Fig. 2

OI-linked L478P PLS3 mutation disrupts the actin-binding surface of ABD2. a Cryo-EM reconstruction with aligned model of ABD2_PLS2-decorated F-actin (PDB ID: 6VEC; EMDB: EMD-21155). Actin is colored in green, CH3 and CH4 are in purple and blue, respectively. The loops linking the CH domains (385–394 and 511–517) are in red (see also Supplementary Fig. S2 and Supplementary Table S1). b Superposition of F-actin decorated by ABD2_PLS2 (green; PDB ID: 6VEC) and pure F-actin (yellow; PDB ID: 5ONV). c Superposition of ABD2_PLS2 in the complex with F-actin (CH3 domain is in purple, CH4 domain is in blue; PDB ID: 6VEC) and crystal structures of fimbrin ABD2 from Arabidopsis thaliana (pink; PDB ID: 1PXY) and Schizosaccharomyces pombe (green; PDB ID: 1RT8). The arrow indicates the rotation of CH4 upon F-actin binding. d The interface of the PLS2 CH3 domain (purple; PDB ID: 6VEC) binding to F-actin, showing that L475 (corresponding to L478 in PLS3) is not directly interacting with F-actin (green). e Superposition of the L475-containing loop of WT-ABD2_PLS2 (in magenta) with the corresponding elements of ABDs from various t-CH-domain proteins (in gray): utrophin, dystrophin, filamin A, α-actinin, and ABD2 of yeast fimbrin (PDB IDs are provided in the Supplementary Table S2). Distances between L475 and the other four members of the cluster (V464, F473, I478, and L494) are consistent with hydrophobic interactions (see Supplementary Table S2). For respective multiple sequence alignment⁸⁷ see Supplementary Fig. S3. f Superposition of F-actin-bound structure of ABD2_PLS2 obtained in the present study (purple; PDB ID: 6VEC) with filamin A structure (gray; PDB ID: 6D8C) produced by aligning actin (gray/green for ABD2_PLS2/filamin A). Actin-binding residues in the ABD of filamin A³³ that are not conserved with PLS2 ABD2 are shown in yellow. The only two actin-binding residues of filamin conserved in PLS2 ABD2 are in red; the respective conserved PLS2 residues are in magenta