Mechanism of actin filament severing and capping by gelsolin | Nature Structural & Molecular Biology

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Gelsolin is the prototypical member of a family of Ca2+-activated F-actin severing and capping proteins. Here we report structures of Ca2+-bound human gelsolin at the barbed end of F-actin. One structure reveals gelsolin’s six domains (G1G6) and interdomain linkers wrapping around F-actin, while another shows domains G1G3—a fragment observed during apoptosis—binding on both sides of F-actin. Conformational changes that trigger severing occur on one side of F-actin with G1G6 and on both sides with G1G3. Gelsolin remains bound after severing, blocking subunit exchange.

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Maps and models generated have been deposited with the EMDB and PDB. G1G6 at the barbed end: consensus map (EMD-43262), local refinements (EMD-43263, EMD-43264, EMD-43268), combined map (EMD-43274) and coordinates (PDB 8VIZ). G1G3 on both sides at the barbed end: consensus map (EMD-43316) and coordinates (PDB 8VKH). Source data are provided with this paper.

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We thank S. Steimle for assistance with data collection and T. Svitkina for providing the gelsolin cDNA. Supported by National Institutes of Health grants R01 GM073791 and R01 GM152412 to R.D. Data collection was performed at The Beckman Center for Cryo-Electron Microscopy, University of Pennsylvania (Research Resource Identifier: SCR_022375).

Department of Physiology and Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

Kyle R. Barrie, Grzegorz Rebowski & Roberto Dominguez

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K.R.B. and R.D. conceived the study and designed the experiments. K.R.B. and G.R. prepared the proteins. K.R.B. performed cryo-EM experiments including sample and grid preparation, data collection and data analysis. K.R.B and R.D. built and refined the atomic models. R.D. procured funding. K.R.B and R.D. wrote the paper and prepared the figures.

Correspondence to Roberto Dominguez.

The authors declare no competing interests.

Nature Structural & Molecular Biology thanks Robert Robinson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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a, Domain organization of gelsolin (as in Fig. 1a). b, Structures of full-length inactive gelsolin (PDB: 3FFN) and Ca2+-bound, active gelsolin from the complex with F-actin. c, Gelsolin domains and their preceding linkers (colored as in part a) in the inactive and active (Ca2+-bound, red) structures, indicating nomenclatures used in the main text. Note the changes in the position of the linkers and the straightening of the long helices of G3 and G6 from the inactive to the active structures. d, Structure of G-actin (PDB: 1J6Z), indicating nomenclatures used in the main text. Numbers in green circles indicate actin subdomains 1 to 4. e, G- to F-actin transition. When the inner domain is superimposed, the outer domain rotates by ~20° from the twisted (G-actin, green) to the flat (F-actin, blue) conformation. f, Structure of F-actin (PDB: 8F8P), indicating nomenclatures used in the main text.

a, SDS-PAGE of purified gelsolin. Gelsolin purity was similar across independent replicates b, Representative micrograph (out of 27,210). c, 2D class averages of F-actin ends. d, Cryo-EM movie processing and particle picking strategy. e, Particle processing workflow and masks (pink, blue, green, and magenta) used in focused 3D classification and local refinement, resulting in the final map at 2.63-Å resolution (see Methods).

Source data

a, Half-map Fourier shell correlation (FSC) resolution analysis of the consensus map (2.63 Å at FSC = 0.143). b, Orientation distribution of particles used in the consensus map determined with the program cryoEF32. The consensus map has a calculated efficiency (Eod) of 0.76. c, 3D Fourier shell correlation calculated using 3DFSC31. The consensus map has a sphericity of 0.981 and a global resolution of 2.77 Å. d, Map to model FSC determined with the program Phenix34. e, Two perpendicular views of the model fit to the cryo-EM map (transparent surface), colored as in Fig. 1b in the main text. f, Two perpendicular views of the cryo-EM map colored by local resolution as indicated by the scale bar (right).

a, Representative cryo-EM micrographs of F-actin (left) and after a 30 sec incubation with Ca2+-gelsolin (right). b, Quantification of filament length as a function of the time after the addition of Ca2+-gelsolin. Individual filament lengths (n = 50 per condition) are depicted as gray dots and the average length is indicated by a red line. c, Cryo-EM map obtained from data collected from a sample vitrified 30 sec after the addition of Ca2+-gelsolin. The map is colored by actin subunits and gelsolin domains as indicated (left) or by local resolution (right). d, Orientation distribution of particles used in the map. e, Half-map Fourier shell correlation (FSC) resolution analysis of the map (3.81 Å at FSC = 0.143). f, 3D Fourier shell correlation calculated using 3DFSC31. The map has a sphericity of 0.729 and a global resolution of 3.91 Å.

Source data

Particle processing workflow and masks (pink) used in focused 3D classification, resulting in the final map at 3.63-Å resolution (see Methods).

a, Half-map Fourier shell correlation (FSC) resolution analysis of the map (3.63 Å at FSC = 0.143). b, Orientation distribution of particles used in the map determined with the program cryoEF32. The map has a calculated efficiency (Eod) of 0.79. c, 3D Fourier shell correlation calculated using 3DFSC31. The map has a sphericity of 0.721 and a global resolution of 3.84 Å. d, Map to model FSC determined with the program Phenix34. e, Two perpendicular views of the model fit to the cryo-EM map (transparent surface), colored as in Fig. 1c in the main text. f, Two perpendicular views of the cryo-EM map colored by local resolution as indicated by the scale bar (right).

a, Type 1 (left) and 2 (right) Ca2+-binding sites of gelsolin in the final refined G1G6 model and map at 2.63-Å resolution (contour level = 0.2) b-c, Nucleotide-binding sites of actin subunits B-0, B-1, and B-2 in the structures of G1G6 (b, contour level = 0.2) and G1G3 (c, contour level = 0.25). Actin subunits and gelsolin domains are colored according to Fig. 1b in the main text.

For each interaction, the contact area in the actin subunit is colored according to the gelsolin region it binds and vice versa. Contact surface areas (given for each interaction) were calculated using the server GetArea (https://curie.utmb.edu/getarea.html).

a, Interactions of actin subunits with gelsolin domains colored according to Fig. 1b in the main text. Note that three of the gelsolin domains (G1, G2, and G4) bind to a similar area on actin subunits. However, G2 cannot insert its long helix into the hydrophobic cleft of B-2, which is occupied by the D-loop of B-0. G2 is also the only gelsolin domain to contact two subunits at the barbed end. Note finally that G5 does not contact any actin subunit. b, Comparisons of the G1G6 cryo-EM structure (PDB: 8VIZ), colored as in Fig. 1b in the main text) with crystal structures of monomeric actin bound to fragmin (PDB: 7W4Z), G1G3 (PDB: 3FFK), and G4G6 (PDB: 1H1V). The crystal structures are colored grey.

a, Actin subunits B-0, B-1, and B-2 (colored in different shades of blue) from the G1G3-bound structure superimposed onto F-actin (PDB: 8F8P). F-actin subunits are colored grey, except those predicted to interact with G1G3 before severing, which are colored purple. b and d, Cartoon putty representation of B-1 and B-0 colored and rendered according to Cα-to-Cα displacement from the corresponding F-actin subunit (as indicated by side bars). Note that the hydrophobic clefts of B-1 and B-0 change substantially due to the binding of G1. The D-loop of these subunits also changes conformation due to interactions with the visible portion of the G3-G4 linker. c and e, B-1 (light blue) and B-0 (blue) are rotated by 6° compared to their corresponding F-actin subunits (grey), with residues at their barbed ends moving by up to 5.5 Å. Note the rotation of B-1 and B-0 occurs in addition to the changes shown in parts b and d.

Proposed mechanism of F-actin severing and capping by gelsolin. The video begins with the structure of inactive (Ca2+ free) gelsolin (PDB 3FFN), transitioning to the Ca2+-bound, activated state observed in the complex with F-actin (PDB 8VIZ). When not bound to F-actin, activated gelsolin is probably flexible.

Uncropped gel corresponding to Extended Data Fig. 2a.

Numerical data used to produce the graph shown in Extended Data Fig. 4b.

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Barrie, K.R., Rebowski, G. & Dominguez, R. Mechanism of actin filament severing and capping by gelsolin. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01412-5

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Received: 19 January 2024

Accepted: 27 September 2024

Published: 24 October 2024

DOI: https://doi.org/10.1038/s41594-024-01412-5

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