Crystal structures of the human Dysferlin inner DysF domain

Abstract

Background: Mutations 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.

Results: We 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.

Conclusions: The high resolution structure of the Dysferlin DysF domain gives a template on which to interpret in detail the pathogenic mutations that lead to disease.

Figure 1

Domain organisation of dysferlin and alignment of DysF domains. a) Multiple domain architecture of human dyferlin protein (Gene3D and pFam). b) Multiple sequence alignment of inner and outer DysF domain of Human Myoferlin, Dysferlin, Fer1l5; human Tecpr1 DysF domains; inner and outer DysF domain of C. elegans FerI protein, and Yeast Pex30 DysF domain (aligned with mafft and drawn with ESPript).

Figure 2

Comparison of DysF domain structures. Ribbon presentation of a) P2₁3 asymmetric unit (the beta strands are coloured in yellow, 3 turn in pink, 4 turn in tan and no structure in grey) and b) P2₁2₁2₁ asymmetric unit coloured by chain. c) Superimposed models of myoferlin NMR DysF domain (blue) with dysferlin crystal DysF domain (red). d) Two areas of very high B factor in dysferlin DysD domain coloured in blue (965-971) and red (1018-1021). Orientation as in Figure 2a). Figure drawn with CCP4mg [19].

Figure 3

The aromatic/arginine stack motif. a) Stick representation of the residues involved in W/R stacks; arginines and lysines are coloured blue, aromatic residues are coloured green, and one cysteine residue in brown. Three forms of stack formation: b) parallel (W1012/R1038/W965), c) perpendicular (R1046/W992), and d) aliphatic stacking (R1040/W1012). Maps are 2mFo-DFc from Phenix.refine contoured at 1.03 Sigma. Figure drawn with CCP4mg [19].

Figure 4

Missense mutations mapped onto DysF domain structure. (a and c) Ribbon representation showing W/R stack rotated 180 degrees about the y axis of the page; mutated residues implicated in stack formation (red), mutated residues in the surface (blue), mutated residues stabilising the arginines involved in stack formation (yellow). Other residues of the W/R stack are in green. (b): Interaction and environment of the most common pathogenic mutations (R959, W999 and R1046). H bonds shown as black dotted lines and stacking interactions as magenta dotted lines. Figure drawn with CCP4mg [19].