Duchenne muscular dystrophy (DMD) is associated with the loss of
dystrophin, which plays an important role in myofiber integrity via interactions with β-
dystroglycan and other members of the transmembrane
dystrophin-associated protein complex. The ZZ domain, a
cysteine-rich zinc-finger domain near the
dystrophin C-terminus, is implicated in forming a stable interaction between
dystrophin and β-
dystroglycan, but the mechanism of pathogenesis of ZZ missense mutations has remained unclear because not all such mutations have been shown to alter β-
dystroglycan binding in previous experimental systems. We engineered three ZZ mutations (p.Cys3313Phe, p.Asp3335His, and p.Cys3340Tyr) into a short construct similar to the Dp71
dystrophin isoform for in vitro and in vivo studies and delineated their effect on
protein expression, folding properties, and binding partners. Our results demonstrate two distinct pathogenic mechanisms for ZZ missense mutations. The
cysteine mutations result in diminished or absent subsarcolemmal expression because of
protein instability, likely due to misfolding. In contrast, the
aspartic acid mutation disrupts binding with β-
dystroglycan despite an almost normal expression at the membrane, confirming a role for the ZZ domain in β-
dystroglycan binding but surprisingly demonstrating that such binding is not required for subsarcolemmal localization of
dystrophin, even in the absence of actin binding domains.