Structural implications of conserved aspartate residues located in tropomyosin's coiled-coil core

Abstract

Polar residues lying between adjacent α-helical chains of coiled-coils often contribute to coiled-coil curvature and flexibility, while more typical core hydrophobic residues anneal the chains together. In tropomyosins, ranging from smooth and skeletal muscle to cytoplasmic isoforms, a highly conserved Asp at residue 137 places negative charges within the tropomyosin coiled-coil core in a position which may affect the conformation needed for tropomyosin binding and regulatory movements on actin. Proteolytic susceptibility suggested that substituting a canonical Leu for the naturally occurring Asp at residue 137 increases inter-chain rigidity by stabilizing the tropomyosin coiled-coil. Using molecular dynamics, we now directly assess changes in coiled-coil curvature and flexibility caused by such mutants. Although the coiled-coil flexibility is modestly diminished near the residue 137 mutation site, as expected, a delocalized increase in flexibility along the overall coiled-coil is observed. Even though the average shape of the D137L tropomyosin is straighter than that of wild-type tropomyosin, it is still capable of binding actin due to this increase in flexibility. We conclude that the conserved, non-canonical Asp-137 destabilizes the local structure resulting in a local flexible region in the middle of tropomyosin that normally is important for tropomyosin steady-state equilibrium position on actin.

Figure 1. Average tropomyosin conformations from MD simulations. (A) Average structures for the wild-type tropomyosin sequence (light blue) and the control C190A mutant sequence (gold). Note the overall curvature of the C190A tropomyosins matches closely to wild-type tropomyosin, which closely matches the long-pitch helical repeat of actin. (B) Average structures for the D137L and D137L/C190A mutant tropomysoin sequences. Overall, the average structure of the mutant tropomyosin deviates from wild-type and control average conformations resulting in a straighter molecule. (C) Tropomyosin curvature is assessed via the mean end-to-end bending angle, θ, which is calculated by comparing the tangent angles of the central coiled-coil axis of the N and C-terminal 15 residues (red arrows). Since tropomyosin is a curved molecule, flexibility is determined by examining δ, the angular deflection of the rod from its averaged curved shape.

Figure 2. C-terminal variation plots. (A–D) x,y coordinates of the tropomyosin C-terminus are plotted for each frame of the simulation after superposing the first 15 residues at the N-terminus, orienting them along the z-axis (out of page), and aligning them at the origin. Each point represents the projection of the position of the C-terminal residue for each conformer observed during the MD simulation. The average C-terminal position of the MD structures for each tropomyosin is indicated in their respective panels (open diamond) for comparison with that of the Lorenz–Holmes model (open square). wild-type (A) and C190A (B) tropomyosins display the characteristic anisotropic bending with the c-terminus frequently in quadrants 3 and 4. On the other hand the straighter D137L (C) and D137L/C190A (D) spend much more time in quadrants 1 and 2.

Figure 3. Local changes in tropomyosin curvature and flexibility induced by the D137L mutation. (A and B)The local curvature of the average structure. Local flexibility (C and D) is determined as the time average of the local deviations from the average structure. Local curvature and flexibility for the region immediately surrounding residue 137 (arrow) are shown in panels B and D respectively. Wild type is shown in cyan, while the C190A control, C190A/D137L and D137L mutant tropomyosins are shown in gold, blue and red respectively.

Figure 4. Comparing the local packing around position 137 (A) Packing of Asp 137 at the d-position residue (B) Packing of Leu 137 at the d-position. Note that the Leu side chains of the D137L mutant stably pack into the hydrophobic core of the α-helical coiled-coil while the Asp side chains are pushed outwards. The average structure of the region surrounding Asp 137 (residues 131–142) for the wild-type and D137L mutant tropomyosin (C) show that the D137L substitution yields a straighter molecule.