identify each of the two stereoisomers