Dynamics of Dystrophin's Actin-Binding Domain.

We have used pulsed electron paramagnetic resonance, calorimetry, and molecular dynamics simulations to examine the structural mechanism of binding for dystrophin's N-terminal actin-binding domain (ABD1) and compare it to utrophin's ABD1. Like other members of the spectrin superfamily, dystrophin's ABD1 consists of two calponin-homology (CH) domains, CH1 and CH2. Several mutations within dystrophin's ABD1 are associated with the development of severe degenerative muscle disorders Duchenne and Becker muscular dystrophies, highlighting the importance of understanding its structural biology. To investigate structural changes within dystrophin ABD1 upon binding to actin, we labeled the protein with spin probes and measured changes in inter-CH domain distance using double-electron electron resonance. Previous studies on the homologous protein utrophin showed that actin binding induces a complete structural opening of the CH domains, resulting in a highly ordered ABD1-actin complex. In this study, double-electron electron resonance shows that dystrophin ABD1 also undergoes a conformational opening upon binding F-actin, but this change is less complete and significantly more structurally disordered than observed for utrophin. Using molecular dynamics simulations, we identified a hinge in the linker region between the two CH domains that grants conformational flexibility to ABD1. The conformational dynamics of both dystrophin's and utrophin's ABD1 showed that compact conformations driven by hydrophobic interactions are preferred and that extended conformations are energetically accessible through a flat free-energy surface. Considering that the binding free energy of ABD1 to actin is on the order of 6-7 kcal/mole, our data are compatible with a mechanism in which binding to actin is largely dictated by specific interactions with CH1, but fine tuning of the binding affinity is achieved by the overlap between conformational ensembles of ABD1 free and bound to actin.

Molecular Therapies for Muscular Dystrophies.

PURPOSE OF REVIEW: To construct a framework to understand the different molecular interventions for muscular dystrophy. RECENT FINDINGS: The recent approval of antisense oligonucleotides treatment for Duchenne muscular dystrophy and spinal muscular atrophy and current clinical trials using recombinant adeno-associated virus for the treatment of those diseases suggests that we are at a tipping point where we are able to treat and potentially cure muscular dystrophies. Understanding the basic molecular pathogenesis of muscular dystrophies and the molecular biology of the treatment allows for critical evaluation of the proposed therapies.

Large-scale opening of utrophin's tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism.

We have used site-directed spin labeling and pulsed electron paramagnetic resonance to resolve a controversy concerning the structure of the utrophin-actin complex, with implications for the pathophysiology of muscular dystrophy. Utrophin is a homolog of dystrophin, the defective protein in Duchenne and Becker muscular dystrophies, and therapeutic utrophin derivatives are currently being developed. Both proteins have a pair of N-terminal calponin homology (CH) domains that are important for actin binding. Although there is a crystal structure of the utrophin actin-binding domain, electron microscopy of the actin-bound complexes has produced two very different structural models, in which the CH domains are in open or closed conformations. We engineered a pair of labeling sites in the CH domains of utrophin and used dipolar electron-electron resonance to determine the distribution of interdomain distances with high resolution. We found that the two domains are flexibly connected in solution, indicating a dynamic equilibrium between two distinct open structures. Upon actin binding, the two domains become dramatically separated and ordered, indicating a transition to a single open and extended conformation. There is no trace of this open conformation of utrophin in the absence of actin, providing strong support for an induced-fit model of actin binding.