Cryo-EM structures of the membrane repair protein dysferlin

Abstract

Plasma membrane repair in response to damage is essential for cell viability. The ferlin family protein dysferlin plays a key role in Ca²⁺-dependent membrane repair in striated muscles. Mutations in dysferlin lead to a spectrum of diseases known as dysferlinopathies. The lack of a structure of dysferlin and other ferlin family members has impeded a mechanistic understanding of membrane repair mechanisms and the development of therapies. Here, we present the cryo-EM structures of the full-length human dysferlin monomer and homodimer at 2.96 Å and 4.65 Å resolution. These structures define the architecture of dysferlin, ferlin family-specific domains, and homodimerization mechanisms essential to function. Furthermore, biophysical and cell biology studies revealed how missense mutations in dysferlin contribute to disease mechanisms. In summary, our study provides a framework for the molecular mechanisms of dysferlin and the broader ferlin family, offering a foundation for the development of therapeutic strategies aimed at treating dysferlinopathies.

Fig. 1. Cryo-EM structure of the dysferlin monomer.

a Domain organization of dysferlin (237 kDa). Individual domains are color-coded. b PageBlue-stained SDS-PAGE gel of purified full-length dysferlin. Three independent experiments were performed. c Representative electron micrograph shows negatively stained dysferlin protein. More than 50 images were collected in two independent sessions. d Cryo-EM micrograph (left) and 2D class average (right) of the dysferlin monomer. e Cryo-EM reconstruction of dysferlin. The front and back views of the cryo-EM maps are shown. Individual domains are color-coded according to panel a. f The front and back view of the molecular model of the dysferlin monomer in cartoon representation. Individual domains are color-coded according to panel a. g Electrostatic potential distribution in the dysferlin monomer. The electrostatic potential of the monomer is calculated using default electrostatic surface potential parameters in ChimeraX, where the acidic surface is shown in red, and the basic surface is in blue. Source data are provided as a Source Data file 2.

Fig. 2. Structures and interactions of resolved domains in dysferlin.

a Structures of the classical C2 domains (C2B-C2G) of dysferlin. Inserts of variable length extend from the consensus β-sandwich of two antiparallel four-stranded β-sheets. b Structure of the composite C2 domain (C2-FerA-FerB-C2). The topology diagram shows the position and orientation of secondary structures. c Structure of the nested DysF region composed of the DysFN, DysFI, and DysFC. d Residue-level interactions between the C2C insert and the FerB. e Residue-level interactions between the C2C and the composite C2 domain. f Residue-level interactions between the DysF region and the C2-FerA domain.

Fig. 3. Key interactions in the dysferlin monomer.

a Two views (front, back) of the pentameric ring formed by the C2B, C2E, C2D, C2C, and composite C2 domain and stabilized by the C2D-C2E linker. Individual domains are color-coded. b Schematic representation of the pentameric ring. Color code according to panel a. c Residue-level interactions at interdomain interfaces in the pentameric ring. d Residue-level interactions of the FerI with the C2B, C2C, and C2E. Color code according to panel a. The electrostatic potential distribution is shown for the C2B, C2C, and C2E (left). e The C2E-C2F (grey) and C2F-C2G (grey) linkers connect the respective domains. The C2E insert interacts with the C2E (yellow), C2F (blue), and C2G (green) domains. f Front view of the electrostatic potential distribution in the pentameric ring. Ca²⁺ ions are colored in green, and C2 domains are indicated. g Back view of the electrostatic potential distribution in the pentameric ring. Ca²⁺ ions are colored in green, and C2 domains are indicated. The FerA/B (purple) is shown in cartoon representation. h Residue-level interactions between the DysFN (orange) and DysFC (purple). Side chains are shown as sticks and their van der Waals radii are shown as light grey spheres. i Residue-level interactions in the DysFI (cyan). Side chains are shown as sticks and their van der Waals radii are shown as light grey spheres.

Fig. 4. Architecture of the dysferlin homodimer.

a Native PAGE of dysferlin shows the presence of monomers, dimers, and higher-order oligomers. M denotes marker. Two independent experiments were performed. b SEC-MALS shows the presence of dysferlin monomers, dimers, and high-order oligomers. The molecular weight of GDN micelles is in the range of ~ 140 to 160 kDa. c Side-view of the asymmetric parallel dysferlin homodimer. The homodimer shows DysF regions on the proximal side and the C2G domains on the distal side of the homodimer. d Close-up view of the dimer interface shows how the two protomers are docked to each other. The homodimer is rotated 180° relative to the overview representation. Individual domains are color coded according to panel c. e Clinically significant dysferlinopathy mutations in the dimer interface. Individual domains are color coded according to panel c. The dimer interface is indicated by a dashed line. f Close-up view of the dimer interface shows the location of select disease-causing mutations. * indicates a terminal codon. Source data are provided as a Source Data file 2.

Fig. 5. Proposed mechanism of dysferlin recruitment to sites of membrane damage.

Upon membrane damage and Ca²⁺ influx, dysferlin containing vesicles are transported to the sites of membrane repair. These vesicles dock to the membrane. Dysferlin then accumulates in the form of dimers and larger oligomers at the sites of membrane damage, where it, together with binding partners, facilitates repair. The precise structural and molecular mechanisms underlying the interaction between dysferlin, and its binding partners are largely unknown. Schematic not drawn to scale. MG53: Mitsugumin 53, also known as TRIM72; A1: Annexin A1; A2: Annexin A2.