Fig. 1
Force-induced unfolding of α1-helix enhances the F-actin-binding activity of the αcat-ABD. a R2/7 cells transiently expressing ABD (residues 663-906) or ABD* (residues 697-906). αcat-ABD/ABD*-FLAG and actin were labeled with the anti-DDDDK antibody and phalloidin, respectively. Scale bar, 10 μm. b Comparison of the ABD crystal structures of αN-catenin, αE-catenin and vinculin. The αcat-ABD contains three distinct structural motifs: α1-helix (α1; red circle), β-hairpin (βH; magenta circle), and C-terminal tail (CT; black circle). PDB ID codes are indicated in parentheses. c Multiple sequence alignment of α-catenin and vinculin primary sequences. The α1-helix and βH sequences are highly conserved among three paralogs of α-catenin (E, N and T; h, human; m, mouse), as well as in Drosophila α-catenin (dα-catenin). The H1 mutation (RAIM670-673GSGS) is indicated. Conservation of three actin-binding site residues in α-catenin, as well as the vinculin actin-binding site residues, I997 and V1001, are marked by purple dots. d Snapshots of the structure of αNcat-ABD at select time points during a constant-force SMD simulations (100-pN pulling force for 120 ns) (Supplementary Movie 1). Cartoon representation shows α1-helix (blue) starts to unfold at ~60 ns. e A close-up view of α1-helix and V796N in the αNcat-ABD crystal structure. During constant-force SMD simulations, V796N in a cryptic state is exposed (V796N*; magenta) at 45 ns, shortly before α1 unfolding occurred at 60 ns (d). Two conserved α1-helix residues, R670N and M673N, engage in critical interactions with the five-helix bundle of ABD to attenuate the ABD-F-actin interaction. f Actin cosedimentation assays comparing WT, H1 and Δα1 variants of αEcat-ABD and αNcat-ABD. Whereas less than half of total ABD-WT (0.37-0.45) cosedimented with F-actin for both αE-catenin and αN-catenin, alterations in α1-helix, either by deletion or unfolding via the H1 mutation, significantly increased the amount of mutant αcat-ABD proteins cosedimented with F-actin (0.71–0.81). Supernatant (S) and pellet (P) fractions are indicated. Data are presented as mean ± standard error of the mean (SEM) (N = 3). Significance by ANOVA: ***P < 0.001
