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Fig. 2. A conserved surface-exposed arginine residue, R30, in coronin 1B is responsible for actin-cable formation. (A) Charged, surface-exposed residues within the first and fourth blades of the coronin 1B β-propeller were mutated to alanine or an amino acid with an opposite charge (i.e. R
D). Cable-formation capability was scored as follows: +++, >99% of transfected cells contain GFP-positive actin cables; +/–, <50% of transfected cells contain actin cables, the rest have phalloidin-positive, small GFP aggregates; –, <1% of transfected cells contain phalloidin-positive GFP aggregates, the majority show high levels of cytoplasmic GFP. (B) S2 cells were transfected with the indicated GFP-tagged coronin 1B mutants and stained with phalloidin. (C) Multiple sequence alignment shows that R30 is a highly conserved charged residue in coronins, and locates between the first and second β-sheet in the propeller structure. M-1B, Mus musculus coronin 1B (gi:12229769); R-1B, Rattus norvegicus coronin 1B (gi:12229732); H-1B, Homo sapiens coronin 1B (gi:21263481); H-1C, Homo sapiens coronin 1C (gi:7656991); Celegans, Caenorhabditis elegans coronin (gi:3121874); Spombe, Schizosaccharomyces pombe coronin (gi:3121869); Scerev, Saccharomyces cerevisiae coronin (gi:3121873); DICDI, Dictyostelium discoideum coronin (gi:116950). The multiple alignment was generated using ClustalX 1.83 and illustrated using ESPRIPT. (D) Top view of coronin 1B homology model. N-terminus is blue, C terminus is red. The side chain of R30 is presented in stick form. The homology model was generated in InsightII-2005 using HOMOLOGY model, and evaluated by Profile_3D function (see supplementary material Fig. S1). Illustration was generated using Pymol (http://www.pymol.org). (E) Side view of coronin 1B structural model showing the postions of R30 and K73.
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