Fig. 2
Crystal structure of αNcat-ABD-H1 reveals a novel ABD dimer interface. a Crystal structure of the αNcat-ABD-H1 dimer in form A (two protomers shown as blue and green). The N and C termini of ABD are indicated by blue and red spheres, respectively. Three actin-binding site residues, L785N, I792N and V796N, are shown as light blue, pink and orange spheres. b A close-up view of the ABD dimer interface. The dashed-line box in a is rotated by ~90° CCW. The βH motif from one protomer covers the hydrophobic patch exposed by α1-helix unfolding in the adjacent protomer (the α1-helix of αNcat-ABD-WT is shown in red). c Concentration-dependent CSPs of αNcat-ABD-H1 are localized to the βH residues. Residues with CSP greater than 8 Hz are indicated on the αNcat-ABD-H1 structure in red. d SEC-MALS analysis of αEcat-ABD. The integrated dimer peak area was plotted against the αEcat-ABD concentration for αEcat-ABD-WT (blue) and αEcat-ABD-H1 (orange). Data are presented as mean ± SEM (N = 3). Significance by ANOVA: *P < 0.05. e In vitro actin cosedimentation assays of αEcat-ABD variants, WT, H1, ΔβH, and H1ΔβH. Actin bundling was analyzed by sedimentation at low RCF (10,000×g). The F-actin-bound ABD was sedimented at high RCF (100,000×g). f TCS experiments with unlabeled F-actin and 15N/2H-labeled αNcat-ABD-WT. Plots of the reduction ratios of the backbone amide signal intensities observed with and without presaturation. Residues with >60% and >35% signal reduction are indicated on the αNcat-ABD-WT structure (right). The affected residues are mostly located in the last four α-helices (α3-α6)
