Mapping of the lipid-binding and stability properties of the central rod domain of human dystrophin

J Mol Biol. 2009 Jun 12;389(3):546-58. doi: 10.1016/j.jmb.2009.04.025. Epub 2009 Apr 18.

Abstract

Dystrophin is a cytoskeletal protein that confers resistance to the sarcolemma against the stress of contraction-relaxation cycles by interacting with cytoskeletal and membrane partners. Apart from several proteins, membrane phospholipids are a partner of the central rod domain made up of 24 spectrin-like repeats, separated into sub-domains by four hinges. We previously showed that repeats 1 to 3 bind to membrane anionic phospholipids, while repeats 20 to 24 are not able to do so. We focus here on the phospholipid-binding properties of the major part of the central rod domain, namely, the sub-domain delineated by hinges 2 and 3 comprising 16 repeats ranging from repeat 4 to 19 (R4-19). We designed and produced multirepeat proteins comprising three to five repeats and report their lipid-binding properties as well as their thermal stabilities. When these proteins are mixed with liposomes including the anionic lipid phosphatidylserine, they form stable protein-vesicle complexes as determined by gel-filtration chromatography. The absence of an anionic lipid precludes the formation of such complexes. Spectroscopic analyses by circular dichroism and tryptophan fluorescence show that, while the alpha-helical secondary structures are not modified by the binding, protein trans conformation leads to the movement of tryptophan residues into more hydrophobic environments. In addition, the decrease in the molar ellipticity ratio at 222/208 nm as observed by circular dichroism indicates that lipid binding reduces the inter-helical interactions of multirepeat proteins, thus suggesting partly "opened" coiled-coil structures. Combining these results with data from our previous studies, we propose a new model of the dystrophin molecule lying along the membrane bilayer, in which the two sub-domains R1-3 and R4-19 interact with lipids and F-actin, while the distal sub-domain R20-24 does not exhibit any interaction. These lipid-binding domains should thus maintain a structural link between cytoskeletal actin and sarcolemma via the membrane phospholipids.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Dystrophin / chemistry
  • Dystrophin / genetics
  • Dystrophin / metabolism*
  • Hot Temperature
  • Humans
  • Lipid Bilayers / chemistry
  • Lipid Bilayers / metabolism*
  • Models, Molecular
  • Protein Folding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Tryptophan / chemistry

Substances

  • Dystrophin
  • Lipid Bilayers
  • Tryptophan