Crystal structures of the human Dysferlin inner DysF domain

Abstract

BackgroundMutations in dysferlin, the first protein linked with the cell membrane repair mechanism, causes a group of muscular dystrophies called dysferlinopathies. Dysferlin is a type two-anchored membrane protein, with a single C terminal trans-membrane helix, and most of the protein lying in cytoplasm. Dysferlin contains several C2 domains and two DysF domains which are nested one inside the other. Many pathogenic point mutations fall in the DysF domain region.ResultsWe describe the crystal structure of the human dysferlin inner DysF domain with a resolution of 1.9 Ångstroms. Most of the pathogenic mutations are part of aromatic/arginine stacks that hold the domain in a folded conformation. The high resolution of the structure show that these interactions are a mixture of parallel ring/guanadinium stacking, perpendicular H bond stacking and aliphatic chain packing.ConclusionsThe high resolution structure of the Dysferlin DysF domain gives a template on which to interpret in detail the pathogenic mutations that lead to disease.

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

“…This corresponds to removal of the C2A domain and linker bearing the Romeo epitope (residues 123-142; see Fig. 2A, Romeo, N [25][26][27][28][29][30] ).…”

“…Left, even at the lowest trypsin concentrations (31.2 g/ml trypsin), full-length dysferlin is detected as a doublet with Hamlet-1 but not Romeo. This represents the removal of a fragment of ϳ25-30 kDa (see Romeo blot, N [25][26][27][28][29][30] ) that necessarily includes the C2A domain and linker region bearing the Romeo epitope. Right, trypsin digest of HEK293 cells transfected with EGFP-DYSF expression construct and probed with Romeo antibody shows removal of EGFP-C2A domain (now ϳ60 kDa).…”

“…Dysferlin contains seven C2 domains coupled via long linker regions. The tertiary structure of dysferlin has not been characterized; only the C2A and DysF domains have been solved by crystallography (25,26). C2A and splice variant C2Av1 possess distinct Ca 2ϩ and phospholipid binding properties.…”

“…In dysferlin, the DysF domain resembles a duplicated module of the yeast Pex proteins, existing as an unusual nested repeat with inner and outer DysF domains (29). The crystal structure of the inner DysF domain was shown to possess six ␤-strands connected by loops and stabilized by arginine-tryptophan stacks (26). Recent studies of a recurrent missense variant within DysF, R959W, suggest the DysF domain may fold into an open or closed state with R959W shifting the conformation toward the open state (30).…”

Caracterização da força e da função muscular nas disferlinopatias em amostra brasileira

“…Ela é um membro da família ferlina e encontra-se ligada à membrana do sarcolema por meio da proteína caveolina (Abdullah et al, 2014;Sula et al, 2014). Sua função está relacionada à contração muscular nos eventos de fusão da membrana mediados por cálcio (Bansal et al, 2003;Abdullah et al, 2014;Sula et al, 2014).…”