Heteronuclear Single Quantum Coherence Spectra Research Articles

HSQC Spectra Simulation and Matching for Molecular Identification.

In the pursuit of improved compound identification and database search tasks, this study explores heteronuclear single quantum coherence (HSQC) spectra simulation and matching methodologies. HSQC spectra serve as unique molecular fingerprints, enabling a valuable balance of data collection time and information richness. We conducted a comprehensive evaluation of the following four HSQC simulation techniques: ACD/Labs (ACD), MestReNova (MNova), Gaussian NMR calculations (DFT), and a graph-based neural network (ML). For the latter two techniques, we developed a reconstruction logic to combine proton and carbon 1D spectra into HSQC spectra. The methodology involved the implementation of three peak-matching strategies (minimum-sum, Euclidean-distance, and Hungarian distance) combined with three padding strategies (zero-padding, peak-truncated, and nearest-neighbor double assignment). We found that coupling these strategies with a robust simulation technique facilitates the accurate identification of correct molecules from similar analogues (regio- and stereoisomers) and allows for fast and accurate large database searches. Furthermore, we demonstrated the efficacy of the best-performing methodology by rectifying the structures of a set of previously misidentified molecules. This research indicates that effective HSQC spectral simulation and matching methodologies significantly facilitate molecular structure elucidation. Furthermore, we offer a Google Colab notebook for researchers to use our methods on their own data (https://github.com/AstraZeneca/hsqc_structure_elucidation.git).

Application of 2D NMR Spectroscopy in Combination with Chemometric Tools for Classification of Natural Lignins.

Lignin is considered a promising renewable source of valuable chemical compounds and a feedstock for the production of various materials. Its suitability for certain directions of processing is determined by the chemical structure of its macromolecules. Its formation depends on botanical origin, isolation procedure and other factors. Due to the complexity of the chemical composition, revealing the structural differences between lignins of various origins is a challenging task and requires the use of the most informative methods for obtaining and processing data. In the present study, a combination of two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and multivariate analysis of heteronuclear single quantum coherence (HSQC) spectra is proposed. Principal component analysis and hierarchical cluster analysis techniques demonstrated the possibility to effectively classify lignins at the level of belonging to classes and families of plants, and in some cases individual species, with an error rate for data classification of 2.3%. The reverse transformation of loading plots into the corresponding HSQC loading spectra allowed for structural information to be obtained about the latent components of lignins and their structural fragments (biomarkers) responsible for certain differences. As a result of the analysis of 34 coniferous, deciduous, and herbaceous lignins, 10 groups of key substructures were established. In addition to syringyl, guaiacyl, and p-hydroxyphenyl monomeric units, they include various terminal substructures: dihydroconiferyl alcohol, balanopholin, cinnamic acids, and tricin. It was shown that, in some cases, the substructures formed during the partial destruction of biopolymer macromolecules also have a significant effect on the classification of lignins of various origins.

Synthesis of Novel Polymers via ROMP of Benzyne‐Derived Monomers and Their NMR Spectroscopic Properties

AbstractDehydrohalogenation of 1,4‐dihalobenzenes and 1,2,4‐trichlorobenzene is performed using n‐BuLi to form benzynes. These benzynes are used as dienophiles in a Diels–Alder reaction with the dienes furan and 2,5‐dimethylfuran to prepare new benzonorbornadiene derivatives. The cycloadducts of furan are polymerized by a ring‐opening metathesis reaction using a Hoveyda–Grubbs ruthenium catalyst. The polymers are characterized by 1H NMR (nuclear magnetic resonance), and unambiguous proton chemical shift assignments are based on the multiplicity pattern of proton resonances. This is confirmed by 2D NMR data from HSQC(Heteronuclear Single Quantum Coherence) and COSY (Correlation Spectroscopy) spectra. The polybenzonorbornadienes have both cis and trans conformation in their chemical structure. The trans conformation is qualitatively in greater proportion observed at 1H NMR experiments. These materials may have syndiotactic microstructure by the lack of correlations in COSY.

GIPMA: Global Intensity-Guided Peak Matching and Alignment for 2D 1H-13C HSQC-Based Metabolomics.

Two-dimensional (2D) 1H-13C heteronuclear single quantum coherence (HSQC) has been increasingly applied to metabolomics studies because it can greatly improve the resolving capability compared with one-dimensional (1D) 1H NMR. However, preprocessing methods such as peak matching and alignment tools for 2D NMR-based metabolomics have lagged behind similar methods for 1D 1H NMR-based metabolomics. Correct matching and alignment of 2D NMR spectral features across multiple samples are particularly important for subsequent multivariate data analysis. Considering different intensity dynamic ranges of a variety of metabolites and the chemical shift variation across the spectra of multiple samples, here, we developed an efficient peak matching and alignment algorithm for 2D 1H-13C HSQC-based metabolomics, called global intensity-guided peak matching and alignment (GIPMA). In GIPMA, peaks identified in all spectra are pooled together and sorted by intensity. Chemical shift of a stronger peak is regarded to be more accurate and reliable than that of a weaker peak. The strongest undesignated peak is chosen as the reference of a new cluster if it is not located within the chemical shift tolerance of any existing peak cluster (PC), or otherwise it is matched to an existing PC and the aligned chemical shift of the PC is updated as the intensity-weighted average of the chemical shifts of all peaks in the cluster. Setting an optimum chemical shift tolerance (Δδo) is critical for the peak matching and alignment across multiple samples. GIPMA dynamically searches for and intelligently selects the Δδo for peak matching to maximize the number of valid peak clusters (vPC), that is, spectral features, among multiple samples. By GIPMA, fully automatic peakwise matching and alignment do not require any spectrum as initial reference, while the chemical shift of each PC is updated as the intensity-weighted average of the chemical shifts of all peaks in the same PC, which is warranted to be statistically more accurate. Accurate chemical shifts for each representative spectral feature will facilitate subsequent peak assignment and are essential for correct metabolite identification and result interpretation. The proposed method was demonstrated successfully on the spectra of six model mixtures consisting of seven typical metabolites, yielding correct matching of all known spectral features. The performance of GIPMA was also demonstrated on 2D 1H-13C HSQC spectra of 87 real extracts of 29 samples of five Dendrobium species. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) of the 87 matched and aligned spectra by GIPMA generates correct classification of the 29 samples into five groups. In summary, the proposed algorithm of GIPMA provided a practical peak matching and alignment method to facilitate 2D NMR-based metabolomics studies.

Exploring Alkyl-O-Alkyl Ether Structures in Softwood Milled Wood Lignins.

Recent studies have suggested that there are significant amounts of various alkyl ether (Alk-O-Alk; Alk = alkyl) moieties in a spruce native lignin preparation, milled wood lignin (SMWL). However, the comprehensive NMR assignment to these moieties has not been addressed yet. This study focused on investigating different types of Alk-O-Alk structures at the α- and γ-positions of the lignin side chain in an heteronuclear single-quantum coherence (HSQC) spectrum of SMWL using experimental NMR data of lignin and synthesized model compounds. Ambiguous structural features were predicted by computer simulation of 1H and 13C NMR spectra to complement the experimental NMR data. As a result, specific regions in the HSQC spectrum were attributed to different Alk-O-Alk moieties of Alk-O-Alk/β-O-4 and Alk-O-Alk/β-β' structures. However, the differences between the specific regions were rather subtle; they were not well separated from each other and some major lignin moieties. Furthermore, SMWL contained a large variety of Alk-O-Alk moieties but in minute individual amounts, resulting in rather broad, superimposing resonances. Thus, evaluation did not allow assigning individual types of Alk-O-Alk moieties from the HSQC spectra; instead, they were quantified as total (α- and γ-linked) Alk-O-Alk based on the balance of structural units in the 13C NMR spectra. At last, potential formation mechanisms of various Alk-O-Alk ether structures in lignin biosynthesis, lignin aging, and during ball milling of wood were hypothesized and discussed.

Correlation between Lignin-Carbohydrate Complex Content in Grass Lignins and Phenolic Aldehyde Production by Rapid Spray Ozonolysis.

We provide strong evidence that the amounts of phenolic aldehydes (vanillin and p-hydroxybenzaldehyde, pHB) selectively released during rapid ozonolysis of grass lignins are correlated with the unsubstituted aryl carbons of lignin-carbohydrate complexes present in these lignins. In the case of acetosolv lignin from corn stover, we observed a steady yield of vanillin and pHB (cumulatively ∼5 wt % of the initial lignin). We demonstrate the continuous ozonolysis of the lignin in a spray reactor at ambient temperature and pressure. In sharp contrast, similar ozonolysis of acetosolv lignin from corn cobs resulted in a twofold increase in the combined yield (∼10 wt %) of vanillin and pHB. Structural analysis with 1H-13C heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance revealed that signals assigned to unsubstituted aryl carbons of lignin-carbohydrate complexes are quantitatively correlated to phenolic aldehyde production from spray ozonolysis. The ratios of the integrated peak volumes corresponding to coumarates and ferulates in the HSQC spectra of cob and corn stover lignins (SLs) are 2.4 and 2.0, respectively. These ratios are nearly identical to the observed 2.3-fold increase in pHB and 1.8-fold increase in vanillin production rates from corn cob lignin compared to corn SL. Considering that the annual U.S. lignin capacity from these grass lignin sources is ∼60 million MT, the value creation potential from these flavoring agents is conservatively ∼$50 million annually from just 10% of the lignin. These new insights into structure/product correlation and spray reactor characteristics provide rational guidance for developing viable technologies to valorize grass lignins.

Structural dynamics of the N-terminal SH2 domain of PI3K in its free and CD28-bound states.

Protein conformational changes with fluctuations are fundamental aspects of protein-protein interactions (PPIs); understanding these motions is required for the rational design of PPI-regulating compounds. Src homology 2 (SH2) domains are commonly found in adapter proteins involved in signal transduction and specifically bind to consensus motifs of proteins containing phosphorylated tyrosine (pY). Here, we analysed the interaction between the N-terminal SH2 domain (nSH2) of the regulatory subunit in phosphoinositide 3-kinase (PI3K) and the cytoplasmic region of the T-cell co-receptor, CD28, using NMR and molecular dynamics (MD) simulations. First, we assigned the backbone signals of nSH2 on 1 H-15 N heteronuclear single quantum coherence spectra in the absence or presence of the CD28 phosphopeptide, SDpYMNMTPRRPG. Chemical shift perturbation experiments revealed allosteric changes at the BC loop and the C-terminal region of nSH2 upon CD28 binding. NMR relaxation experiments showed a conformational exchange associated with CD28 binding in these regions. The conformational stabilisation of the C-terminal region correlated with the regulation of PI3K catalytic function. Further, using 19 F- and 31 P-labelled CD28 phosphopeptide, we analysed the structural dynamics of CD28 and demonstrated that the aromatic ring of the pY residue fluctuated between multiple conformations upon nSH2 binding. Our MD simulations largely explained the NMR results and the structural dynamics of nSH2 and CD28 in both bound and unbound states. Notably, in addition to its major conformation, we detected a minor conformation of nSH2 in the CD28 bound state that may explain the allosteric conformational change in the BC loop.

Structural stability of Cutibacterium acnes acyl carrier protein studied using CD and NMR spectroscopy

To survive in diverse environments, bacteria adapt by changing the composition of their cell membrane fatty acids. Compared with aerobic bacteria, Cutibacterium acnes has much greater contents of branched-chain fatty acids (BCFAs) in the cell membrane, which helps it survive in anaerobic environments. To synthesize BCFAs, C. acnes acyl carrier protein (CaACP) has to transfer growing branched acyl intermediates from its hydrophobic cavity to fatty acid synthases. CaACP contains an unconserved, distinctive Cys50 in its hydrophobic pocket, which corresponds to Leu in other bacterial acyl carrier proteins (ACPs). Herein, we investigated the substrate specificity of CaACP and the importance of Cys50 in its structural stability. We mutated Cys50 to Leu (C50L mutant) and measured the melting temperatures (Tms) of both CaACP and the C50L mutant by performing circular dichroism experiments. The Tm of CaACP was very low (49.6 °C), whereas that of C50L mutant was 55.5 °C. Hydrogen/deuterium exchange experiments revealed that wild-type CaACP showed extremely fast exchange rates within 50 min, whereas amide peaks of the C50L mutant in the heteronuclear single quantum coherence spectrum remained up to 200 min, thereby implying that Cys50 is the key residue contributing to the structural stability of CaACP. We also monitored chemical shift perturbations upon apo to holo, apo to butyryl, and apo to isobutyryl conversion, confirming that CaACP can accommodate isobutyryl BCFAs. These results provide a preliminary understanding into the substrate specificity of CaACPs for the production of BCFAs necessary to maintain cell membrane fluidity under anaerobic environments.

Boosting the resolution of multidimensional NMR spectra by complete removal of proton spin multiplicities

Over decades multidimensional NMR spectroscopy has become an indispensable tool for structure elucidation of natural products, peptides and medium sized to large proteins. Heteronuclear single quantum coherence (HSQC) spectroscopy is one of the work horses in that field often used to map structural connectivity between protons and carbons or other hetero nuclei. In overcrowded HSQC spectra, proton multiplet structures of cross peaks set a limit to the power of resolution and make a straightforward assignment difficult. In this work, we provide a solution to improve these penalties by completely removing the proton spin multiplet structure of HSQC cross peaks. Previously reported sideband artefacts are diminished leading to HSQC spectra with singlet responses for all types of proton multiplicities. For sideband suppression, the idea of restricted random delay (RRD) in chunk interrupted data acquisition is introduced and exemplified. The problem of irreducible residual doublet splitting of diastereotopic CH2 groups is simply solved by using a phase sensitive JRES approach in conjunction with echo processing and real time broadband homodecoupling (BBHD) HSQC, applied as a 3D experiment. Advantages and limitations of the method is presented and discussed.

Protein-Ligand Binding Volume Determined from a Single 2D NMR Spectrum with Increasing Pressure.

Proteins undergo changes in their partial volumes in numerous biological processes such as enzymatic catalysis, unfolding–refolding, and ligand binding. The change in the protein volume upon ligand binding—a parameter termed the protein–ligand binding volume—can be extensively studied by high-pressure NMR spectroscopy. In this study, we developed a method to determine the protein–ligand binding volume from a single two-dimensional (2D) 1H–15N heteronuclear single quantum coherence (HSQC) spectrum at different pressures, if the exchange between ligand-free and ligand-bound states of a protein is slow in the NMR time-scale. This approach required a significantly lower amount of protein and NMR time to determine the protein–ligand binding volume of two carbonic anhydrase isozymes upon binding their ligands. The proposed method can be used in other protein–ligand systems and expand the knowledge about protein volume changes upon small-molecule binding.

Fragment-Based Nuclear Magnetic Resonance Screen against a Regulator of G Protein Signaling Identifies a Binding "Hot Spot".

Regulator of G protein signaling (RGS) proteins have attracted attention as a result of their primary role in directing the specificity as well as the temporal and spatial aspects of G protein-coupled receptor signaling. In addition, alterations in RGS protein expression have been observed in a number of disease states, including certain cancers. In this area, RGS17 is of particular interest. It has been demonstrated that, while RGS17 is expressed primarily in the central nervous system, it has been found to be inappropriately expressed in lung, prostate, breast, cervical, and hepatocellular carcinomas. Overexpression of RGS17 leads to dysfunction in inhibitory G protein signaling and an overproduction of the intracellular second messenger cAMP, which in turn alters the transcription patterns of proteins known to promote various cancer types. Suppressing RGS17 expression with RNA interference (RNAi) has been found to decrease tumorigenesis and sufficiently prevents cancer cell migration, leading to the hypothesis that pharmacological blocking of RGS17 function could be useful in anticancer therapies. We have identified small-molecule fragments capable of binding the RGS homology (RH) domain of RGS17 by using a nuclear magnetic resonance fragment-based screening approach. By chemical shift mapping of the two-dimensional 15 N,1 H heteronuclear single quantum coherence (HSQC) spectra of the backbone-assigned 15 N-labeled RGS17-RH, we determined the fragment binding sites to be distant from the Gα interface. Thus, our study identifies a putative fragment binding site on RGS17 that was previously unknown.

Same analytical method for both (bio)assay and zone isolation to identify/quantify bioactive compounds by quantitative nuclear magnetic resonance spectroscopy

Differing sensitivity is the main obstacle for a direct combination of HPTLC with NMR spectroscopy. A sufficient amount of the isolated compound zone must be provided by HPTLC for subsequent offline NMR detection (HPTLC//NMR). To fill the gap, a straightforward procedure was developed using the same analytical HPTLC system for both bioprofiling and isolation of bioactive zones from multicomponent mixtures. The HPTLC-effect-directed analysis (EDA) revealed several bioactive compounds in five botanical extracts, i.e. Salvia officinalis, Thymus vulgaris and Origanum vulgare, all Lamiaceae, and peels of red and green apples (Jonagored and Granny Smith, respectively), both Rosaceae. A tricky case study was designed to show how to deal with potentially coeluting bioactive structural isomers, e.g., ursolic (UA), oleanolic (OA) and betulinic acids (all C30H48O3), which are most difficult to identify and assign. A multipotent bioactive HPTLC zone showed the same hRF value and mass signal in HPTLCHRMS, though containing the coeluting structural isomers UA and OA. After zone isolation from the HPTLC plate, first the 1H NMR spectrum allowed to distinguish distinct allylic H-18 protons, i.e. 2.20ppm for UA and 2.85ppm for OA, and at the same time, to quantify the two isomers by using the PUlse Length-based CONcentration methodology (HPTLC//1H qNMR-PULCON). In case of a partial overlap of the diagnostic signal with that of the matrix, results were corroborated with those obtained by using the 1H deconvoluted or 2D 1H-13C Heteronuclear Single Quantum Coherence spectra. The comparison of the quantitative results showed a good correlation (R2=0.9718) between the two orthogonal methods HPTLC-Vis and HPTLC//1H qNMR-PULCON. A sufficient zone isolation from the HPTLC plate (mean isolation rate of 82%) for both UA and OA (0.27 - 4.67mM) was achieved for HPTLC//qNMR, comparing the isolated bioactive compound zone with the respective zone in the botanical extract via HPTLC-Vis densitometry. The HPTLC-EDA-Vis//1H qNMR-PULCON procedure for bioprofiling and quantification/identification/confirmation of bioactive compounds in botanical extracts is considered as straightforward, eco-friendly (only 16mL solvent required), simple (NMR calibration used over weeks) and reliable new alternative to the status quo of bioactivity-guided fractionation.

Rapid Quantification of Protein-Ligand Binding via 19F NMR Lineshape Analysis

Fluorine incorporation is ideally suited to many NMR techniques, and incorporation of fluorine into proteins and fragment libraries for drug discovery has become increasingly common. Here, we use one-dimensional 19F NMR lineshape analysis to quantify the kinetics and equilibrium thermodynamics for the binding of a fluorine-labeled Src homology 3 (SH3) protein domain to four proline-rich peptides. SH3 domains are one of the largest and most well-characterized families of protein recognition domains and have a multitude of functions in eukaryotic cell signaling. First, we showe that fluorine incorporation into SH3 causes only minor structural changes to both the free and bound states using amide proton temperature coefficients. We then compare the results from lineshape analysis of one-dimensional 19F spectra to those from two-dimensional 1H-15N heteronuclear single quantum coherence spectra. Their agreement demonstrates that one-dimensional 19F lineshape analysis is a robust, low-cost, and fast alternative to traditional heteronuclear single quantum coherence-based experiments. The data show that binding is diffusion limited and indicate that the transition state is highly similar to the free state. We also measured binding as a function of temperature. At equilibrium, binding is enthalpically driven and arises from a highly positive activation enthalpy for association with small entropic contributions. Our results agree with those from studies using different techniques, providing additional evidence for the utility of 19F NMR lineshape analysis, and we anticipate that this analysis will be an effective tool for rapidly characterizing the energetics of protein interactions.

2D HSQC Chemical Shifts of Impurities from Biomass Pretreatment

AbstractTwo dimensional (2D) heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) is a powerful analytical method which can be used to elucidate the structure of biomass. During processing, biomass is typically subjected to some form of chemical treatment which can be performed with a variety of compounds. The presence of these compounds, even in trace amounts, has the potential to contaminate the sample and lead to misinterpretation of the HSQC spectra. Here we report the chemical shifts of 29 compounds commonly used in biomass processing which have the potential to contaminate the biomass samples and lead to the misinterpretation of peaks associated with biomass (Populus trichocarpa) pretreated via autohydrolysis. The identification of these chemical shifts could serve as a valuable tool in preventing errors in characterizing biomass via HSQC.

Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.

COLMAR Lipids Web Server and Ultrahigh-Resolution Methods for Two-Dimensional Nuclear Magnetic Resonance- and Mass Spectrometry-Based Lipidomics

Accurate identification of lipids in biological samples is a key step in lipidomics studies. Multidimensional nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool for this purpose as it provides comprehensive structural information on lipid composition at atomic resolution. However, the interpretation of NMR spectra of complex lipid mixtures is currently hampered by limited spectral resolution and the absence of a customized lipid NMR database along with user-friendly spectral analysis tools. We introduce a new two-dimensional (2D) NMR metabolite database "COLMAR Lipids" that was specifically curated for hydrophobic metabolites presently containing 501 compounds with accurate experimental 2D 13C-1H heteronuclear single quantum coherence (HSQC) chemical shift data measured in CDCl3. A new module in the public COLMAR suite of NMR web servers was developed for the (semi)automated analysis of complex lipidomics mixtures (http://spin.ccic.osu.edu/index.php/colmarm/index2). To obtain 2D HSQC spectra with the necessary high spectral resolution along both 13C and 1H dimensions, nonuniform sampling in combination with pure shift spectroscopy was applied allowing the extraction of an abundance of unique cross-peaks belonging to hydrophobic compounds in complex lipidomics mixtures. As shown here, this information is critical for the unambiguous identification of underlying lipid molecules by means of the new COLMAR Lipids web server, also in combination with mass spectrometry, as is demonstrated for Caco-2 cell and lung tissue cell extracts.

The influenza NS1 protein modulates RIG-I activation via a strain-specific direct interaction with the second CARD of RIG-I

A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid–inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function. A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid–inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function. The influenza A virus (IAV) 3The abbreviations used are: IAVinfluenza A virusRBDRNA-binding domainEDeffector domainCARDcaspase activation and recruitment domainHSQCheteronuclear single-quantum coherencem.o.i.multiplicity of infectionLucluciferaseGAGSH-agaroseIACUCInstitutional Animal Care and Use CommitteeMDCKMadin-Darby canine kidneySFserum-freeqPCRquantitative PCRFPfluorescence polarizationEMEMeagle's minimum essential medium6-FAM6-carboxyfluorescein. is a serious public health concern that causes annual epidemics and occasional pandemics, resulting in significant levels of morbidity and mortality each year (1Thompson W.W. Comanor L. Shay D.K. Epidemiology of seasonal influenza: use of surveillance data and statistical models to estimate the burden of disease.J. Infect. Dis. 2006; 194 (17163394): S82-S9110.1086/507558Crossref PubMed Scopus (211) Google Scholar, 2Molinari N.A. Ortega-Sanchez I.R. Messonnier M.L. Thompson W.W. Wortley P.M. Weintraub E. Bridges C.B. 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One such protein encoded by IAV is nonstructural protein 1 (NS1). influenza A virus RNA-binding domain effector domain caspase activation and recruitment domain heteronuclear single-quantum coherence multiplicity of infection luciferase GSH-agarose Institutional Animal Care and Use Committee Madin-Darby canine kidney serum-free quantitative PCR fluorescence polarization eagle's minimum essential medium 6-carboxyfluorescein. NS1 is highly multifunctional and ranges from 215–237 amino acids in length, depending on the strain from which it is derived. During infection, NS1 plays a critical role in antagonizing the cellular innate immune response (3Ayllon J. García-Sastre A. The NS1 protein: a multitasking virulence factor.Curr. Top Microbiol. Immunol. 2015; 386 (25007846): 73-107PubMed Google Scholar, 4Hale B.G. Randall R.E. Ortín J. Jackson D. The multifunctional NS1 protein of influenza A viruses.J. Gen. 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IFNβ induction by influenza A virus is mediated by RIG-I, which is regulated by the viral NS1 protein.Cell Microbiol. 2007; 9 (17140406): 930-93810.1111/j.1462-5822.2006.00841.xCrossref PubMed Scopus (228) Google Scholar, 11Pichlmair A. Schulz O. Tan C.P. Näslund T.I. Liljeström P. Weber F. Reis e Sousa C. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates.Science. 2006; 314 (17038589): 997-100110.1126/science.1132998Crossref PubMed Scopus (1767) Google Scholar). RIG-I is an ATP-dependent cytoplasmic helicase whose primary function is to induce an antiviral signaling cascade in response to RNA virus infections (12Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses.Nat. Immunol. 2004; 5 (15208624): 730-73710.1038/ni1087Crossref PubMed Scopus (3112) Google Scholar, 13Yoneyama M. Onomoto K. Jogi M. 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Species-specific inhibition of RIG-I ubiquitination and IFN induction by the influenza A virus NS1 protein.PLoS Pathog. 2012; 8 (23209422): e100305910.1371/journal.ppat.1003059Crossref PubMed Scopus (234) Google Scholar, 27Gack M.U. Kirchhofer A. Shin Y.C. Inn K.S. Liang C. Cui S. Myong S. Ha T. Hopfner K.P. Jung J.U. Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction.Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (18948594): 16743-1674810.1073/pnas.0804947105Crossref PubMed Scopus (197) Google Scholar). This step in the signaling pathway is inhibited during influenza infection by a direct interaction between NS1 and TRIM25 (6Gack M.U. Albrecht R.A. Urano T. Inn K.S. Huang I.C. Carnero E. Farzan M. Inoue S. Jung J.U. García-Sastre A. Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral RNA sensor RIG-I.Cell Host Microbe. 2009; 5 (19454348): 439-44910.1016/j.chom.2009.04.006Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 26Rajsbaum R. Albrecht R.A. Wang M.K. Maharaj N.P. Versteeg G.A. Nistal- Villán E. García-Sastre A. Gack M.U. Species-specific inhibition of RIG-I ubiquitination and IFN induction by the influenza A virus NS1 protein.PLoS Pathog. 2012; 8 (23209422): e100305910.1371/journal.ppat.1003059Crossref PubMed Scopus (234) Google Scholar). Although it is has been shown that this interaction is a critical mechanism through which NS1 antagonizes RIG-I 2CARD ubiquitination (6Gack M.U. Albrecht R.A. Urano T. Inn K.S. Huang I.C. Carnero E. Farzan M. Inoue S. Jung J.U. García-Sastre A. Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral RNA sensor RIG-I.Cell Host Microbe. 2009; 5 (19454348): 439-44910.1016/j.chom.2009.04.006Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 26Rajsbaum R. Albrecht R.A. Wang M.K. Maharaj N.P. Versteeg G.A. Nistal- Villán E. García-Sastre A. Gack M.U. Species-specific inhibition of RIG-I ubiquitination and IFN induction by the influenza A virus NS1 protein.PLoS Pathog. 2012; 8 (23209422): e100305910.1371/journal.ppat.1003059Crossref PubMed Scopus (234) Google Scholar), it has not been shown whether additive antagonistic effects could result from additional direct interactions between NS1 and RIG-I. Recently, we reported that the NS1RBD from 1918H1N1 IAV (A/Brevig Mission/1/1918) interacts directly with the second CARD of RIG-I and that a naturally occurring mutation in the NS1RBD (R21Q) abrogated this direct interaction (8Jureka A.S. Kleinpeter A.B. Cornilescu G. Cornilescu C.C. Petit C.M. Structural basis for a novel interaction between the NS1 protein derived from the 1918 influenza virus and RIG-I.Structure. 2015; 23 (26365801): 2001-201010.1016/j.str.2015.08.007Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Because of the critical role the 2CARDs play in RIG-I activation, we hypothesized that this direct interaction would enhance the ability of NS1 to inhibit ubiquitination of the RIG-I 2CARDs. Furthermore, this inhibition would be independent of other previously studied NS1 interactions involving TRIM25 (6Gack M.U. Albrecht R.A. Urano T. Inn K.S. Huang I.C. Carnero E. Farzan M. Inoue S. Jung J.U. García-Sastre A. Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral RNA sensor RIG-I.Cell Host Microbe. 2009; 5 (19454348): 439-44910.1016/j.chom.2009.04.006Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 7Guo Z. Chen L.M. Zeng H. Gomez J.A. Plowden J. Fujita T. Katz J.M. Donis R.O. Sambhara S. NS1 protein of influenza A virus inhibits the function of intracytoplasmic pathogen sensor, RIG-I.Am. J. Respir. Cell Mol. Biol. 2007; 36 (17053203): 263-26910.1165/rcmb.2006-0283RCCrossref PubMed Scopus (243) Google Scholar, 8Jureka A.S. Kleinpeter A.B. Cornilescu G. Cornilescu C.C. Petit C.M. Structural basis for a novel interaction between the NS1 protein derived from the 1918 influenza virus and RIG-I.Structure. 2015; 23 (26365801): 2001-201010.1016/j.str.2015.08.007Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 9Mibayashi M. Martínez-Sobrido L. Loo Y.M. Cárdenas W.B. Gale Jr., M. García-Sastre A. Inhibition of retinoic acid-inducible gene I-mediated induction of beta interferon by the NS1 protein of influenza A virus.J. Virol. 2007; 81 (17079289): 514-52410.1128/JVI.01265-06Crossref PubMed Scopus (493) Google Scholar, 10Opitz B. Rejaibi A. Dauber B. Eckhard J. Vinzing M. Schmeck B. Hippenstiel S. Suttorp N. Wolff T. IFNβ induction by influenza A virus is mediated by RIG-I, which is regulated by the viral NS1 protein.Cell Microbiol. 2007; 9 (17140406): 930-93810.1111/j.1462-5822.2006.00841.xCrossref PubMed Scopus (228) Google Scholar, 11Pichlmair A. Schulz O. Tan C.P. Näslund T.I. Liljeström P. Weber F. Reis e Sousa C. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates.Science. 2006; 314 (17038589): 997-100110.1126/science.1132998Crossref PubMed Scopus (1767) Google Scholar) and dsRNA structures (3Ayllon J. García-Sastre A. The NS1 protein: a multitasking virulence factor.Curr. Top Microbiol. Immunol. 2015; 386 (25007846): 73-107PubMed Google Scholar) also known to inhibit activation of RIG-I. In this study, we demonstrate that the naturally occurring R21Q mutation in NS1 (NS1R21Q) significantly impacts NS1's ability to control RIG-I signaling. Specifically, we used reverse genetics to generate WT A/Puerto Rico/8/1934 IAV (rPR8WT) and a mutant IAV encoding the R21Q mutation in NS1 (rPR8R21Q) to test the effects of the mutation on aspects of the viral life cycle. We observed that rPR8R21Q induced significantly more IRF3 phosphorylation and IFN-β expression compared with rPR8WT. These data indicate that rPR8R21Q is a significantly more potent RIG-I activator compared with rPR8WT. Further analysis revealed that NS1R21Q is significantly less efficient at inhibiting TRIM25-dependent ubiquitination of the RIG-I 2CARDs, a critical step in activation of RIG-I signaling. Additionally, we determined that the R21Q mutation markedly diminished the ability of NS1 to interact with the RIG-I 2CARDs. Finally, we confirmed that the R21Q mutation did not have an effect on any of the other NS1 functions known to inhibit the RIG-I pathway. Taken together, this is the first study to identify a natural polymorphism in the NS1RBD that mitigates its ability to control RIG-I 2CARD ubiquitination by TRIM25. This study demonstrates that strain-specific polymorphisms in NS1 not only have a significant impact on its ability to efficiently antagonize RIG-I activation but that the effects of these polymorphisms may be conditional to species-specific activation of RIG-I. Previously, we demonstrated that an R21Q mutation in the NS1RBD abrogated its ability to interact with the second CARD domain of RIG-I. Residue 21 is distal to other residues in the NS1RBD (Arg35, Arg38, and Lys41) that are known to have functions in RNA binding and NS1 cellular localization (Fig. 1) (28Cheng A. Wong S.M. Yuan Y.A. Structural basis for dsRNA recognition by NS1 protein of influenza A virus.Cell Res. 2009; 19 (18813227): 187-19510.1038/cr.2008.288Crossref PubMed Scopus (100) Google Scholar, 29Melén K. Kinnunen L. Fagerlund R. Ikonen N. Twu K.Y. Krug R.M. Julkunen I. Nuclear and nucleolar targeting of influenza A virus NS1 protein: striking differences between different virus subtypes.J. Virol. 2007; 81 (17376915): 5995-600610.1128/JVI.01714-06Crossref PubMed Scopus (155) Google Scholar, 30Greenspan D. Palese P. Krystal M. Two nuclear location signals in the influenza virus NS1 nonstructural protein.J. Virol. 1988; 62 (2969057): 3020-3026Crossref PubMed Google Scholar). Disrupting these residues (Arg35, Arg38, and Lys41) by mutation is well-known to cause significant defects in viral fitness, including decreased replication and inability to control the cellular antiviral response. What is not known, however, is the effect of the R21Q mutation on the viral life cycle, as mutations in this region of the NS1RBD have yet to be studied. Given that residue 21 is in an unstudied region of the NS1RBD, we first wanted to ensure that there were no gross structural changes in the NS1RBD associated with the R21Q mutation. To assess any potential structural changes caused by the mutation, we used NMR to analyze the 1H-15N heteronuclear single-quantum coherence (HSQC) spectra of both the WT NS1RBD (RBDWT) and the R21Q mutation (RBDR21Q). Both HSQC spectra showed a single set of dispersed peaks, indicative of a well-folded protein with a single, major conformation (Fig. 2A). Because the NS1RBD is a symmetric homodimer, only 72 amide resonances were expected and ultimately observed (not including the side-chain amides of glutamine and asparagine) for both the RBDWT and RBDR21Q. We then obtained resonance assignments for RBDR21Q using three-dimensional NMR experiments such as HNCACB and CBCA(CO)NH for further analysis. High-quality triple-resonance spectra allowed backbone resonance assignments to be obtained for 70 of the 72 (97%) possible assigned residues of the RBDR21Q. Analysis of the 13Cα chemical shifts indicated that each monomer of the RBDR21Q mutant is composed of α1–turn–α2–turn–α3 (Fig. 2B) (31Wishart D.S. Sykes B.D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data.J. Biomol. NMR. 1994; 4 (8019132): 171-180Crossref PubMed Scopus (1908) Google Scholar). This six-helix dimeric structure of the RBDR21Q homodimer is consistent with both 13Cα chemical shift analysis of the RBDWT (Fig. 2B) and our previously solved solution structure of the 1918H1N1 NS1RBD. Because the NS1 R21Q mutation had not been characterized previously, it was critical to determine whether the mutation impacted basic aspects of the viral life cycle, such as IAV replication and NS1 cellular localization. To accomplish this, we used a previously described IAV reverse genetics system (32Quinlivan M. Zamarin D. García-Sastre A. Cullinane A. Chambers T. Palese P. Attenuation of equine influenza viruses through truncations of the NS1 protein.J. Virol. 2005; 79 (15956587): 8431-843910.1128/JVI.79.13.8431-8439.2005Crossref PubMed Scopus (200) Google Scholar) to generate and rescue recombinant A/Puerto Rico/8/1934 viruses encoding WT NS1 (rPR8WT) and R21Q NS1 mutant (rPR8R21Q). Upon infecting adenocarcinomic human alveolar basal epithelial (A549) cells at m.o.i. 0.01, we observed no significant differences in viral replication between rPR8WT and rPR8R21Q (Fig. 3A). Similarly, we observed no significant change in the cellular localization of NS1 in A549 cells infected with rPR8WT or rPR8R21Q 12 h post-infection (m.o.i. 2) (Fig. 3B). Given that R21Q is a naturally occurring mutation, it is not surprising that replication and intracellular localization are not affected; numerous viruses capable of productive infection in humans also possess a Gln at position 21. However, the observation of similar replication kinetics does not adjudicate the question of whether the two viruses will result in differential activation of the RIG-I signaling pathway upon infection, nor is it prognostic of potential differences in virulence between the two viruses, as influenza replication levels do not necessarily correlate with virulence (33Askovich P.S. Sanders C.J. Rosenberger C.M. Diercks A.H. Dash P. Navarro G. Vogel P. Doherty P.C. Thomas P.G. Aderem A. Differential host response, rather than early viral replication efficiency, correlates with pathogenicity caused by influenza viruses.PLoS ONE. 2013; 8 (24073225): e7486310.1371/journal.pone.0074863Crossref PubMed Scopus (22) Google Scholar, 34Fan S. Hatta M. Kim J.H. Halfmann P. Imai M. Macken C.A. Le M.Q. Nguyen T. Neumann G. Kawaoka Y. Novel residues in avian influenza virus PB2 protein affect virulence in mammalian hosts.Nat. Commun. 2014; 5 (25289523): 502110.1038/ncomms6021Crossref PubMed Scopus (50) Google Scholar). Activation of the RIG-I pathway results in phosphorylation of the antiviral transcription factor IRF3, which then translocates to the nucleus and induces transcription of type I interferons (e.g. IFN-β). IAV NS1 has been shown previously to inhibit IRF3-mediated signaling through its antagonism of the RIG-I pathway (9Mibayashi M. Martínez-Sobrido L. Loo Y.M. Cárdenas W.B. Gale Jr., M. García-Sastre A. Inhibition of retinoic acid-inducible gene I-mediated induction of beta interferon by the NS1 protein of influenza A virus.J. Virol. 2007; 81 (17079289): 514-52410.1128/JVI.01265-06Crossref PubMed Scopus (493) Google Scholar, 35Talon J. Horvath C.M. Polley R. Basler C.F. Muster T. Palese P. García-Sastre A. Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein.J. Virol. 2000; 74 (10933707): 7989-799610.1128/JVI.74.17.7989-7996.2000Crossref PubMed Scopus (502) Google Scholar). Based on our previous data demonstrating that NS1R21Q is unable to interact with the second CARD of RIG-I, we hypothesized that rPR8R21Q would be less efficient at antagonizing RIG-I signaling. To test this hypothesis, we infected A549 cells with rPR8WT and rPR8R21Q (m.o.i. 2) to determine the respective levels of IRF3 phosphorylation 6, 12, and 24 h post-infection. We observed that infection with rPR8R21Q resulted in significantly increased IRF3 phosphorylation (Fig. 4, A and B) 12 and 24 h post-infection relative to rPR8WT levels. Furthermore, these observed differences are not due to disparities in NS1 expression levels between the two viruses, as NS1 expression was determined to be equal across all time points measured (Fig. 4A, quantification not shown). These data suggest that a Gln at position 21 in NS1 results in significantly less efficient antagonism of the RIG-I signaling pathway without altering NS1 expression. Given that phosphorylated IRF3 acts as a transcription factor for up-regulation of type I IFNs, a corresponding increase in IFN-β expression upon infection with rPR8R21Q compared with rPR8WT was expected. We observed significantly increased IFN-β mRNA expression at all time points (Fig. 4C) and significantly increased IFN-β protein expression 12 and 24 h post-infection (Fig. 4D) using identical experimental conditions as those described previously. Increased IRF3 phosphorylation and IFN-β mRNA and protein expression suggest that rPR8R21Q is a significantly more potent activator of the RIG-I pathway during infection. Although increased IRF3 phosphorylation and IFN-β

Purification and characterization of an exopolysaccharide from Leuconostoc lactis L2

An exopolysaccharide (EPS)-producing bacterium was isolated from the homemade fermentation product of hami melon (Cucumis melo var. saccharinus) and identified as Leuconostoc lactis L2 mainly based on 16S rDNA sequence analysis and the API 50 CHL test. The monosaccharide composition of Leu. lactis L2 EPS was determined to be glucose by gas chromatography (GC), and its molecular weight was 3.7 × 103 kDa by high-performance size exclusion chromatography (HPSEC). The EPS microscopic characteristics were compact, branched and tubular under scanning electron microscopy (SEM). Atomic force microscopy (AFM) showed a smooth and consistent polymeric matrix. The EPS was a linear polymer composed of α-(1 → 6)-linked D-pyranose residues, based on the Fourier transform infrared (FT-IR), 1H, 13C nuclear magnetic resonance (NMR) and heteronuclear single quantum coherence (HSQC) spectra. This EPS exhibited a high water solubility index (WSI) and water holding capacity (WHC) (91.90 ± 2.45% and 509.45 ± 28.59%, respectively), as well as good emulsifying properties with many organic solvents. These results collectively suggest the applicable potential of this EPS in the food, feed and pharmaceutical fields.

Biophysical and functional characterizations of recombinant RimI acetyltransferase from Mycobacterium tuberculosis.

Nα-acetylation is a universal protein modification related to a wide range of physiological processes in eukaryotes and prokaryotes. RimI, an Nα-acetyltransferase in Mycobacterium tuberculosis, is responsible for the acetylation of the α-amino group of the N-terminal residue in the ribosomal protein S18. Despite growing evidence that protein acetylation may be correlated with the pathogenesis of tuberculosis, no structural information is yet available for mechanistically understanding the MtRimI acetylation. To enable structural studies for MtRimI, we constructed a serial of recombinant MtRimI proteins and assessed their biochemical properties. We then chose an optimal construct MtRimIC21A4-153 and expressed and purified the truncated high-quality protein for further biophysical and functional characterizations. The 2D 1H-15N heteronuclear single quantum coherence spectrum of MtRimIC21A4-153 exhibits wider chemical shift dispersion and favorable peak isolation, indicating that MtRimIC21A4-153 is amendable for further structural determination. Moreover, bio-layer interferometry experiments showed that MtRimIC21A4-153 possessed similar micromolar affinity to full-length MtRimI for binding the hexapeptide substrate Ala-Arg-Tyr-Phe-Arg-Arg. Enzyme kinetic assays also exhibited that MtRimIC21A4-153 had almost identical enzymatic activity to MtRimI, indicating insignificant influence of the recombinant variations on enzymatic functions. Furthermore, binding sites of the peptide were predicted by molecular docking approach, suggesting that this substrate binds to MtRimI primarily through electrostatic and hydrogen bonding interactions. Our results lay a foundation for the further structural determination and dynamics detection of MtRimI.

Conformational Complexity and Dynamics in a Muscarinic Receptor Revealed by NMR Spectroscopy.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.