Fig. 2: Structural, biochemical, and antiviral analyses of SspE and its variants.
From: Nicking mechanism underlying the DNA phosphorothioate-sensing antiphage defense by SspE
a The distribution of the PT-sensing cavity and D96GQQR100-containing NTP binding pocket are shown as black and yellow boxes, respectively, in the NTD of SspE. Key residues are represented as sticks. The magenta dotted line represents the distance of side chains between residues K40 and R100 in the absence of PT-DNA. b Assessment of the GTPase activity of SspE and its mutants in the presence of 40-bp 5′-CCA-3′- or 5′-CPSCA-3′-containing DNA fragments. The responsiveness of SspE GTPase activity to 5′-CCA-3′-containing single-stranded DNA or RNA was also measured. The mean ± SEM values of three independent experiments are shown. Statistical significance was calculated by unpaired two-sided Student t tests. **P < 0.01. *P < 0.05. NS, not significant. c Plaque assays were carried out to compare the antiphage activities of SspE and variants in S. lividans HXY6 using 2 μL of each serial tenfold dilution (10−1–10−6) of phage JXY1 at 28 °C. The data shown here are representative images of three independent experiments. d Docking of double-stranded DNA (5′-GCGTCCA-3′/5′-TGGACGC-3′ derived from PDB entry 2XHI) into the hydrophobic patch on the SspENTD surface. The surface of SspENTD is colored according to the electrostatic potential. Red and blue indicate negative and positive potentials, respectively. The sulfur atom on the DNA skeleton is colored green. The magenta dotted line represents the distance of side chains between residues K40 and R100 in the docking structure of SspENTD with 5′-CPSCA-3′-containing DNA. e Growth curve analysis of S. lividans HXY6 cells expressing wild-type SspE or its mutants grown in TSBY medium (3% tryptone soy broth medium, 0.5% yeast extract, and 10.3% sucrose) at 28 °C for 72 h (upper panel). A 1-mL aliquot was collected at the indicated time points and centrifuged for 30 min at 16,000 × g. The supernatant was discarded, and the pellet was dried at 70 °C for 48 h and weighed. SspEK40A alone attenuated the growth of PT-lacking S. lividans HXY6. Effects of SspE and its mutants on cell growth in the presence of SspABCD (lower panel). Values represent the mean of triplicate experiments for each time point tested. Error bars represent the SD from n = 3 replicates. Source data are provided as a Source Data file.
