Dynamics of Dystrophin’s Actin-Binding Domain

Abstract

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.

Steric Regulation of Tandem Calponin Homology Domain Actin-Binding Affinity

“…We extend this model by showing not only that the 'open' utrophin CH1-CH2 can be made 'more open' and higher affinity but also that an unstructured linker can reduce CH2 steric clash and increase CH1-CH2 affinity, offering a mechanism for titrating 'openness' to vary affinity. This notion is consistent with MD simulations and DEER measurements that have shown that the CH domains from dystrophin can adopt a range of conformations (Fealey et al, 2018). We show that these mechanisms of affinity modulation can be additively combined with others, such as N-terminal region modifications, to create domains with diverse affinities and levels of openness.…”

Steric regulation of tandem calponin homology domain actin-binding affinity

“…We speculate that the large difference in off-rate implies that reduced inter-CH domain interactions allow the domain to adopt a high-affinity state when bound to F-actin, potentially through reduced steric interactions between CH2 and F-actin. This notion is consistent with measurements that have shown that the CH domains from dystrophin and utrophin can adopt a range of conformations in solution, only some of which are potentially compatible with F-actin binding (Fealey et al, 2018).…”

The Dystrophin Node as Integrator of Cytoskeletal Organization, Lateral Force Transmission, Fiber Stability and Cellular Signaling in Skeletal Muscle

“…The full-length dystrophin isoform Dp427-M belongs to the class of giant muscle proteins [ 50 ] and consists of several distinct molecular domains as illustrated in the upper panel of Figure 1 . This includes amino-terminal and central actin-binding domains, proline-rich hinge regions, spectrin-like rod domains and crucial carboxy-terminal binding sites for interactions with plasmalemmal and cytosolic components [ 51 , 52 , 53 , 54 ]. Dystrophin closely interacts with the integral proteins beta-dystroglycan, alpha/beta/gamma/delta-sarcoglyan and sarcospan of the sarcolemma, the extracellular receptor alpha-dystroglycan and laminin-211, the cytosolic components alpha/beta-dystrobrevin and alpha/beta-syntrophin, and the cortical actin cytoskeleton [ 9 , 10 , 11 , 12 , 13 ], as shown in the lower panel of Figure 1 .…”