Human dystrophin tandem calponin homology actin-binding domain crystallized in a closed-state conformation

Abstract

The structure of the N-terminal actin-binding domain of human dystrophin was determined at 1.94 Å resolution. Each chain in the asymmetric unit exists in a `closed' conformation, with the first and second calponin homology (CH) domains directly interacting via a 2500.6 Å<sup>2</sup> interface. The positioning of the individual CH domains is comparable to the domain-swapped dimer seen in previous human dystrophin and utrophin actin-binding domain 1 structures. The CH1 domain is highly similar to the actin-bound utrophin structure and structural homology suggests that the `closed' single-chain conformation opens during actin binding to mitigate steric clashes between CH2 and actin.

Keywords: DMD; actin binding; calponin homology; cytoskeleton; dystrophin.

Figure 1

Crystallization and modeling of hDys-C10S-C188S-ABD1. (a) SDS–PAGE gel stained with Coomassie staining showing representative recombinant hDys-C10S-C188S-ABD1 used for crystallization (magenta arrow, 3 µg of protein loaded, molecular weight 28.6 kDa). (b) Size-exclusion chromatography of hDys-C10S-C188S-ABD1. The magenta brackets indicate the fractions pooled to obtain the material in (a). (c) Representative crystal of hDys-C10S-C188S-ABD1. (d) Molecular replacement with the AlphaFold2 model of hDys-C10S-C188S-ABD1 residues 2–246 showing the backbone (lines) and electron density (2F_o − F_c map contoured at 1.5σ). (e) Asymmetric unit of the final refined model: chains A, B, C and D are shown in green, cyan, magenta and yellow, respectively. Crystallographic waters are shown as red dots.

Figure 2

Backbone homology. (a) Alignment of PDB entry 9d58 chain A (green) with PDB entry 1dxx chains A (pink) and chain B (violet). Backbone homology is shown between PDB entry 9d58 (green) and (b) the CH1 and CH2 domains of domain-swapped dystrophin ABD1 (PDB entry 1dxx, violet) and utrophin (PDB entry 1qag, light blue), (c) α-actinin (PDB entry 2eyi, purple) and filamin (PDB entry 2wa5, yellow) and (d) fimbrin (PDB entry 1aoa, brick) and plectin (PDB entry 1mb8, cyan). (e) Electron density in PDB entry 9d58 for residues 125–144 of chain A connecting the CH1 and CH2 domains (2F_o − F_c map contoured at 1.5σ). (f) Interacting residues (PDB entry 9d58 in green, PDB entry 1dxx in violet) in the interface between the CH1 and CH2 domains.

Figure 3

(a, b) B factors of the refined model PDB entry 9d58 for aligned chains A–D viewed from two orthogonal viewpoints. The B factor is scaled from low (cyan) to high (red). (c) General case Ramachandran plot for peptide backbone φ–ψ angles of all chains. (d) Isoleucine and valine Ramachandran plot of φ–ψ angles of all chains.

Figure 4

Actin-bound homology models. (a) PDB entry 9d58 (green) and PDB entry 1dxx (violet) aligned with the CH1 domain of utrophin bound to actin (PDB entry 6m5g). The box indicates the region of CH2 that clashes with actin in PDB entry 1dxx. (b) The inset box in (a) showing the position of loops in CH2 that are positioned further from the surface of actin. In the closed CH1–CH2 conformation, actin residue Thr6 clashes with dystrophin residues 157–162. The resolved N-terminal residues of PDB entries 9d58 and 1dxx also clash with actin in this model, indicating that structural changes occur in CH2 upon actin binding.