doi:10.1016/j.jmb.2008.04.046 
Copyright © 2008 Elsevier Ltd All rights reserved.
Solution Structure of the Inner DysF Domain of Myoferlin and Implications for Limb Girdle Muscular Dystrophy Type 2B
Pryank Patel1, Richard Harris2, Stella M. Geddes1, Eugen-Matthias Strehle1, 3, James D. Watson4, Rumaisa Bashir5, Katharine Bushby3, Paul C. Driscoll2 and Nicholas H. Keep1, 
, 
1Institute of Structural and Molecular Biology and School of Crystallography, Birkbeck University of London, London WC1E 7HX, UK
2Institute of Structural and Molecular Biology and Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, UK
3Institute of Human Genetics, University of Newcastle, Newcastle-upon-Tyne NE1 3BZ, UK
4European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK
5School of Biological and Biomedical Sciences, South Road, Durham DH1 3LE, UK
Received 15 February 2008;
revised 11 April 2008;
accepted 21 April 2008.
Edited by M. F. Summers.
Available online 26 April 2008.
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Abstract
Mutations in the protein dysferlin, a member of the ferlin family, lead to limb girdle muscular dystrophy type 2B and Myoshi myopathy. The ferlins are large proteins characterised by multiple C2 domains and a single C-terminal membrane-spanning helix. However, there is sequence conservation in some of the ferlin family in regions outside the C2 domains. In one annotation of the domain structure of these proteins, an unusual internal duplication event has been noted where a putative domain is inserted in between the N- and C-terminal parts of a homologous domain. This domain is known as the DysF domain. Here, we present the solution structure of the inner DysF domain of the dysferlin paralogue myoferlin, which has a unique fold held together by stacking of arginine and tryptophans, mutations that lead to clinical disease in dysferlin.
Keywords: muscular dystrophy; dysferlin; myoferlin; Fer domain; NMR
Abbreviations: LGMD, limb girdle muscular dystrophy; PDB, Protein Data Bank; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser enhancement; TSR, thrombospondin repeat; NOESY, nuclear Overhauser enhancement spectroscopy
Fig. 1. Domain structure and sequence of the ferlins. Pfam and SMART domain representations of human myoferlin.
Fig. 2. Structure of myoferlin DysF domain. (a) Ensemble of the 20 lowest-energy structures of the myoferlin inner DysF domain, with the DysFN domain in red and the DysFC domain in green. (b) Stereo pair of the backbone of the lowest-energy structure. The figure was drawn using PyMOL.21
Fig. 3. Sequence alignment of DysF domains of human dysferlin and myoferlin, C. elegans fer-1, yeast peroxisomal protein PEX30 and human β-propeller protein DKFZP434B0335. The rows correspond to the DysFN and DysFC regions. Secondary structure of the myoferlin inner domain is shown. The figure was drawn using ESPript.22
Fig. 4. Conserved and mutated residues. (a) Conserved residues in ball and stick. Arginines are in blue, tryptophans in yellow, tyrosines in red and other residues in green. (b) Close-up of residues Q936, E938, W946, Y993, R1019 and R1021. (c) Disease-causing mutations in ball and stick. Tryptophans and histidines are in yellow, arginines in blue, tyrosines in red and alanines in green. This view is 180° away from Fig. 5a to show the opposite face of the β-sheet. The figure was drawn using PyMOL.21
Fig. 5. Electrostatic surface of DysF domain (positive, blue; negative, red). Left and centre views, approximately 180° rotation apart; right view, approximately 90° with respect to centre. The figure was drawn using PyMOL.21
Fig. 6. 1H–15N HSQC of the inner DysF domain of human myoferlin showing assignments. Spectrum recorded in 20 mM 4-morpholineethanesulfonic acid, 100 mM NaCl and 90% H2O/10% D2O at 298 K.
Table 1.
Refinement statistics
Table 2.
Disease-causing mutations in the inner DysF domain of dysferlin
MM, Myoshi myopathy; DACM, distal anterior compartment myopathy; LMDD, Leiden Muscular Dystrophy Database (but not published).
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