¹H, ¹⁵N, ¹³C backbone resonance assignments of human phosphoglycerate kinase in a transition state analogue complex with ADP, 3-phosphoglycerate and magnesium trifluoride

Human phosphoglycerate kinase (PGK) is an energy generating glycolytic enzyme that catalyses the transfer of a phosphoryl group from 1,3-bisphosphoglycerate (BPG) to ADP producing 3-phosphoglycerate (3PG) and ATP. PGK is composed of two α/β Rossmann-fold domains linked by a central α-helix and the active site is located in the cleft formed between the N-domain which binds BPG or 3PG, and the C-domain which binds the nucleotides ADP or ATP. Domain closure is required to bring the two substrates into close proximity for phosphoryl transfer to occur, however previous structural studies involving a range of native substrates and substrate analogues only yielded open or partly closed PGK complexes. X-ray crystallography using magnesium trifluoride (MgF3(-)) as a isoelectronic and near-isosteric mimic of the transferring phosphoryl group (PO3(-)), together with 3PG and ADP has been successful in trapping human PGK in a fully closed transition state analogue (TSA) complex. In this work we report the (1)H, (15)N and (13)C backbone resonance assignments of human PGK in the solution conformation of the fully closed PGK:3PG:MgF3:ADP TSA complex. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97% of all backbone resonances were assigned in the complex, with 385 out of a possible 399 residues assigned in the (1)H-(15)N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS-N webserver is in good agreement with the published X-ray crystal structure of this complex

Lectin-like bacteriocins from pseudomonas spp. utilise D-rhamnose containing lipopolysaccharide as a cellular receptor

Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins

Has the DOTS Strategy Improved Case Finding or Treatment Success? An Empirical Assessment

Background: Nearly fifteen years after the start of WHO's DOTS strategy, tuberculosis remains a major global health problem. Given the lack of empirical evidence that DOTS reduces tuberculosis burden, considerable debate has arisen about its place in the future of global tuberculosis control efforts. An independent evaluation of DOTS, one of the most widely-implemented and longest-running interventions in global health, is a prerequisite for meaningful improvements to tuberculosis control efforts, including WHO's new Stop TB Strategy. We investigate the impact of the expansion of the DOTS strategy on tuberculosis case finding and treatment success, using only empirical data. Methods and Findings: We study the effect of DOTS using time-series cross-sectional methods. We first estimate the impact of DOTS expansion on case detection, using reported case notification data and controlling for other determinants of change in notifications, including HIV prevalence, GDP, and country-specific effects. We then estimate the effect of DOTS expansion on treatment success. DOTS programme variables had no statistically significant impact on case detection in a wide range of models and specifications. DOTS population coverage had a significant effect on overall treatment success rates, such that countries with full DOTS coverage benefit from at least an 18% increase in treatment success (95% CI: 5–31%). Conclusions: The DOTS technical package improved overall treatment success. By contrast, DOTS expansion had no effect on case detection. This finding is less optimistic than previous analyses. Better epidemiological and programme data would facilitate future monitoring and evaluation efforts

Accurate and efficient gp120 V3 loop structure based models for the determination of HIV-1 co-receptor usage

<p>Abstract</p> <p>Background</p> <p>HIV-1 targets human cells expressing both the CD4 receptor, which binds the viral envelope glycoprotein gp120, as well as either the CCR5 (R5) or CXCR4 (X4) co-receptors, which interact primarily with the third hypervariable loop (V3 loop) of gp120. Determination of HIV-1 affinity for either the R5 or X4 co-receptor on host cells facilitates the inclusion of co-receptor antagonists as a part of patient treatment strategies. A dataset of 1193 distinct gp120 V3 loop peptide sequences (989 R5-utilizing, 204 X4-capable) is utilized to train predictive classifiers based on implementations of random forest, support vector machine, boosted decision tree, and neural network machine learning algorithms. An <it>in silico </it>mutagenesis procedure employing multibody statistical potentials, computational geometry, and threading of variant V3 sequences onto an experimental structure, is used to generate a feature vector representation for each variant whose components measure environmental perturbations at corresponding structural positions.</p> <p>Results</p> <p>Classifier performance is evaluated based on stratified 10-fold cross-validation, stratified dataset splits (2/3 training, 1/3 validation), and leave-one-out cross-validation. Best reported values of sensitivity (85%), specificity (100%), and precision (98%) for predicting X4-capable HIV-1 virus, overall accuracy (97%), Matthew's correlation coefficient (89%), balanced error rate (0.08), and ROC area (0.97) all reach critical thresholds, suggesting that the models outperform six other state-of-the-art methods and come closer to competing with phenotype assays.</p> <p>Conclusions</p> <p>The trained classifiers provide instantaneous and reliable predictions regarding HIV-1 co-receptor usage, requiring only translated V3 loop genotypes as input. Furthermore, the novelty of these computational mutagenesis based predictor attributes distinguishes the models as orthogonal and complementary to previous methods that utilize sequence, structure, and/or evolutionary information. The classifiers are available online at <url>http://proteins.gmu.edu/automute</url>.</p

Pub1p C-Terminal RRM Domain Interacts with Tif4631p through a Conserved Region Neighbouring the Pab1p Binding Site

Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to β-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1–402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role

Structural Biology by NMR: Structure, Dynamics, and Interactions

The function of bio-macromolecules is determined by both their 3D structure and conformational dynamics. These molecules are inherently flexible systems displaying a broad range of dynamics on time-scales from picoseconds to seconds. Nuclear Magnetic Resonance (NMR) spectroscopy has emerged as the method of choice for studying both protein structure and dynamics in solution. Typically, NMR experiments are sensitive both to structural features and to dynamics, and hence the measured data contain information on both. Despite major progress in both experimental approaches and computational methods, obtaining a consistent view of structure and dynamics from experimental NMR data remains a challenge. Molecular dynamics simulations have emerged as an indispensable tool in the analysis of NMR data