Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function

Abstract

In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomers are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. These phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients.

Keywords: Actin; Contractile dysfunction; In vitro motility assay; Molecular dynamics; Myopathy; Small-angle X-ray scattering.

Fig. 1

Molecular dynamics simulation setup. (A) Actin subunit structure with rigid portions of SG1, SG2, SG3, and SG4 shown in blue, red, cyan, and magenta, respectively, and the COG of each rigid group is represented in a sphere of the same colour, labelled as R1, R2, R3, and R4. Upper panel is the subunit crystal structure (PDB ID: 2ZWH) and the lower panel is the equilibrated H40Y structure after molecular dynamics simulation. Subunit structure D-loop is shown and labelled in yellow. (B) An effective infinitely long H40Y actin filament in a periodic boundary condition (PBC) water box under physiological conditions.

Fig. 2

Actin-layer line (ALL) spacing and intensity profiles. X-ray diffraction patterns (specimen-to-detector distance: 2.50 m) of membrane-permeabilised myofibres set at a sarcomere length of 2.50 μm from WT (first row) and from H40Y (second row) mice in resting (pCa 9.0, first column) and maximal activating (pCa 4.5, second column) conditions. The ALL6 and ALL7 actin spacing increments (in %) as well as the ALL2 intensity increment (in fold-change) during activation are presented. Five arrays of approximately 30 fibres were tested per mouse (four knock-in and four wild-type mice) for these experiments.

Fig. 3

TN3 intensity profiles. X-ray diffraction patterns (specimen-to-detector distance: 3.47 m) of membrane-permeabilised myofibres set at a sarcomere length of 2.50 μm from WT (first column) and from H40Y (second column) mice in resting (pCa 9.0, blue lines on graphs) and maximal activating (pCa 4.5, red lines on graphs) conditions. The thinner red and blue curves represent the observed data, and the thicker red and blue curves represent the fitted Gaussian function with a range of fitting ± 4 pixels or ± 3 pixels. For these experiments, five arrays of approximately 30 fibres were mounted per mouse (four knock-in and four wild-type mice).

Fig. 4

Actin sliding speed. This figure displays the means/standard errors as well as distribution of speeds for individual filaments coming from WT and H40Y mouse muscles. The star denotes a significant difference between WT and H40Y (p < 0.05).