The Molecular Mechanism and Structural Analysis of Membrane Interaction Via Fera and C2 Domains in Ferlins Associated with Muscular Dystrophy and Cancer

Ferlins are a family of proteins involved in a variety of biological processes such as exocytosis, membrane repair mechanism, and other membrane fusion events. Dysferlin and myoferlin are two members of human Ferlins with the highest similarity, and are mostly expressed in skeletal muscle cells. Dysferlin is involved in membrane repair mechanism, and lack of functional dysferlin can cause Muscular Dystrophy. Although the role of myoferlin in membrane interaction has been proven, its main function is not well understood. It has been shown however that myoferlin is overexpressed in several types of cancer including Triple-Negative Breast Cancer (TNBC), and TNBC patients with overexpressed myoferlin show a worse overall survival. Dysferlin and myoferlin employ seven C2 domains to interact with the membrane in a Ca2+ dependent manner. C2A domain is believed to function as the main Ca2+ sensor, playing an essential and indispensable role in their function. In this study, we solved the structure of Ca2+ bound myoferlin C2A using X-ray crystallography and compared that to dysferlin C2A structure which we previously solved. We also determined their specificities for Ca2+ and different lipid compositions. In addition, we identified residues that are critical in membrane interaction via these domains. Finally, we present the first structure of the FerA domain solved by 3D NMR and provide evidences of its interaction with the membrane using confocal microscopy, electron microscopy, and a co-sedimentation assay. Studying FerA pathogenic mutations by Circular Dichroism and Differential Scanning Calorimetry allowed us to have a better understanding of the pathogenicity of the FerA mutations. Outcome of this study will further advance our understanding of the function of these proteins and their relevance to human diseases.