![Figure 1. Characterization of the actin-uncoupled αABD mutants. (a) Structure of αABD in the context of α-catenin and in isolation in both open and closed conformations (PDB ID 4IGG, chain A and B, respectively). αABD is colored in orange, whereas the other domains of α-catenin (αH, M1, M2, and M3) are in gray. The residues that decrease αABD–actin binding in vitro upon mutation to alanine (Fig. 1 c) are shown as spheres and colored according to conservation score estimated via the ConSurf algorithm (see legend). The conserved W859 is in stick representation. The structurally resolved parts of the C-terminal extension of αABD are colored in blue. I792A is exposed on the surface of αABD in isolation but is buried in the interface between αABD and M1 of full-length α-catenin (pink stars). (b) SDS-PAGE showing the results of actin cosedimentation assays with GST-α(671–883) and its deletion mutant GST-α(671–864), each at 3 µM. Pellet (P) and supernatant (S) fractions are shown. Note that the GST-α(671–883) mutant (one asterisk) cosedimented with the actin filaments, whereas its deletion mutant, GST-α(671–864), marked by two asterisks, remains in the supernatant. Bar shows position of ovalbumin (45 kD). (c) In vitro actin binding assays of αABD mutants. The K842-K883 region of αABD (top line) was divided into triplets, and binding of each triple alanine mutant (blue bars) was plotted as the quantity of mutant protein in the pellet relative to total protein (pellet + supernatant). Based on these data, several point mutations were selected (black bars). For comparison, the binding of control proteins, such as GST, GST-α(671–883), and GST-α(671–864), as well as the point mutant GST-α(671–883)-I792A, is also shown. (d) Cosedimentation assays with GST-α(671–883) and its K866A point mutant at constant F-actin concentration (1 µM) while varying the amount of GST-tagged proteins (ligand [Lig]) from 1.5 to 24 µM. (e) Actin binding curves of GST-α(671–883), GST-α(671–864), GST-α(671–883)-K866A, GST-α(671–883)-K842A, and GST-α(671–883)-I792A. Binding affinities were approximated only for two recombinant proteins that showed evidence of saturation at higher ligand concentrations: Kd(GST-α(671–883)) = 1 µM and Kd(GST-α(671–883)-K842A) = ∼30 µM.](https://cdn.rupress.org/rup/content_public/journal/jcb/210/4/10.1083_jcb.201412064/7/jcb_201412064_fig1.jpeg?Expires=1773553896&Signature=CD--qGqYpF2vSLZWhuMhw8ooJxsFxLvxeDhBBhu8g4lbUyzKQw8EAQzLOIbYsGZnKJsH26FTiLU-RPM-PMghn4hMeckmdTVhMf2iyLIJRmjf6XRnBR6GyJun5oOUIqYIyYIUBqlY~2GpZMwmDf~fvz5Ux-EST9gFsx1PzeY3L8n7PX5XBzcH3gbIDIz147OtNgDi4FKba2iJ4IPgMSACiGMBfZxQP06Fb~0VAgLUVErTVdTqMWxyaSYtVlHIqc3rwx0UqSv0KxEDvRFapMymGV8DI2NFelnpjnYx16ztJ5687up7a89e0-ckWC64M5QyezIeFSZX6H8pg3rnNvSzzA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Characterization of the actin-uncoupled αABD mutants. (a) Structure of αABD in the context of α-catenin and in isolation in both open and closed conformations (PDB ID 4IGG, chain A and B, respectively). αABD is colored in orange, whereas the other domains of α-catenin (αH, M1, M2, and M3) are in gray. The residues that decrease αABD–actin binding in vitro upon mutation to alanine (Fig. 1 c) are shown as spheres and colored according to conservation score estimated via the ConSurf algorithm (see legend). The conserved W859 is in stick representation. The structurally resolved parts of the C-terminal extension of αABD are colored in blue. I792A is exposed on the surface of αABD in isolation but is buried in the interface between αABD and M1 of full-length α-catenin (pink stars). (b) SDS-PAGE showing the results of actin cosedimentation assays with GST-α(671–883) and its deletion mutant GST-α(671–864), each at 3 µM. Pellet (P) and supernatant (S) fractions are shown. Note that the GST-α(671–883) mutant (one asterisk) cosedimented with the actin filaments, whereas its deletion mutant, GST-α(671–864), marked by two asterisks, remains in the supernatant. Bar shows position of ovalbumin (45 kD). (c) In vitro actin binding assays of αABD mutants. The K842-K883 region of αABD (top line) was divided into triplets, and binding of each triple alanine mutant (blue bars) was plotted as the quantity of mutant protein in the pellet relative to total protein (pellet + supernatant). Based on these data, several point mutations were selected (black bars). For comparison, the binding of control proteins, such as GST, GST-α(671–883), and GST-α(671–864), as well as the point mutant GST-α(671–883)-I792A, is also shown. (d) Cosedimentation assays with GST-α(671–883) and its K866A point mutant at constant F-actin concentration (1 µM) while varying the amount of GST-tagged proteins (ligand [Lig]) from 1.5 to 24 µM. (e) Actin binding curves of GST-α(671–883), GST-α(671–864), GST-α(671–883)-K866A, GST-α(671–883)-K842A, and GST-α(671–883)-I792A. Binding affinities were approximated only for two recombinant proteins that showed evidence of saturation at higher ligand concentrations: Kd(GST-α(671–883)) = 1 µM and Kd(GST-α(671–883)-K842A) = ∼30 µM.
