Fig. 3

Aip5-C interacts with G-actin through a stable loop region to deliver actin assembly activity. a The elution profile on Aip5-C and protein standards from calibrated-Superdex 75 10/300 GL. The molecular weights of the protein are indicated. Calculated molecular weight of Aip5-C suggested a dimer state in solution. b The crystal structure of Aip5-C. The two protomers A and B are colored with bright orange and violet purple, respectively. Dimerization of Aip5-C involves β2 strands from both protomers. c An enlarged view of hydrogen bond within the dimer interface of Aip5-C. The β2 strands from both protomers of Aip5-C dimer form hydrogen bonds with water molecules, in particular water molecules 3, 5, 10, 18, 20, and 101 are involved. d The electron density map wrapping around the loop region in protomer A of Aip5-C, which reinforces the stability of this loop and assisted in its structural determination. 2Fo-Fc electron density maps are contoured at 1σ. e Structural comparison of IxxT motif in Aip5-C with cGrx2 at the corresponding position. f Comparison between protomers A and B in Aip5-C dimer. Structures of protomers A and B are vastly similar except for lack of electron density for the loop region in protomer B. g Domain schematics of Aip5-C mutation variants. The dashed rectangular box represents a truncated loop region from S1149 to H1156. The red lines represent the mutated residues. h Pyrene-actin polymerization by an increasing concentration of Aip5-C-LD. i Fluorescence anisotropic binding measurements of Alexa-488-labeled 30 nM Aip5-C-LD that was titrated by non-polymerized actin (NP-actin). Data are represented by circles using the average value of three biological replicates. j Microscale thermophoresis binding curves of 2 μM G-actin titrated with peptides of Aip5 loop. Three biological replicates are shown with an error bar of ±S.D. k Relative actin assembly rate with the indicated Aip5-C variants after being normalized to the actin-alone reaction. The plot shows the mean value from at least two independent experiments. l Pyrene-actin polymerization reaction of Aip5-C and Aip5-C-LD in the presence of Bni1FH1COOH. Source data are provided as a Source Data file