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Fig. 3. CMS domains contribute cooperatively to F-actin binding. (A) In order to map the actin-binding site(s) on CMS, a series of CMS deletion mutants fused to GST were purified and used in G- and/or F-actin binding assays. Schematic representation of constructs (black bars represent sequences rich in prolines, white regions represent the linker sequence, gray bars represent the CC domain, black lines mark the locations of the putative actin-binding motifs, ovals represent the GST peptides, numbers mark the first and last aa of the CMS peptides and Coomassie-stained gels of purified GST fusion peptides and G-actin are shown. (B) CMS does not bind to G-actin. G-actin-binding by CMS was assayed in a GST pull-down reaction. The GST peptide, GST-tagged actin-binding domain of talin, and the GST-tagged CMS C-terminus (CMS CT) were incubated with G-actin at different pH conditions, and subjected to pull-down and western blot analysis. (C) Mapping of the F-actin binding domain(s) of CMS. GST-tagged CMS peptides along with GST alone or GST-talin were assayed by F-actin pull-down reactions. Samples were washed and analyzed by western blotting to detect precipitated F-actin (left panel). The graph represents the relative values ± standard deviation (s.d.) of bound F-actin to CMS CT, CT CC, CT 524 and CT PR of three independent experiments (n=3). P values were calculated using Student's t test.

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