Structural basis underlying the autoinhibition of the formin FHOD1 and its phosphorylation-dependent activation

Figures

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  Figure 1. Prediction of a structural model of the autoinhibitory complex of FHOD1.A, schematic representation of the domain architecture of human FHOD1. The fragments used for model prediction are indicated by white boxes. B, sequence alignment of DAD of FHOD and DIAPH proteins: dm, Drosophila melanogaster; dr, Danio rerio; and h, Homo sapiens. The DAD core motif (ΦMDxLΦxxΦ) is shaded in yellow. Basic residues and phosphorylatable Ser/Thr residues are shown in blue and orange, respectively. C, the predicted model of the complex structure of the N terminus and DAD of FHOD1 by AlphaFold3. D, the model with colors of pLDDT values. Very high (pLDDT >90), high (90 > pLDDT > 70), low (70 > pLDDT > 50), and very low (pLDDT <50) are indicated by blue, cyan, yellow, and orange, respectively. E, electrostatic surface representation of the predicted complex between the N terminus and DAD. Left, view from the DAD side with reduced opacity for the DAD domain, highlighting the negative charge on the binding surface of the N terminus. The green lines represent the surface contour of the DAD domain. Right, view from the N terminus side with reduced opacity for the N terminus, highlighting the positive…

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  Figure 2. Basic residues in the extended polybasic region of FHOD1 are essential for the autoinhibitory interactions.A, structures of the interfaces of the N terminus with DAD core (left) and with the extended polybasic region (right). B, effect of amino acid substitutions on the interaction of FHOD1–DAD with the N-terminal region. His-tagged FHOD1-N3 (1–360) was incubated with GST-fused FHOD1–DAD (1081–1145) carrying the indicated amino acid substitution: 3×D, the S1131D/S1137D/T1141D substitution. Proteins were pulled down with glutathione-Sepharose-4B, and the precipitants were subjected to SDS-PAGE, followed by CBB staining. C, quantification of the band intensities of FHOD1-N pulled down with DAD mutants relative to that with DAD-WT from three independent pull-down experiments. Values are means ± SD. ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001. D, effect of amino acid substitutions on stress fiber formation. HeLa cells were transfected with a plasmid encoding GFP-FHOD1-FL carrying the indicated substitution. Cells were fixed, followed by visualization by GFP fluorescence (green) or phalloidin staining (red). The scale bars represent 10 μm. E and F, quantitative analysis of the stress…

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  Figure 3. Conserved acidic residues in the N-terminal region mediate autoinhibitory interactions of FHOD1.A–C, structures of the interfaces between the N terminus and DAD. D, sequence alignment of the area in the N terminus of FHOD that contacts the extended polybasic region: dm, Drosophila melanogaster; dr, Danio rerio; and h, Homo sapiens. Conserved acidic residues D290, D292, D296, and E331 are shown in the same color as (A–C). E, effect of amino acid substitutions on the interaction of FHOD1–DAD with the N-terminal region. His-tagged FHOD1-N3 (1–360) proteins carrying the indicated amino acid substitution (left panel) were incubated with GST-fused FHOD1–DAD (1081–1145). Proteins were pulled down with glutathione-Sepharose-4B, and the precipitants were subjected to SDS-PAGE, followed by CBB staining (right panel). F, quantification of the band intensities of FHOD1-N mutants pulled down with DAD relative to that of FHOD1-N-WT from three independent pull-down experiments. Values are means ± SD. ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001. G, effect of amino acid substitutions on stress fiber formation. HeLa cells were transfected with a plasmid encoding GFP-FHOD1-FL carrying the indicated…

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  Figure 4. Effect of substitutions for hydrophobic residues in the DAD core motif of FHOD1.A, structures of the interfaces between the DAD core motif and the N terminus of FHOD1. B, the effect of amino acid substitutions on the interaction of FHOD1–DAD with the N-terminal region. His-tagged FHOD1-N3 (1–360) was incubated with GST-fused mutant FHOD1–DAD (1081–1145) carrying the indicated amino acid substitution. Proteins were pulled down with glutathione-Sepharose-4B, and the precipitants were subjected to SDS-PAGE, followed by CBB staining. C, quantification of the band intensities of FHOD1-N pulled down with DAD mutants relative to that with DAD-WT from three independent pull-down experiments. Values are means ± SD. D, effect of amino acid substitutions on stress fiber formation. HeLa cells were transfected with a plasmid encoding GFP-FHOD1-FL carrying the indicated substitution. Cells were fixed followed by visualization by GFP fluorescence (green) or phalloidin staining (red). The scale bars represent 10 μm. E and F, quantitative analysis of the stress fiber formation. Cells showing stress fiber formation exceeding 50% of the cell area (E) and cells showing more than five thick stress…

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  Figure 5. Effect of phosphorylation at S1131, S1137, and T1141 within the extended polybasic region of FHOD1.A, structures of the interfaces between the extended polybasic region and the N terminus of FHOD1. The relative positions of the basic residues R1128, R1135, R1139, and the phosphorylatable residues S1131, S1137, T1141 are indicated. B and C, an overlay of 15 predicted models of the complex of unmodified DAD (B) and those of phosphorylated DAD at S1131, S1137, and T1141 (C). D and E, residue-specific interaction energy in the complex of the N terminus and DAD. The interaction energies of the basic residues R1128, R1135, and R1139 in the extended polybasic region (D) and the hydrophobic residues M1108, L1111, V1112, and V1115 (E) are calculated by the INTAA web server using 15 predicted models for unmodified DAD (WT) and DAD phosphorylated at S1131, S1137, and T1141 (pSST), respectively. Data are presented as dot plots and mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ns, not significant. F, the sum of interaction energies between the residues in the N terminus and DAD was calculated by the INTAA web server using 15 predicted models for unmodified DAD (WT) and DAD…

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  Figure 6. Role of the GBD of FHOD1 in the regulation of stress fiber formation.A, schematic representation of the domain architecture of human FHOD1. The fragments used are indicated by white boxes. B, effect of the N-terminal deletion on stress fiber formation. HeLa cells were transfected with a plasmid encoding the indicated fragments. Cells were fixed, followed by visualization by GFP fluorescence (green) or phalloidin staining (red). The scale bars represent 10 μm. C and D, quantitative analysis of the stress fiber formation. Cells showing stress fiber formation exceeding 50% of the cell area (C) and cells showing more than five thick stress fibers (D) were counted. For each mutant, the percentages of positive cells were calculated from a total of 20 to 30 cells in a single transfection experiment, and the mean ± SD was obtained from three independent transfections. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ns, not significant. E, effect of a single substitution of critical residues R1128, R1135, and R1139 into the ΔN93 mutant on stress fiber formation. HeLa cells were transfected with a plasmid encoding the indicated mutants. Cells were fixed, followed by visualization by GFP…

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  Figure 7. A proposed model for the activation process of human FHOD1.A, schematic model of the stepwise process of DAD release from the FH3 domain of FHOD1 by ROCK-mediated phosphorylation (upper part) and that of DIAPH1 by Rho binding (lower part), as described in the text. B, schematic representation of the integrated model of domain organization and molecular regulation of human FHOD1. The autoinhibitory interaction between FH3 and the bipartite DAD is relieved by ROCK-induced phosphorylation within the extended polybasic region, leading to FH2-mediated actin polymerization and GBD-mediated colocalization of FHOD1 with stress fibers, resulting in the formation of bundled stress fibers. The surface of the complex is shown. DAD, diaphanous autoregulatory domain; FHOD, formin homology domain–containing protein; GBD, GTPase-binding domain; ROCK, Rho-associated kinase.