Abstract

Skeletal muscle dystrophin is a 427 kDa protein thought to act as a link between the actin cytoskeleton and the extracellular matrix. Perturbations of the dystrophin-associated complex, for example, between dystrophin and the transmembrane glycoprotein β-dystroglycan, may lead to muscular dystrophy. Previously, the cysteine-rich region and first half of the carboxy-terminal domain of dystrophin were shown to interact with β-dystroglycan through a stretch of fifteen amino acids at the carboxy-terminus of β-dystroglycan. This region of dystrophin implicated in binding β-dystroglycan contains four modular protein domains: a WW domain, two putative Ca²⁺-binding EF-hand motifs, and a putative zinc finger ZZ domain. The WW domain is a globular domain of 38–40 amino acids with two highly conserved tryptophan residues spaced 20–22 amino acids apart. A subset of WW domains was shown to bind ligands that contain a Pro-Pro-x-Tyr core motif (where x is any amino acid). Here we elucidate the role of the WW domain of dystrophin and surrounding sequence in binding β-dystroglycan. We show that the WW domain of dystrophin along with the EF-hand motifs binds to the carboxy-terminus of β-dystroglycan. Through site-specific mutagenesis and in vitro binding assays, we demonstrate that binding of dystrophin to the carboxyterminus of β-dystroglycan occurs via a β-dystroglycan Pro-Pro-x-Tyr core motif. Targeted mutagenesis of conserved WW domain residues reveals that the dystrophin/β-dystroglycan interaction occurs primarily through the WW domain of dystrophin. Precise mapping of this interaction could aid in therapeutic design.