FHL2 production enabled new insight into its flexibility and unexpected binding stoichiometry with β-catenin

Figures

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  Fig. 1. FHL2 LIM domain organization, composition and structure. Grey beads represent individual amino acid residues. Colored circles represent Cys (yellow) and His (blue) residues coordinating zinc ions in the zinc fingers (ZFs). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 2. Expression analysis of FHL2 with different fusion tags (left) and analysis of sfGFP-FHL2 expression at different ZnCl2 concentrations (right). (A) Western blot analysis of FHL2 expression with varying fusion tags. SF, soluble fraction; IF, insoluble fraction. Relative amounts of FHL2 cell lysate fractions (normalized to soluble fraction of HT-FHL2) were calculated based on western blot signal intensities: (B) soluble and insoluble fractions, (C) soluble fraction only. (D) Comparison of sfGFP-FHL2 amount (normalized to soluble fraction without ZnCl2) in soluble and insoluble cell lysate. (E) OD600 values at the end of expression, for different ZnCl2 concentrations in the expression medium. (F) A comparison of the relative amount of soluble sfGFP-FHL2 (normalized to the condition without ZnCl2). Column heights represent average values of biological triplicates, with individual measurements denoted by circles. One sfGFP-FHL2 (B and C) outlier was excluded from the average and standard error of the mean (SE) calculations due to significant deviations. Error bars indicate ±2 SE; *, p ≤ 0.5; **, p ≤ 0.1; ***, p ≤ 0.001.

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  Fig. 3. Characterization of purified FHL2 without a tag with (A) CD, (B) SEC-SLS, (C) DLS at constant temperature (25 °C), (D) temperature-ramped DLS (25–95 °C), (E) nanoDSF and (F) turbidity assay.

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  Fig. 4. Characterization of purified FHL2 with SEC-SAXS analysis. (A) Kratky plot for FHL2 in comparison with globular protein (BSA; SASDA32). (B) AlphaFold model of FHL2 (AF-Q14192-F1-v6). (C) Experimental SAXS plot (blue dots) with fitted AlphaFold model theoretical scattering curve, calculated with FoXS (black line). (D) Pair-distance distribution plot. (E) Guinier fit plot. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 5. Modeling and experimental validation of FHL2 conformational ensembles. (A) Five ensemble families are represented as trees, with the top node corresponding to the best-scoring (i.e. representative) ensemble and their score indicated above the node. (B) Characterization of the representative ensembles. SAXS (left), experimental SAXS plot (blue dots) with fitted representative ensemble's theoretical scattering curve (black line) calculated with FoXS. XL-MS (middle), XL-MS restraints mapped onto the representative ensembles. Black arcs show satisfied and grey arcs show unsatisfied XL-MS restraints. The percentage indicates the fraction of satisfied cross-links within each representative ensemble. Models (right), conformational models within the representative ensembles with their relative populations indicated below each model, highlighted according to the assigned conformational groups: yellow, extended; grey, intermediate; blue, bent. The colors of domains match the colored bars in the XL-MS representation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 6. Distribution of conformational states (extended, intermediate, bent) within ensemble representatives. Ensemble representative numbers (1, 2, 4, 14, 25) are depicted in a grey circle on the y-axis. Roman numerals (I—V) denote conformational model indices corresponding to those in Fig. 5 B.

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  Fig. 7. Interaction analysis between FHL2 and β-catenin. (A) SEC analysis of β-catenin + FHL2, β-catenin, and FHL2 samples. The grey area under the curve represents fractions analyzed with SDS-PAGE. (B) Comparison of experimental (SEC-SLS analyses) and calculated molecular weight. Absorbance (green, yellow, and blue curves) is on the left axis, and molecular weight (black curves) is on the right axis. An asterisk (*) marks the molecular weight calculated for 2 FHL2 + 1 β-catenin. Molecular weight on chromatogram is rounded. (C) ITC thermogram of heat flow over time for forward titration of FHL2 into β-catenin. (D) Corresponding titration plot obtained by integration of the peak areas and normalization. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)