Journal: Nucleic Acids Research

Article Title: Structural basis for sensitivity and acquired resistance of fungal cap guanine-N7 methyltransferases to the antifungal antibiotic sinefungin

doi: 10.1093/nar/gkaf538

Figure Lengend Snippet: Structures of K. lactis RNA cap methyltransferase in complexes with products SAH and m 7 GTP. ( A ) Schmatic outline of the experimental setup for methylation assays. Methylation of substrate (m 7 GpppA) was detected by separating reactions products (in red and blue) using a high-performance liquid chromatography (HPLC) column coupled to a triple quadrupole (QQQ) MS, which detects product ions (see the “Materials and methods” section). Quantification was achieved by normalizing ion counts to an internal standard, l -tryptophan. The ratio of m 7 GpppA ion counts to l -tryptophan ion counts yielded the internal standard ratio (ISTD). A range of m 7 GpppA calibration standards (with constant l -tryptophan) provided a linear calibration curve between ISTD and m 7 GpppA concentration. Sample ISTDs were then used to interpolate a discrete m 7 GpppA concentration from the calibration curve. ( B ) Methylation activities of wild-type (purple circles) and two truncated Kl Abd1 variants ( Kl Abd1Δ116, pink circles; and Kl Abd1Δ137, dark pink circles) with GpppA substrate (see the “Materials and methods” section). Error bars (one standard deviation) are calculated from three independent experiments performed in triplicate. ( C ) A view of the Kl Abd1 structure (colored purple) as a ribbon with arrows for β-strands and wide ribbons for helices. A transparent molecular surface envelops the structure. N and C denote amino and carboxyl termini, respectively. Bound SAH in the methyl donor site is shown in stick representation (green). ( D ) A closeup view of the methyl donor site of Kl Abd1–SAH complex. ( E ) A view of the methyl acceptor site of Kl Abd1 bound to m 7 GTP (stick representation in cyan). Bound adenine in the methyl donor site is shown in stick representation (green) and rest of the unresolved substrate/product is depicted in thin gray line. Side chains shown in stick representation, and waters are denoted by red spheres. Atomic contacts are indicated by dashed lines with distances. ( F ) Aligned primary structures of cap methyltransferases from K. lactis ( Kl Abd1), S. cerevisiae ( Sc Abd1), E. cuniculi (Ecm1), and Homo sapiens (RNMT). The secondary structure elements of Kl Abd1 are shown above the amino acid sequences, with α-helices depicted as cylinders and β-strand as arrows. Gaps in the alignments are indicated by “•”. Side-chain identity/similarity is denoted by shading and letter color (purple shade conserved in all; purple letters conserved in most). A predicted disordered region N-terminal to the catalytic domain is denoted by a purple dashed line above the alignment. Kl Abd1 amino acids that contact SAH and m 7 GTP are indicated by green and orange circles, respectively. A conserved tyrosine in β11 that interacts with the cap guanosine is outlined in cyan.

Article Snippet: Solutions of SAM (Cayman Chemical), SAH (Millipore-Sigma), SFG (Santa Cruz Biotechnology), GTP (Jena Bioscience), m 7 GTP (Millipore-Sigma), GpppA (Jena Bioscience), and m 7 GpppA (Jena Bioscience) were prepared in 300 mM HEPES buffer (pH 7.5), and concentrations were determined from the absorbance at 260 nm using the following extinction coefficients (M −1 cm −1 ): SAM, 16000; SAH, 15400; SFG, 16000; GTP, 13700; m 7 GTP, 9800; GpppA, 27000; and m 7 GpppA, 22700.

Techniques: Methylation, High Performance Liquid Chromatography, Targeted Proteomics, Concentration Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Structural basis for sensitivity and acquired resistance of fungal cap guanine-N7 methyltransferases to the antifungal antibiotic sinefungin

doi: 10.1093/nar/gkaf538

Figure Lengend Snippet: Activity and inhibition of S. cerevisiae Abd1. ( A ) Methylation activities of full-length wild-type (WT) Sc Abd1 (light gray triangles), two truncated Sc Abd1 variants ( Sc Abd1Δ119, gray circles; and Sc Abd1Δ140, dark gray squares) and SFG-resistant Sc Abd1-E124K-K163R-K311R-F387Y-Y416F variant (red squares) with GpppA substrate (see the “Materials and methods” section). Error bars (one standard deviation) are calculated from three independent experiments performed in triplicate. ( B ) Inhibition of methylation activities (three independent experiments) of Sc Abd1WT (light gray circles), SFG-resistant variants of Sc Abd1 ( Sc Abd1-L59P-Y416C, green circles; Sc Abd1-E124K-K163-K311R-F387Y-Y416, red circles; Sc Abd1-Y416N-R422H, black circles; Sc Abd1-Y416A, orange circles) with SAH. Reaction mixtures containing 250 nM enzyme preparations were incubated with 100 μM SAM, 250 μM GpppA, and variable concentrations (0, 3.125, 12.5, 25, 50, 100, 200, and 800 μM; see the “Materials and methods” section) of SAH at 30°C in buffer consisting of 50 mM Tris (pH 8.2), 200 mM NaCl, and 5 mM βME. After 20 min, 10 μl reaction samples were quenched with 10 μl of quenching solution containing 100 mM H 2 SO 4 and 50 μM l -tryptophan. Concurrently, m 7 GpppA standards were quenched in quenching solution. ( C ) Inhibition of methylation activities of wild-type and SFG-resistant variants of Sc Abd1 with SFG as shown in panel (B). Reaction mixture containing 250 nM enzyme preparations was incubated with 100 μM SAM, 250 μM GpppA, and variable concentrations (0, 15.6, 31.25, 62.5, 125, 250, and 500 nM for the wild-type proteins; 0, 100 nM, 500 nM, 750 nM, 1.2 μM, 1.7 μM, 2.2 μM, and 4.0 μM for the variants; see the “Materials and methods” section) of SFG at 30°C in buffer consisting of 50 mM Tris (pH 8.2), 200 mM NaCl, and 5 mM βME. After 20 min, 10 μl reaction samples were quenched with 10 μl of quenching solution containing 100 mM H 2 SO 4 and 50 μM l -tryptophan. ( D ) Ratios of IC 50 values of wild-type and variant Sc Abd1 and Kl Abd1 between SAH and SFG with GpppA substrate. Significance between the wild-type and resistant variants determined by ordinary one-way ANOVA (**** signifies adjusted P -value <.0001). ( E ) IC 50 values for SAH (μM) and SFG (nM) obtained from panels (B) and (C) for Sc Abd1WT (gray), Sc Abd1-L59P-Y416C (green), Sc Abd1-E124K-K163-K311R-F387Y-Y416 (red), Sc Abd1-Y416N-R422H (black), and Sc Abd1-Y416A (orange).

Article Snippet: Solutions of SAM (Cayman Chemical), SAH (Millipore-Sigma), SFG (Santa Cruz Biotechnology), GTP (Jena Bioscience), m 7 GTP (Millipore-Sigma), GpppA (Jena Bioscience), and m 7 GpppA (Jena Bioscience) were prepared in 300 mM HEPES buffer (pH 7.5), and concentrations were determined from the absorbance at 260 nm using the following extinction coefficients (M −1 cm −1 ): SAM, 16000; SAH, 15400; SFG, 16000; GTP, 13700; m 7 GTP, 9800; GpppA, 27000; and m 7 GpppA, 22700.

Techniques: Activity Assay, Inhibition, Methylation, Variant Assay, Standard Deviation, Incubation

Journal: Nucleic Acids Research

Article Title: Structural basis for sensitivity and acquired resistance of fungal cap guanine-N7 methyltransferases to the antifungal antibiotic sinefungin

doi: 10.1093/nar/gkaf538

Figure Lengend Snippet: Structure of K. lactis Abd1 bound to inhibitor SFG and SFG plus GTP. ( A ) SAH inhibition. The extent of methylation of GpppA by wild-type Kl Abd1 was quantified (see the “Materials and methods” section) in the presence of increasing concentrations (0, 3.125, 12.5, 25, 50, 100, 200, and 800 μM) of SAH and the obtained relative activities (%) for the enzyme (green circles) were plotted as a function SAH concentrations to determine the IC 50 value (indicated). ( B ) SFG inhibition. The extent of methylation of GpppA by wild-type Kl Abd1 was quantified (see the “Materials and methods” section) in the presence of increasing concentrations (0, 15.6, 32.25, 62.5, 125, 250, and 500 nM) of SFG and the relative activities (%) for Kl Abd1 (light yellow circles) were plotted as a function SFG concentrations to determine the IC 50 value (indicated). Error bars (one standard deviation) are calculated from three independent experiments performed in triplicate. ( C ) A view of the methyl donor site of Kl Abd1 bound to SFG shown in stick representation and colored light yellow. ( D ) A closeup view of the methyl acceptor site of Kl Abd1 bound to SFG (as in panel B) and GTP (stick representation in cyan). Side chains are shown in stick representation (as in Fig. ), and waters are denoted by red spheres. Atomic contacts are indicated by dashed lines with distances.

Article Snippet: Solutions of SAM (Cayman Chemical), SAH (Millipore-Sigma), SFG (Santa Cruz Biotechnology), GTP (Jena Bioscience), m 7 GTP (Millipore-Sigma), GpppA (Jena Bioscience), and m 7 GpppA (Jena Bioscience) were prepared in 300 mM HEPES buffer (pH 7.5), and concentrations were determined from the absorbance at 260 nm using the following extinction coefficients (M −1 cm −1 ): SAM, 16000; SAH, 15400; SFG, 16000; GTP, 13700; m 7 GTP, 9800; GpppA, 27000; and m 7 GpppA, 22700.

Techniques: Inhibition, Methylation, Standard Deviation