Actin-binding Domain Of Dystrophin Research Articles

Probing Conformational Change in the First Actin-Binding Domain of Dystrophin

We have used time-resolved EPR and fluorescence to resolve structural transitions of dystrophin upon actin binding. Dystrophin (Dys) is a muscle cytoskeletal protein that binds to filamentous actin (F-actin) and the dystroglycan complex in the sarcolemmal membrane. Dys acts to dissipate mechanical forces generated during the contraction and relaxation of muscle thereby maintaining sarcolemmal membrane integrity and protecting from tears. The protein-protein interactions and allostery underlying this function of Dys have not been well studied in the context of conformational change and thermodynamics, partly because acquisition of structural and thermodynamic detail on large and flexible proteins is difficult. Two techniques capable of measuring large-scale conformational changes are dipolar electron-electron resonance (DEER) and time-resolved fluorescence resonance energy transfer (TR-FRET). Using a combination of DEER and TR-FRET, we placed a single label (nitroxide or fluorescent) in each CH domain Dys ABD1 and subsequently measured the interprobe distance to assess conformational change upon association with F-actin. To probe the allosteric network of Dys ABD1, we also subjected the protein to differential scanning calorimetry.

The N-Terminal Actin Binding Domain of Dystrophin is in a Closed Conformation in Solution and When Bound to F-Actin

Deficiency of the vital muscle protein dystrophin triggers Duchenne/Becker muscular dystrophy. Dystrophin stabilizes muscle cells against forces associated with muscle contraction and stretch. It interacts with F-actin using its N-terminal actin-binding domain (N-ABD), which is comprised of tandem calponin-homology (CH) domains. A previously determined X-ray crystal structure indicated that the N-ABD is a domain-swapped dimer, with each monomer adopting an ‘open’ conformation in which the two CH domains do not interact. This structure is controversial as it contradicts known structures of similar domains in other muscle proteins. Here we investigated the conformation of dystrophin N-ABD in solution using pyrene excimer fluorescence. Our results clearly indicate that it is a monomer in solution and is in a ‘closed’ conformation in which the two CH domains appear to interact. Upon binding to F-actin, this domain transitions into a more compact closed conformation. This study demonstrates for the first time the conformational transition of a dystrophin domain during function, and suggests that such structural dynamics play important role in controlling dystrophin function.

Missense Mutations in N-Terminal Actin Binding Domain of Dystrophin that Trigger Muscular Dystrophy Decrease Protein Stability and Lead to Cross-β Aggregates

A deficiency of functional dystrophin protein in muscle cells causes muscular dystrophy (MD). More than 50% of missense mutations that trigger the disease occur in the N-terminal actin binding domain (N-ABD or ABD1). We examined the effect of four disease-causing mutations - L54R, A168D, A171P, and Y231N - on the structural and biophysical properties of isolated N-ABD. Our results indicate that N-ABD is a monomeric, well-folded α-helical protein in solution, as is evident from its α-helical circular dichroism spectrum, blue shift of the native state tryptophan fluorescence, well-dispersed amide crosspeaks in 2D NMR 15N-1H HSQC fingerprint region, and its rotational correlation time calculated from NMR longitudinal (T1) and transverse (T2) relaxation experiments. Compared to WT, three mutants - L54R, A168D, and A171P - show a decreased α-helicity and do not show a cooperative sigmoidal melt with temperature, indicating that these mutations exist in a wide range of conformations or in a ‘molten globule’ state. In contrast, Y231N has an α-helical content similar to WT and shows a cooperative sigmoidal temperature melt but with a decreased stability. All four mutants experience serious misfolding and aggregation. FT-IR, circular dichroism, increase in thioflavin T fluorescence, and congo red absorption spectral shift and birefringence show that these aggregates contain intermolecular cross-β structure similar to that found in amyloid diseases. These results indicate that disease-causing mutants affect N-ABD structure by decreasing its thermodynamic stability and increasing its misfolding, thereby decreasing the net functional dystrophin concentration.

Detection of a fast isoform of C-protein with an antiserum directed against the N-terminal portion of dystrophin

An antiserum raised against the N-terminal actin-binding portion of dystrophin cross-reacted with a 130-kDa protein in fast skeletal muscle. The results of purification and two-dimensional gel electrophoresis and its immunological properties demonstrated that this protein is identical to a 130-kDa basic isoform of fast-C-protein. These results suggest that the actin-binding domain of dystrophin shares one or more antigenic determinants with those of C-protein.