Spectroscopic and Computational Analysis of Dystrophin Regulation of Actin Dynamics

Dystrophin is a filamentous muscle protein that links the cytoskeleton to the extracellular matrix, dampening the laterally transduced force of sarcomere shortening and lengthening and thus limiting the mechanical stress reaching the sarcolemma membrane. Dystrophin's homologue, utrophin, fulfills a similar role in developing muscle but is ultimately replaced by dystrophin. Previous spectroscopic measurements on full-length dystrophin and utrophin showed that each significantly restrict the amplitude of actin filament rotational dynamics upon binding, but utrophin restricts this motion to a much greater extent. We hypothesize that part of this functional difference between homologous proteins stems from distinct structural binding modes and dynamics in the first actin-binding domain (ABD1). Using double electron-electron resonance (DEER) and time-resolved FRET, we characterized the closed-to-open structural transition of dystrophin's ABD1 and compared it to the previously studied utrophin. Dystrophin exhibits more disorder and a greater propensity to be in a closed (compact) state. To understand and complement these spectroscopic findings, we tested the thermal stability of dystrophin's ABD1 and examined possible driving forces for ABD1 closure with molecular dynamics (MD) simulations. We found that dystrophin is only marginally stable, consistent with the structural disorder measured spectroscopically. In MD simulations, the ABD1 crystal structure relaxed to a closed compact structural state and thus protected several hydrophobic residues. This suggests that location of hydrophobic residues is an important dictator of dystrophin's open-closed structural equilibrium. Together these results indicate that dystrophin's ABD1 forms an unstable complex upon binding to F-actin, due to energetically unfavorable exposure of hydrophobic surfaces within ABD1. These features may contribute to the reduced overall restriction of actin dynamics in comparison to utrophin. Funding to DDT (NIH grant AR63007).