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.

Figure 1

Proposed structural model for Dys ABD1 upon binding F-actin. In the absence of actin, the two adjacent CH domains are closely packed (closed state, blue). In the presence of actin, CH1 and CH2 can become more separated (open state, red) and sample multiple structural states. To see this figure in color, go online.

Figure 2

DEER data on 80 μM 4-maleimido-2,2,6,6-tetramethyl-1-piperidinylo-labeled Dys ABD1 (blue data sets) in the presence of increasing F-actin from top to bottom. The molar ratio of F-actin (FA) to Dys ABD1 is shown above the time domain data. The Utr ABD1 DEER reported previously in (7) (red data sets) is overlaid for comparison. The left shows the time-domain decays. The right shows the derived distance distributions. Tikhonov distributions for Dys ABD1 (black dotted lines) were fitted to two discrete Gaussian distributions (blue solid lines) and indicate that F-actin shifts interprobe distance toward a more open structural state, but there is considerable structural disorder. Note that this contrasts with Utr ABD1’s Gaussian distributions (shown as red solid lines), which are ordered. Moreover, Utr ABD1’s structural opening is complete. For complete time domains, see Fig. S7. To see this figure in color, go online.

Figure 3

Free-energy landscape of Dys ABD1 (A) and Utr ABD1 (B) projected on the first two principal components. The two principal components describe a “bending” motion of the two CH domains around a central swivel (PC1) that allows the extended-to-compact transition and the “revolution” motion of one CH domain around the other (PC2). A representative structure for each of the major conformational minima is also plotted. Structural heterogeneity correlates with the measured structural disorder present in DEER distributions and low unfolding free energy. To see this figure in color, go online.

Figure 4

Structural collapse of dystrophin and utrophin ABD1 derived from MD simulations. (A) The Dys ABD1 Shannon entropy calculated for macrodiehedrals formed by four consecutive Cα carbons is shown. (B) The SASA averaged over the ensemble of structures defining each minimum identified in Fig. 3 is shown, with error bars representing one SD. Representative closed structural states of (C) Dys and (D) Utr ABD1 are shown. Hydrophobic residues that promote closure are highlighted. To see this figure in color, go online.

Figure 5

Modeling of MD simulation-derived Dys ABD1 conformers on actin filament. When the CH1 domain of Dys ABD1 conformers in free-energy minima of Fig. 3 is aligned with the CH1 domain of β-III-spectrin ABD from a recent 6.9 Ǻ cryo-EM structure (6ANU), some of the structural models are devoid of steric clashes (green, yellow, red). The open conformation (magenta) is similarly free of steric clashes. Although some closed conformations of Dys ABD1 exhibit significant steric clashes with actin (blue), the fact that others do not suggests there are binding-compatible closed states for Dys ABD1, in agreement with previous structural measurements using pyrene excimer fluorescence (8). To see this figure in color, go online.