Fig 1.
Scaled schematic diagram of the human ferlin family members.
The length of the total protein is in proportion to the length of each domain. The six ferlin proteins are segregated by type. Type-1 ferlins possess a DysF domain; Type-2 ferlins lack a DysF domain [37]. ‘TM’ indicates the transmembrane span. *Fer1L4 in humans is currently characterized as a pseudogene.
Fig 2.
RoseTTAFold model of the full-length dysferlin.
The full-length model indicated super-tertiary [53] domain interactions in the dysferlin model. The RoseTTAFold models that were used in this study were flexibly aligned using FATCAT [54]. Inconsistencies in the 3D models that were generated as a result of the elastic alignment process were repaired using PyMod [55]. Figures were rendered with PyMol and displayed as 180° views of the model. The various domains of dysferlin are shown as colored surfaces and similarly colored labels. The other ferlin full-length models can be found in the supplemental information (S1-S17 Figs and S1-S7 Tables in S1 File).
Fig 3.
C2A structures and model of C2A from Fer1L5.
Dysferlin C2A (green, 4IHB), otoferlin C2A (blue, 3L9B), myoferlin C2A (cyan, 6EEL), and Fer1L5 C2A (yellow). The gray spheres are the divalent cations from the crystal structures of dysferlin C2A (4IHB, chain E) and myoferlin C2A (6EEL). The amino acid boundaries from each respective C2 domain are listed with the domain assignment. The superposition of all four C2A domains is labeled as ‘Overlay’. The ferlins without a C2A domain are listed as Not Applicable (N/A).
Fig 4.
C2B models from dysferlin C2B (green), otoferlin C2B (blue), myoferlin C2B (cyan), Fer1L4 C2B (orange), Fer1L5 C2B (yellow), and Fer1L6 (magenta).
Amino acid ranges for each domain are listed under the domain label. The superposition of all six C2B domains is labeled as ‘Overlay’.
Table 1.
Primary sequence alignment of the FerI region of the six human ferlin proteins.
Fig 5.
C2C models for dysferlin C2C (green), otoferlin C2C (blue), myoferlin C2C (cyan), Fer1L4 C2C (orange), Fer1L5 C2C(yellow), and Fer1L6 C2C (magenta).
Amino acid ranges for each domain are listed under the domain label. The β1–2, β6–7, and β7–8 subdomains for C2C are labeled and shown in red. The superposition of all six C2C domains is labeled as ‘Overlay’.
Fig 6.
C2-FerA models for dysferlin C2-FerA (green), otoferlin C2-FerA (blue), myoferlin C2-FerA (cyan), Fer1L3 C2-FerA (orange), Fer1L5 C2-FerA (yellow), Fer1L6 C2-FerA (magenta).
The FerA subdomain (β4–5) is shown as red α-helices, as is the β2–3 subdomain unique to Type-2 ferlins. The superposition of all six C2-FerA models is labeled as ‘Overlay’.
Fig 7.
A. C2-FerA domain schematic showings the secondary structure connectivity and the large insertion of the β4–5 FerA subdomain. B. SDS-PAGE showing the purified dysferlin C2-FerA domain versus molecular weight size markers. C. Far-UV CD spectrum of purified dysferlin C2-FerA (red curve) and predicted C2-FerA spectrum derived from the model (blue curve). The inset table reports the secondary structure summary of the purified dysferlin C2-FerA domain (Experimental), and the dysferlin C2-FerA model (Predicted).
Fig 8.
Models of the complete DysF region of the Type-1 ferlins; dysferlin, myoferlin, and Fer1L5.
The inner DysF domain is positioned at the left of each model, whereas the outer domain is positioned on the right side. The models of the complete DysF region have been colored as a gradient from blue to red to highlight the crossover topology of the outer DysF domain’s fold. The color bar illustrates the progression of colors from N-terminus to C-terminus. The superposition of each of the Type-1 Ferlin DysF domains is labeled as ‘Overlay’.
Fig 9.
C2D models for dysferlin C2D (green), otoferlin C2D (blue), myoferlin C2D (cyan), Fer1L3 C2D (orange), Fer1L5 C2D (yellow), Fer1L6 C2D (magenta).
The superposition of all six C2D models is labeled as ‘Overlay’.
Fig 10.
C2E models for dysferlin C2E (green), otoferlin C2E (blue), myoferlin C2E (cyan), Fer1L3 C2E (orange), Fer1L5 C2E (yellow), Fer1L6 C2E (magenta).
The β6–7 subdomain of C2E is labeled and colored in red. The superposition of all six C2E models is labeled as ‘Overlay’.
Fig 11.
Dysferlin C2F (green), otoferlin C2F (blue), myoferlin C2F (cyan), Fer1L4 C2F (orange), Fer1L5 C2F (yellow), Fer1L6 (magenta).
The β6–7 subdomain of C2F is labeled and colored red. The overlay of each domain is to the right of the figure.
Fig 12.
Dysferlin C2F β6–7 subdomain aligned with known dsRNA binding domains.
A. Superimposed β6–7 subdomain of Dysferlin C2F (green) and RNA binding domain of Staufen (1STU) in cyan; RMSD = 2.7 Å. B. Superimposed β6–7 subdomain of Dysferlin C2F (green) and dsRNA binding domain of TARBP2 (4WYQ) in magenta; RMSD = 0.86 Å.
Fig 13.
C2G models for dysferlin C2G (green), otoferlin C2G (blue), myoferlin C2G (cyan), Fer1L3 C2G (orange), Fer1L5 C2G (yellow), Fer1L6 C2G (magenta).
The β4–5 and β6–7 subdomains of C2G are labeled and colored red. The superposition of all six C2G models is labeled as ‘Overlay’.
Table 2.
Predicted ferlin transmembrane span boundaries and extracellular residues.
Fig 14.
Schematic of Type-II C2 domain.
A: Dysferlin C2A structure (4IHB) colored to highlighted the various insertions of subdomains. B:The schematic highlights the loops with embedded subdomains. The Type-2 C2 domain β-strand topology is shown as grey arrows. The colored loops: β1–2 (green, C2C), β2–3 (orange, C2-FerA), β4–5 (blue, C2-FerA, C2G), β6–7 (red, C2C, C2E, C2G, C2F), and β7–8 (purple, C2C) show where conserved subdomains are present.