Structural mechanisms of drebrin-mediated F-actin network modulation

Figures

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  Fig. 1. Drebrin’s ABDs each bind across two F-actin protomers and multiple subdomains, with ABD1 adopting two conformations. a Cryo-EM reconstruction (from our drebrin-E135-355 dataset) and b corresponding atomic model for the ABD1a conformation. c Cryo-EM reconstruction (from our drebrin-E135-355 dataset) and d corresponding atomic model for the ABD1b conformation. e Schematic model showing ABD1 assuming both ABD1a and ABD1b conformations on F-actin, such that the stoichiometry of ABD1 alone varies between 1:1-2 for F-actin. f Cryo-EM reconstruction (from our drebrin-E135-355 dataset) and g corresponding atomic model for ABD2. h Schematic model showing ABD2 alone assuming a single conformation with 1:1 stoichiometry with actin. Superimposition of the ABD2-bound F-actin model on to each protomer of the (i) ABD1a and (j) ABD1b-bound F-actin models, showing steric clashes (calculated with ChimeraX’s ‘clashes’ tool101 using default settings) with drebrin’s peptide backbone (cyan density) or side chains only (green density). Clashing side chains are displayed. Cryo-EM density for the drebrin ABD-bound F-actin protomer pair (A-1 and A1) is coloured according to actin subdomain (SD1-4 as…

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  Fig. 2. F-actin-interacting residues in Drebrin ABDs are highly conserved and important for drebrin function. a A multiple sequence alignment of human drebrin-E residues 135–308 with canonical drebrin orthologue sequences from 113 other vertebrate species (acquired from the Ensembl database111) was performed with the MUSCLE algorithm112. The Homo sapiens sequence alongside a reduced set of five vertebrate class model species (mammals, reptiles, birds, amphibians and fish) is shown, with Homo sapiens sequence numbering, secondary structure elements (SSEs) identified by cryo-EM and sequence conservation from the larger 113 species alignment shown above. F-actin-contacting residues are highlighted in red and orange for ABD1 and ABD2, respectively. Those contacting F-actin in both ABD1 conformations are highlighted in dark red; those in one or the other conformation are highlighted in light red, with the conformation indicated below (a = ABD1a, b = ABD1b). Residues within black boxes were mutated for the confocal analysis presented in Supplementary Fig. 12, while those with asterisks below were included in ABD double mutations presented in Supplementary Fig. 11. Our cryo-EM-derived…

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  Fig. 3. Drebrin’s ABD1 and 2 can adopt a limited number of relative positions along F-actin, or can act to loosely bundle F-actin. a Schematic models indicating the 6 permitted positions of ABD1 relative to ABD2 on a single actin filament. Solvent Accessible Surface Distances (SASDs)116 determined by JWalk78 between the ABD1 C-terminus and ABD2 N-terminus (i.e L3) are shown below each model. L3 length (SASD) cannot exceed a ~ 150 Å physical length limit determined by Cα-Cα distance restraints (~3.5 Å × 43 amino acids) assuming adoption of an extended linear peptide. Micrographs and domain schematics of (b) F-actin alone or F-actin decorated with saturating concentrations of (c) Drebrin- E135-256, (d) Drebrin- E256-355, (e) Drebrin- E135-355, (f) Drebrin- E1-431 or (g) bundling protein fascin-1. Representative micrographs are shown from datasets of 100 micrographs. h Low-speed co-sedimentation assay with F-actin and Drebrin-E135-355, where any bundled F-actin sediments in the low-speed pellet (Pl), unbundled F-actin sediments in the high-speed pellet (Ph) and soluble protein remains in the supernatant (S). This assay was independently repeated three times, with a single representative…

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  Fig. 4. Drebrin sterically competes with key ABPs for F-actin association. Models of F-actin-bound ABPs are superimposed onto our drebrin ABD-bound F-actin models (actin portion as the reference), and resultant clashes (detected with default settings in ChimeraX101) are shown as green density. Superimposed ABPs; (a) α-actinin CH1 domain (PDB: 3LUE117), (b) myosin-11 motor domain (MD, PDB: 6BIH118), (c) fascin binding mode 1 (PDB: 8VO5119), (d) fascin binding mode 2 (PDB: 8VO6119), (e) tropomyosin (Tpm1.1, apo-state, PDB: 5JLF120) and (f) cofilin ADFH domain (PDB: 6UBY87). The cofilin model used was the structure of a single cofilin ADFH domain bound to F-actin, representative of an initial association event. The cofilin-bound unmodified ‘straight’ actin protomer in this model was used for superimpositions.

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  Fig. 5. Models of multimodal drebrin-F-actin interactions, F-actin stabilisation and ABP displacement. a Drebrin ABD1 and ABD2, separated by a long flexible L3, can assume multiple relative positions on single or adjacent protofilaments on a single actin filament, but can also loosely cross-link two actin filaments. b Drebrin ABD binding across two actin protomers and their subdomain pairs SD1 & SD2 and SD3 & SD4 cross-links F-actin and stabilises the ‘flattened’ conformation in the ultimate pointed end protomers, respectively, preventing depolymerisation. c ABD association sterically displaces a number of other important ABPs, including cofilin, an F-actin disassembly-promoting factor, further contributing to F-actin stabilisation. d By associating with F-actin via a minor portion of the sequence, drebrin’s ADFH domain, L3, PP domain and BB domain are free to link F-actin to other cellular and molecular components. e At the F-actin barbed end, drebrin may bind to penultimate or ultimate actin protomers due to their ‘flattened’ conformation. Here, ABDs may sterically compete with formins to inhibit actin promotion of assembly. Mobile regions C-terminal to ABD2 are projected away from the…