The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes.

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.

Orientation of the Dysferlin C2A Domain is Responsive to the Composition of Lipid Membranes.

Dysferlin is a 230 kD protein that plays a critical function in the active resealing of micron-sized injuries to the muscle sarcolemma by recruiting vesicles to patch the injured site via vesicle fusion. Muscular dystrophy is observed in humans when mutations disrupt this repair process or dysferlin is absent. While lipid binding by dysferlin's C2A domain (dysC2A) is considered fundamental to the membrane resealing process, the molecular mechanism of this interaction is not fully understood. By applying nonlinear surface-specific vibrational spectroscopy, we have successfully demonstrated that dysferlin's N-terminal C2A domain (dysC2A) alters its binding orientation in response to a membrane's lipid composition. These experiments reveal that dysC2A utilizes a generic electrostatic binding interaction to bind to most anionic lipid surfaces, inserting its calcium binding loops into the lipid surface while orienting its ß-sheets 30-40° from surface normal. However, at lipid surfaces, where PI(4,5)P2 is present, dysC2A tilts its ß-sheets more than 60° from surface normal to expose a polybasic face, while it binds to the PI(4,5)P2 surface. Both lipid binding mechanisms are shown to occur alongside dysC2A-induced lipid clustering. These different binding mechanisms suggest that dysC2A could provide a molecular cue to the larger dysferlin protein as to signal whether it is bound to the sarcolemma or another lipid surface.

Calcium binds and rigidifies the dysferlin C2A domain in a tightly coupled manner.

The membrane protein dysferlin (DYSF) is important for calcium-activated plasma membrane repair, especially in muscle fibre cells. Nearly 600 mutations in the DYSF gene have been identified that are causative for rare genetic forms of muscular dystrophy. The dysferlin protein consists of seven C2 domains (C2A-C2G, 13%-33% identity) used to recruit calcium ions and traffic accessory proteins and vesicles to injured membrane sites needed to reseal a wound. Amongst these, the C2A is the most prominent facilitating the calcium-sensitive interaction with membrane surfaces. In this work, we determined the calcium-free and calcium-bound structures of the dysferlin C2A domain using NMR spectroscopy and X-ray crystallography. We show that binding two calcium ions to this domain reduces the flexibility of the Ca2+-binding loops in the structure. Furthermore, calcium titration and mutagenesis experiments reveal the tight coupling of these calcium-binding sites whereby the elimination of one site abolishes calcium binding to its partner site. We propose that the electrostatic potential distributed by the flexible, negatively charged calcium-binding loops in the dysferlin C2A domain control first contact with calcium that promotes subsequent binding. Based on these results, we hypothesize that dysferlin uses a 'calcium-catching' mechanism to respond to calcium influx during membrane repair.

AMPK Complex Activation Promotes Sarcolemmal Repair in Dysferlinopathy.

Mutations in dysferlin are responsible for a group of progressive, recessively inherited muscular dystrophies known as dysferlinopathies. Using recombinant proteins and affinity purification methods combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found that AMP-activated protein kinase (AMPK)γ1 was bound to a region of dysferlin located between the third and fourth C2 domains. Using ex vivo laser injury experiments, we demonstrated that the AMPK complex was vital for the sarcolemmal damage repair of skeletal muscle fibers. Injury-induced AMPK complex accumulation was dependent on the presence of Ca2+, and the rate of accumulation was regulated by dysferlin. Furthermore, it was found that the phosphorylation of AMPKα was essential for plasma membrane repair, and treatment with an AMPK activator rescued the membrane-repair impairment observed in immortalized human myotubes with reduced expression of dysferlin and dysferlin-null mouse fibers. Finally, it was determined that treatment with the AMPK activator metformin improved the muscle phenotype in zebrafish and mouse models of dysferlin deficiency. These findings indicate that the AMPK complex is essential for plasma membrane repair and is a potential therapeutic target for dysferlinopathy.

Defects in G-Actin Incorporation into Filaments in Myoblasts Derived from Dysferlinopathy Patients Are Restored by Dysferlin C2 Domains.

Dysferlin is a transmembrane C-2 domain-containing protein involved in vesicle trafficking and membrane remodeling in skeletal muscle cells. However, the mechanism by which dysferlin regulates these cellular processes remains unclear. Since actin dynamics is critical for vesicle trafficking and membrane remodeling, we studied the role of dysferlin in Ca2+-induced G-actin incorporation into filaments in four different immortalized myoblast cell lines (DYSF2, DYSF3, AB320, and ER) derived from patients harboring mutations in the dysferlin gene. As compared with immortalized myoblasts obtained from a control subject, dysferlin expression and G-actin incorporation were significantly decreased in myoblasts from dysferlinopathy patients. Stable knockdown of dysferlin with specific shRNA in control myoblasts also significantly reduced G-actin incorporation. The impaired G-actin incorporation was restored by the expression of full-length dysferlin as well as dysferlin N-terminal or C-terminal regions, both of which contain three C2 domains. DYSF3 myoblasts also exhibited altered distribution of annexin A2, a dysferlin partner involved in actin remodeling. However, dysferlin N-terminal and C-terminal regions appeared to not fully restore such annexin A2 mislocation. Then, our results suggest that dysferlin regulates actin remodeling by a mechanism that does to not involve annexin A2.

Structural Basis for the Distinct Membrane Binding Activity of the Homologous C2A Domains of Myoferlin and Dysferlin.

Dysferlin has been implicated in acute membrane repair processes, whereas myoferlin's activity is maximal during the myoblast fusion stage of early skeletal muscle cell development. Both proteins are similar in size and domain structure; however, despite the overall similarity, myoferlin's known physiological functions do not overlap with those of dysferlin. Here we present for the first time the X-ray crystal structure of human myoferlin C2A to 1.9 Å resolution bound to two divalent cations, and compare its three-dimensional structure and membrane binding activities to that of dysferlin C2A. We find that while dysferlin C2A binds membranes in a Ca2+-dependent manner, Ca2+ binding was the rate-limiting kinetic step for this interaction. Myoferlin C2A, on the other hand, binds two calcium ions with an affinity 3-fold lower than that of dysferlin C2A; and, surprisingly, myoferlin C2A binds only marginally to phospholipid mixtures with a high fraction of phosphatidylserine.

Distinct amino acid motifs carrying multiple positive charges regulate membrane targeting of dysferlin and MG53.

Dysferlin (Dysf) and mitsugumin53 (MG53) are two key proteins involved in membrane repair of muscle cells which are efficiently recruited to the sarcolemma upon lesioning. Plasma membrane localization and recruitment of a Dysf fragment to membrane lesions in zebrafish myofibers relies on the presence of a short, polybasic amino acid motif, WRRFK. Here we show that the positive charges carried by this motif are responsible for this function. In mouse MG53, we have identified a similar motif with multiple basic residues, WKKMFR. A single amino acid replacement, K279A, leads to severe aggregation of MG53 in inclusion bodies in HeLa cells. This result is due to the loss of positive charge, as shown by studying the effects of other neutral amino acids at position 279. Consequently, our data suggest that positively charged amino acid stretches play an essential role in the localization and function of Dysf and MG53.

FerA is a Membrane-Associating Four-Helix Bundle Domain in the Ferlin Family of Membrane-Fusion Proteins.

Ferlin proteins participate in such diverse biological events as vesicle fusion in C. elegans, fusion of myoblast membranes to form myotubes, Ca2+-sensing during exocytosis in the hair cells of the inner ear, and Ca2+-dependent membrane repair in skeletal muscle cells. Ferlins are Ca2+-dependent, phospholipid-binding, multi-C2 domain-containing proteins with a single transmembrane helix that spans a vesicle membrane. The overall domain composition of the ferlins resembles the proteins involved in exocytosis; therefore, it is thought that they participate in membrane fusion at some level. But if ferlins do fuse membranes, then they are distinct from other known fusion proteins. Here we show that the central FerA domain from dysferlin, myoferlin, and otoferlin is a novel four-helix bundle fold with its own Ca2+-dependent phospholipid-binding activity. Small-angle X-ray scattering (SAXS), spectroscopic, and thermodynamic analysis of the dysferlin, myoferlin, and otoferlin FerA domains, in addition to clinically-defined dysferlin FerA mutations, suggests that the FerA domain interacts with the membrane and that this interaction is enhanced by the presence of Ca2+.

Limited proteolysis as a tool to probe the tertiary conformation of dysferlin and structural consequences of patient missense variant L344P.

Dysferlin is a large transmembrane protein that plays a key role in cell membrane repair and underlies a recessive form of inherited muscular dystrophy. Dysferlinopathy is characterized by absence or marked reduction of dysferlin protein with 43% of reported pathogenic variants being missense variants that span the length of the dysferlin protein. The unique structure of dysferlin, with seven tandem C2 domains separated by linkers, suggests dysferlin may dynamically associate with phospholipid membranes in response to Ca2+ signaling. However, the overall conformation of the dysferlin protein is uncharacterized. To dissect the structural architecture of dysferlin, we have applied the method of limited proteolysis, which allows nonspecific digestion of unfolded peptides by trypsin. Using five antibodies spanning the dysferlin protein, we identified a highly reproducible jigsaw map of dysferlin fragments protected from digestion. Our data infer a modular architecture of four tertiary domains: 1) C2A, which is readily removed as a solo domain; 2) midregion C2B-C2C-Fer-DysF, commonly excised as an intact module, with subdigestion to different fragments suggesting several dynamic folding options; 3) C-terminal four-C2 domain module; and 4) calpain-cleaved mini-dysferlinC72, which is particularly resistant to proteolysis. Importantly, we reveal a patient missense variant, L344P, that largely escapes proteasomal surveillance and shows subtle but clear changes in tertiary conformation. Accompanying evidence from immunohistochemistry and flow cytometry using antibodies with conformationally sensitive epitopes supports proteolysis data. Collectively, we provide insight into the structural topology of dysferlin and show how a single missense mutation within dysferlin can exert local changes in tertiary conformation.

Structure-Based Designed Nano-Dysferlin Significantly Improves Dysferlinopathy in BLA/J Mice.

Dysferlinopathy is an autosomal recessive muscular dystrophy characterized by the progressive loss of motility that is caused by mutations throughout the DYSF gene. There are currently no approved therapies that ameliorate or reverse dysferlinopathy. Gene delivery using adeno-associated vectors (AAVs) is a leading therapeutic strategy for genetic diseases; however, the large size of dysferlin cDNA (6.2 kB) precludes packaging into a single AAV capsid. Therefore, using 3D structural modeling and hypothesizing dysferlin C2 domain redundancy, a 30% smaller, dysferlin-like molecule amenable to single AAV vector packaging was engineered (termed Nano-Dysferlin). The intracellular distribution of Nano-Dysferlin was similar to wild-type dysferlin and neither demonstrated toxicity when overexpressed in dysferlin-deficient patient myoblasts. Intramuscular injection of AAV-Nano-Dysferlin in young dysferlin-deficient mice significantly improved muscle integrity and decreased muscle turnover 3 weeks after treatment, as determined by Evans blue dye uptake and central nucleated fibers, respectively. Systemically administered AAV-Nano-Dysferlin to young adult dysferlin-deficient mice restored motor function and improved muscle integrity nearly 8 months after a single injection. These preclinical data are the first report of a smaller dysferlin variant tailored for AAV single particle delivery that restores motor function and, therefore, represents an attractive candidate for the treatment of dysferlinopathy.

Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling.

Myoferlin and dysferlin are closely related members of the ferlin family of Ca2+-regulated vesicle fusion proteins. Dysferlin is proposed to play a role in Ca2+-triggered vesicle fusion during membrane repair. Myoferlin regulates endocytosis, recycling of growth factor receptors and adhesion proteins, and is linked to the metastatic potential of cancer cells. Our previous studies establish that dysferlin is cleaved by calpains during membrane injury, with the cleavage motif encoded by alternately-spliced exon 40a. Herein we describe the cleavage of myoferlin, yielding a membrane-associated dual C2 domain 'mini-myoferlin'. Myoferlin bears two enzymatic cleavage sites: a canonical cleavage site encoded by exon 38 within the C2DE domain; and a second cleavage site in the linker adjacent to C2DE, encoded by alternately-spliced exon 38a, homologous to dysferlin exon 40a. Both myoferlin cleavage sites, when introduced into dysferlin, can functionally substitute for exon 40a to confer Ca2+-triggered calpain cleavage in response to membrane injury. However, enzymatic cleavage of myoferlin is complex, showing both constitutive or Ca2+-enhanced cleavage in different cell lines, that is not solely dependent on calpains-1 or -2. The functional impact of myoferlin cleavage was explored through signalling protein phospho-protein arrays revealing specific activation of ERK1/2 by ectopic expression of cleavable myoferlin, but not an uncleavable isoform. In summary, we molecularly define two enzymatic cleavage sites within myoferlin and demonstrate 'mini-myoferlin' can be detected in human breast cancer tumour samples and cell lines. These data further illustrate that enzymatic cleavage of ferlins is an evolutionarily preserved mechanism to release functionally specialized mini-modules.