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Cryo-EM reveals different coronin binding modes for ADP– and ADP–BeFx actin filaments

Nature Structural & Molecular Biology volume 21, pages 1075–1081 (2014)Cite this article

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Abstract

Essential cellular processes involving the actin cytoskeleton are regulated by auxiliary proteins that can sense the nucleotide state of actin. Here we report cryo-EM structures for ADP-bound and ADP–beryllium fluoride (ADP–BeFx, an ADP-Pi mimic)-bound actin filaments in complex with the β-propeller domain of yeast coronin 1 (crn1), at 8.6-Å resolution. Our structures reveal the main differences in the interaction of coronin with the two nucleotide states of F-actin. We derived pseudoatomic models by fitting the atomic structures of actin and coronin into the EM envelopes and confirmed the identified interfaces on actin by chemical cross-linking, fluorescence spectroscopy and actin mutagenesis. The models offer a structural explanation for the nucleotide-dependent effects of coronin on cofilin-assisted remodeling of F-actin.

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Figure 1: Cryo-EM reconstruction of coronin-decorated actin filaments in both ADP and ADP–BeFx states.
Figure 2: Different interaction modes between coronin and actin in ADP and ADP–BeFx states.
Figure 3: Interactions between coronin and filamentous actin in the ADP state.
Figure 4: Charge and shape complementarity between actin and coronin.
Figure 5: Crn1ΔCC and ADP–F-actin are cross-linked by EDC mainly, but not only, through actin's N terminus.
Figure 6: Modeling of cofilin on the coronin-decorated F-actin in ADP–BeFx and ADP states.

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Acknowledgements

This work was supported by US National Institutes of Health (US NIH) grants GM077190 (to E.R.), GM071940 and AI094386 (to Z.H.Z.) and F32HL119069 (to Z.A.O.D.); an American Heart Association postdoctoral fellowship 13POST17340020 (to P.G.); and a startup fund from The Ohio State University (to D.S.K.). The authors acknowledge the use of instruments at the Electron Imaging Center for NanoMachines supported by US NIH grant 1S10RR23057 (to Z.H.Z.) and CNSI at UCLA. The authors also acknowledge the use of computer time at the Extreme Science and Engineering Discovery Environment (XSEDE) resources (MCB130126 to Z.H.Z.) and thank B. Goode (Brandeis University) for coronin-expression plasmids. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Author notes

  1. Peng Ge and Zeynep A Oztug Durer: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, California, USA

    Peng Ge & Z Hong Zhou

  2. California NanoSystems Institute (CNSI), UCLA, Los Angeles, California, USA

    Peng Ge & Z Hong Zhou

  3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA

    Zeynep A Oztug Durer & Emil Reisler

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

    Dmitri Kudryashov

  5. Molecular Biology Institute, UCLA, Los Angeles, California, USA

    Z Hong Zhou & Emil Reisler

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Contributions

P.G., Z.A.O.D., D.K. and E.R. designed experiments; P.G., Z.A.O.D. and D.K., collected EM data; P.G. and Z.H.Z. processed, analyzed and interpreted EM data; Z.A.O.D. collected and analyzed biochemical data; all authors wrote and reviewed the manuscript.

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Correspondence to Emil Reisler.

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Integrated supplementary information

Supplementary Figure 1 Actin array generated by full-length coronin.

Representative cryoEM fields of full-length coronin decorated ADP-actin.

Full-length coronin bundles actin into thick bundles (left) and meshes (right). Bar: 100nm.

Supplementary Figure 2 Stereo views.

Stereo views of Figure 1, panels b and f.

Supplementary Figure 3 Comparison of all possible docking modes of the coronin density and its model.

(a-f) The density corresponding to coronin in our ADP state structure is fitted with our homology model of coronin. Due to the pseudo-seven-fold symmetry of coronin’s seven-bladed propeller, there are seven possible modes of docking. The docking that we chose in our paper (a) is significantly (>5 SD) better than other possible dockings. The last possible mode (model rotated by 6/7×360°) cannot be generated, since the docking program automatically fits it towards the best model (a).

Supplementary Figure 4 Crn1ΔCC binding to wild-type and mutant yeast ADP–actins.

(a) Coomassie-stained gels of supernatant (S) and pellet (P) fractions of 1 μM Crn1ΔCC cosedimented with 0-30 μM pre-polymerized wild type and mutant actins. (b) Quantification of Crn1ΔCC binding by wild type and select mutant actins obtained for analysis of gels shown in (a). Fractions of Crn1ΔCC bound to various F-actins was determined by densitometry and plotted versus the concentration of F-actin.

Supplementary Figure 5 Distribution of key residues that mediate actin-coronin interaction.

Key residues that are shown in this study to be responsible for actin-coronin interaction are mapped on the surfaces of actin (a, c) and coronin (b, d). The key groups of interacting residues are colored as in Table 1, regardless of the interacting actin subunits.

Supplementary Figure 6 Competition between coronin and Arp2/3 complex at high coronin concentration.

The pseudo-atomic model of actin-Arp2/3 complex (a) is superimposed with that of ADP-F-actin-coronin, similarly to Figure 6. (b) Relative position of coronin in the superimposed model (the actin model from the actin-Arp2/3 complex is shown to simplify the comparison). (c-d) Coronin blocks binding of Arp2/3 complex at high coronin concentrations, when Arp2/3 complex and coronin compete for actin binding (c), but allows binding of Arp2/3 complex to adjacent unoccupied actins (d) at low coronin concentrations. In all panels, actin subunits are delineated by grey transparent surfaces derived from their atomic models.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Table 1 (PDF 4069 kb)

Comparison between the pseudoatomic models of F-actin–coronin complexes in ADP and ADP–BeFx states.

The two pseudo-atomic models are morphed back and forth as a movie in two orthogonal views. The movie starts with the viewpoint similar to Figure 2a, showing the model in ADP state. It then morphs to ADP-BeFx state and back to ADP state. The model then rotates 90° about its helical axis, and performs the same morphing again. (MP4 17572 kb)

41594_2014_BFnsmb2907_MOESM4_ESM.mp4

Animation of the competition between coronin and cofilin in ADP–BeFx state, not in ADP state (based on Figure 6). (MP4 2011 kb)

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Ge, P., Durer, Z., Kudryashov, D. et al. Cryo-EM reveals different coronin binding modes for ADP– and ADP–BeFx actin filaments. Nat Struct Mol Biol 21, 1075–1081 (2014). https://doi.org/10.1038/nsmb.2907

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