Conformational dependence of <Superscript>13</Superscript>C shielding and coupling constants for methionine methyl groups

Methionine residues fulfill a broad range of roles in protein function related to conformational plasticity, ligand binding, and sensing/mediating the effects of oxidative stress. A high degree of internal mobility, intrinsic detection sensitivity of the methyl group, and low copy number have made methionine labeling a popular approach for NMR investigation of selectively labeled protein macromolecules. However, selective labeling approaches are subject to more limited information content. In order to optimize the information available from such studies, we have performed DFT calculations on model systems to evaluate the conformational dependence of ³ J _CSCC, ³ J _CSCH, and the isotropic shielding, σ_iso. Results have been compared with experimental data reported in the literature, as well as data obtained on [methyl-¹³C]methionine and on model compounds. These studies indicate that relative to oxygen, the presence of the sulfur atom in the coupling pathway results in a significantly smaller coupling constant, ³ J _CSCC/³ J _COCC ~ 0.7. It is further demonstrated that the ³ J _CSCH coupling constant depends primarily on the subtended CSCH dihedral angle, and secondarily on the CSCC dihedral angle. Comparison of theoretical shielding calculations with the experimental shift range of the methyl group for methionine residues in proteins supports the conclusion that the intra-residue conformationally-dependent shift perturbation is the dominant determinant of δ¹³Cε. Analysis of calmodulin data based on these calculations indicates that several residues adopt non-standard rotamers characterized by very large ~100° χ³ values. The utility of the δ¹³Cε as a basis for estimating the gauche/trans ratio for χ³ is evaluated, and physical and technical factors that limit the accuracy of both the NMR and crystallographic analyses are discussed.     

Amberlite IR-120H catalyzed MCR: Design,synthesis and crystal structure analysis of 1,8-dioxodecahydroacridines as potential inhibitors of sirtuins

A rapid, inexpensive and high yielding method has been developed for the synthesis of 1,8-dioxodecahydroacridines using Amberlite IR-120H as a reusable catalyst under open air. These compounds were designed as potential inhibitors of sirtuins and prepared via the MCR of 5,5-dimethyl-1,3-cyclohexanedione, (hetero)aryl aldehydes and (hetero)aromatic amines under mild conditions. Further structure elaboration of a representative compound was performed via Pd catalyzed C–C bond forming reactions. The crystal structure analysis and H-bonding patterns along with in vitro inhibitory activity against yeast Sir2 of the same compound is presented. Docking studies indicated that the compound interacts well with the yeast Sir2.     

Characterization of SARS2 Nsp15 nuclease activity reveals it's mad about U

Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3′ of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here, we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3′ of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA, and will aid in the development of new anti-viral therapeutics.     

Nutrient resorption from senescing leaves of epiphytes,hemiparasites and their hosts in tropical forests of Sri Lanka

Aims Epiphytes and hemiparasites do not have direct access to soil nutrients. Epiphytes acquire nutrients through symbiosis, foliar leachates and throughfall, whilst hemiparasites have specialized structures (haustoria) to acquire nutrients from their host. Irrespective of the green leaf nutrient concentrations of epiphytes, hemiparasites and their hosts, nutrient-resorption efficiency and proficiency are expected to be the greater in epiphytes than in their hosts and in hemiparasites. These hypotheses were tested.     

Tropane alkaloids from Erythroxylum zeylanicum O.E. Schulz (Erythroxylaceae)

Six tropane alkaloids were isolated from the Sri Lankan endemic plant Erythroxylum zeylanicum O.E. Schulz (Erythroxylaceae) and structurally elucidated by NMR and MS measurements. Three of them, erythrozeylanines A [1R,3R,5S,6R-6-acetoxy-3-(3',4',5'-trimethoxybenzoyloxy)tropane], B [cis-3 beta-(cinnamoyloxy)tropane], and C [cis-6 beta-acetoxy-3 alpha-(cinnamoyloxy)tropane] are new, whereas the others have already been found in other Erythroxylum species. For the first time, the absolute configuration of a tropane alkaloid (erythrozeylanine A) has been determined by quantum chemical CD calculations.     

Dephosphorylation of Threonine 38 Is Required for Nuclear Translocation and Activation of Human Xenobiotic Receptor CAR (NR1I3)

Upon activation by therapeutics, the nuclear xenobiotic/ constitutive active/androstane receptor (CAR) regulates various liver functions ranging from drug metabolism and excretion to energy metabolism. CAR can also be a risk factor for developing liver diseases such as hepatocellular carcinoma. Here we have characterized the conserved threonine 38 of human CAR as the primary residue that regulates nuclear translocation and activation of CAR. Protein kinase C phosphorylates threonine 38 located on the α-helix spanning from residues 29–42 that constitutes a part of the first zinc finger and continues into the region between the zinc fingers. Molecular dynamics study has revealed that this phosphorylation may destabilize this helix, thereby inactivating CAR binding to DNA as well as sequestering it in the cytoplasm. We have found, in fact, that helix-stabilizing mutations reversed the effects of phosphorylation. Immunohistochemical study using an anti-phospho-threonine 38 peptide antibody has, in fact, demonstrated that the classic CAR activator phenobarbital dephosphorylates the corresponding threonine 48 of mouse CAR in the cytoplasm of mouse liver and translocates CAR into the nucleus. These results define CAR as a cell signal-regulated constitutive active nuclear receptor. These results also provide phosphorylation/dephosphorylation of the threonine as the primary drug target for CAR activation.     

The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling

Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ～10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a 'defective' docking domain may be a primary structural role of H2A.Bbd in chromatin.