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Allosteric regulation controls actin-bundling properties of human plastins

Nature Structural & Molecular Biology volume 29, pages 519–528 (2022)Cite this article

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Abstract

Plastins/fimbrins are conserved actin-bundling proteins contributing to motility, cytokinesis and other cellular processes by organizing strikingly different actin assemblies as in aligned bundles and branched networks. We propose that this ability of human plastins stems from an allosteric communication between their actin-binding domains (ABD1/2) engaged in a tight spatial association. Here we show that ABD2 can bind actin three orders of magnitude stronger than ABD1, unless the domains are involved in an equally strong inhibitory engagement. A mutation mimicking physiologically relevant phosphorylation at the ABD1–ABD2 interface greatly weakened their association, dramatically potentiating actin cross-linking. Cryo-EM reconstruction revealed the ABD1–actin interface and enabled modeling of the plastin bridge and domain separation in parallel bundles. We predict that a strong and tunable allosteric inhibition between the domains allows plastins to modulate the cross-linking strength, contributing to remodeling of actin assemblies of different morphologies defining the unique place of plastins in actin organization.

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Fig. 1: ABD2 of PLS2 binds to F-actin with nanomolar affinity and nucleates actin.
Fig. 2: ABD1 of PLS2 interacts with and suppresses ABD2.
Fig. 3: F-actin binding destabilizes PLS2 due to ABD1–ABD2 disentanglement.
Fig. 4: Cryo-EM reconstruction of ABD1-decorated F-actin.
Fig. 5: S406E PLS2 mutation mimicking physiologically relevant phosphorylation of S406 releases inhibition of ABD2 by uncoupling it from ABD1.
Fig. 6: Intracellular localization of human plastins.
Fig. 7: Hypothetical experimental model of actin bundling by plastins.

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Data availability

The cryo-EM map of ABD1PLS3–F-actin complex was deposited with accession code EMD-25371 in the EMDB. Figures using protein structures were generated using PDB accession numbers: 6ANU, 1AOA, and 6VEC and AlphaFold entry: https://alphafold.ebi.ac.uk/entry/P13797. Source data are provided with this paper.

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Acknowledgements

We thank F. Wang (University of Virginia) for assistance in modeling parallel actin filaments linked by a plastin bridge and S. Dong (OSU) and L. Runyan (OSU) for assistance in protein purification. This work was supported by the National Institute of General Medical Sciences of the NIH under award numbers R01GM114666 (D.S.K.), R35GM122510 (E.H.E.) and a 2018 Pelotonia Graduate Fellowship Award at The Ohio State University Comprehensive Cancer Center (C.L.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. Richa Agrawal

    Present address: Biochemistry, Molecular, and Cell Biology (BMCB) program, Cornell University, Ithaca, NY, USA

  2. Weili Zheng

    Present address: NanoImaging Services, San Diego, CA, USA

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA

    Christopher L. Schwebach, Elena Kudryashova, Richa Agrawal & Dmitri S. Kudryashov

  2. Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, USA

    Weili Zheng & Edward H. Egelman

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Contributions

D.S.K. conceptualized the study. D.S.K., E.H.E. and C.L.S. acquired funding. D.S.K. and E.H.E. supervised the study and C.L.S, E.K., R.A. and W.Z carried out the investigations. C.L.S., E.K. and W.Z. carried out formal analysis and visualization. C.L.S, D.S.K. and E.K. wrote the original draft. All authors reviewed and edited the final manuscript.

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Correspondence to Dmitri S. Kudryashov.

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Extended data

Extended Data Fig. 1 ABD2PLS2 exists in solution as a monomer and rescues polymerization of ACD-crosslinked actin oligomers.

(a-c) Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of ABD2PLS2. Raw sedimentation profiles of absorbance at 280 nm versus radius (a) and residual plots (b) are shown. The distribution of sedimentation coefficients (c) indicates the presence of only monomeric species of ABD2PLS2 protein. (d) Actin nucleation activity of ABD2 of PLS2 was tested by TIRFM in the presence of profilin (PFN1)/F-actin. Error bars represent the SD of the mean; n is number of biologically independent samples examined over two independent experiments. ANOVA followed by multiple comparison tests (two-sided Student’s t-test) with Bonferroni correction was applied: asterisks indicate statistically significant difference (*p<0.017). Scale bars are 10 µm. (e) ABD2PLS2 rescues polymerization of ACD-cross-linked actin oligomers. G-actin was covalently cross-linked by addition of ACD toxin in the absence (Actin) or presence (Actin +ABD2) of ABD2. Following ACD treatment, cross-linked actin was allowed to polymerize by addition of Mg2+ and KCl and subjected to ultracentrifugation to separate non-polymerized soluble (S) and polymerized pellet (P) fractions on SDS-PAGE. ABD2 alone sample (ABD2) treated identically served as a negative control. Data for graph in d are available as source data.

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Extended Data Fig. 2 ABD1/ABD2 interaction is not affected by salt.

The ABD1–ABD2 affinity in the presence of 30 and 130 mM KCl was determined by fluorescence anisotropy assays. Error bars represent the SD of the mean; n=3 independent experiments. Data for graph are available as source data.

Source data

Extended Data Fig. 3 L475P mutation diminishes F-actin bundling ability of PLS2.

(a) F-actin bundling and binding by PLS2WT and PLS2L475P was assessed by low- (bundling) and high- (binding) speed sedimentation. Low-speed co-sedimentation (panel Actin Bundling) detected F-actin bundles formed only in the presence of PLS2WT in the absence of Ca2+ (PLS2WT Actin Bundling 3s/3p vs 4s/4p), while PLS2L475P was unable to bundle F-actin regardless of Ca2+ [F-actin remained mainly in the supernatant (PLS2L475P Actin Bundling 3s/3p and 4s/4p)]. In high-speed co-sedimentation assays (panel Actin Binding), both constructs were co-pelleted with F-actin in the absence (Actin Binding 3s/3p) and presence of Ca2+ (Actin Binding 4s/4p) implying that L475P mutation does not affect F-actin binding mediated through ABD1. (b,c) For DSF experiments, protein preps of PLS constructs (lines ‘T’ on SDS-PAGE (b)) were further cleared to remove minor contamination of truncated fragments corresponding to RD–ABD1 (red arrow on SDS-PAGE (b)). This contamination resulted in an additional melting peak (Tm2, dotted lines on DSF graphs (c)) but was neither removable by ion exchange chromatography (due to similar pIs) nor by gel filtration (due to insufficiently different sizes). However, following heating to 55 °C and ultracentrifugation at 300,000g for 30 min at 4 °C to remove precipitate (lines ‘P’ on SDS-PAGE (b)), the supernatants (lines ‘S’ on SDS-PAGE (b)) containing cleared FL PLS constructs were free of the contaminants, displayed single DSF peaks (Tm1) and were otherwise indistinguishable from the originally prepped proteins (c). The cleared samples (lines ‘S’ on SDS-PAGE (b)) were used in DSF experiments (Fig. 3). Data for graphs in c are available as source data.

Source data

Extended Data Fig. 4 Cryo-EM reconstruction of ABD1/F-actin.

(a-d) Preparation of ABD1/F-actin sample for cryo-EM. To produce F-actin decorated with ABD1, recombinant human β-Actin carrying K50C and C374A mutations was purified from Pichia pastoris and polymerized as described in online Methods. Individual Cys residues were introduced on the Cys-null RD–ABD1PLS3 background (Supplementary Table 1) at the indicated positions resulting in constructs containing single cysteines (Q194C, A216C, L226C, or G229C). Asterisks indicate activation of either actin (Actin*) or RD–ABD1 constructs (194*, 216*, 226*, or 229*) using a corresponding cross-linking reagent [MTS-8-MTS (a), oPDM (c), or pPDM (d)]. Following the activation, 2.5 µM actin was mixed with 10-molar excess of an RD–ABD1 construct. The resulting cross-linked samples were resolved on 9% SDS-PAGE (a, c, d). Note that non-crosslinked actin (42 kDa) and RD–ABD1 constructs (43 kDa) have similar molecular weights resulting in similar mobility on SDS-PAGE. Anti-actin and anti-PLS3 western blotting was performed to confirm identity of the resulting bands; only the immunoblots for the samples boxed in a are shown in b. Red arrowheads indicate RD–ABD1/RD–ABD1 cross-links. Green arrowheads indicate successful formation of RD–ABD1/F-actin cross-links. The cross-linking of MTS-8-MTS-activated actin with the RD–ABD1 (boxed in a) was the most efficient as compared to other tested reagents/combinations, and the sample of MTS-8-MTS-activated actin cross-linked with RD–ABD1Q194C was subjected to cryo-EM. (e) ABD1/F-actin density map is colored according to the resolution in angstroms.

Extended Data Fig. 5 S406E phospho-mimetic mutation moderately increases nucleation activity of PLS2.

Actin nucleation activity of PLS2S406E at 50-nM (a; n=5) and 1-µM (b; n=8) was tested by TIRFM (n is number of biologically independent samples examined over two independent experiments). Error bars represent the SD of the mean. ANOVA followed by multiple comparison tests with Bonferroni correction was applied: asterisk indicates statistically significant difference (*p<0.025) compared to the actin control. Data for graphs in a,b are available as source data.

Source data

Extended Data Fig. 6 Intracellular localization of human plastins.

U2OS cells transiently co-transfected with the indicated mEmerald-tagged plastin constructs and a focal adhesion marker mCardinal-paxillin were fixed and counter-stained with TRITC-phalloidin and Hoechst. Boxed areas in (a) are enlarged in (b). Scale bars are 20 µm in (a) and 5 µm in (b).

Extended Data Fig. 7 Intracellular localization of human plastins.

To calculate the lamellipodial and total cell fluorescence density (fluorescence intensity per cell area), XTC cells were transiently transfected with either mEmerald-tagged PLS2-WT or PLS2-S406E (a). Cells were fragmented in ImageJ and the obtained masks were eroded by 12 px=2 µm (using Process → Binary → Options → Erode ImageJ tool); the resulting masks were used to generate selections to outline and measure the total cell fluorescence (b) and lamellipodial fluorescence, that is, fluorescence in the 2-µm-thick band at the cell edge (c). Scale bars are 10 µm.

Supplementary information

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Supplementary Table 1

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Supplementary Video 1

Human plastin isoforms undergo retrograde flow in the lamellipodia. SiMS TIRFM time-lapse imaging of XTC cells transiently transfected with mEmerald-tagged human plastin isoforms. Scale bars, 5 µm.

Supplementary Video 2

The domain reorientation is required for parallel, inregister actin bundle. Two modes of plastin are shown: the AlphaFold model of plastin core in the unbound state and the model for a crossbridge in actin bundle. CH1 is light blue, CH2 is dark blue, CH3 is light red, CH4 is dark red and actin is gray.

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Schwebach, C.L., Kudryashova, E., Agrawal, R. et al. Allosteric regulation controls actin-bundling properties of human plastins. Nat Struct Mol Biol 29, 519–528 (2022). https://doi.org/10.1038/s41594-022-00771-1

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