Journal: PLoS ONE

Article Title: Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin

doi: 10.1371/journal.pone.0171278

Figure Lengend Snippet: A, the positions of amino acid residues selected for the mutational study. The contact surfaces interacting with actin in CPILE-a (top) and Ia (bottom) are shown. They are color-coded as in . The side chain of Glu-270 of actin is shown as sphere in Ia. B, comparison of the ADP-ribosylation activity of wildtype (WT) and mutants in CPILE-a (left) and Ia (right). The ADP-ribosylation assay was performed using biotin-NAD + and the biotin-ADP-ribosylated actin was detected by streptavidin-FITC. The SDS-page and the representative scanned images are shown on the top. In α-actin case, two bands were observed [α-actin (top) and protease cleaved α-actin (down)]. Thus each FITC- labeled actin from two bands are calculated and shown (white: non-cleaved black: cleaved). C, comparison of the sensitivity of CPILE-a and Ia against α-actin and β/γ-actin. The SDS-PAGE and the representative scanned images are shown on the top. In α-actin case, total FITC-labeled actin from two bands were calculated and shown. The results of three independent experiments were averaged and Error bar represents S.D. in (B) and (C).

Article Snippet: This time, however, all of the assays were completed against both α- and β/γ-actin. α-Actin (rabbit skeletal muscle) and β/γ-actin (human platelet) were purchased from Cytoskeleton (Denver, CO, USA).

Techniques: Activity Assay, TNKS1 Histone Ribosylation Assay, SDS Page, Labeling

Journal: PLoS ONE

Article Title: Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin

doi: 10.1371/journal.pone.0171278

Figure Lengend Snippet: A, Model structure of the CPILE-a–α-actin complex (blue, N-terminal domain; yellow, C-terminal domain; green, actin). B, E270 A on the convex surface and the deep concave area around it. C, Superimposed structures of CPILE-a and Ia (cyan, N-terminal domain; gray, C-terminal domain) complexed with α-actin. The NAD–Ia–actin structure used was the post-reaction state structure from 4H03. D, Y375 of the Ia convex surface and the shallow concave area around it. Y377 of CPILE-a (yellow) shall change upon binding actin as does the corresponding identical residue on Ia, Y375 (gray; also see text).

Article Snippet: This time, however, all of the assays were completed against both α- and β/γ-actin. α-Actin (rabbit skeletal muscle) and β/γ-actin (human platelet) were purchased from Cytoskeleton (Denver, CO, USA).

Techniques: Binding Assay