Phosphorylation of Ser283 enhances the stiffness of the tropomyosin head-to-tail overlap domain

Abstract

The ends of coiled-coil tropomyosin molecules are joined together by nine to ten residue-long head-to-tail "overlapping domains". These short four-chained interconnections ensure formation of continuous tropomyosin cables that wrap around actin filaments. Molecular Dynamics simulations indicate that the curvature and bending flexibility at the overlap is 10-20% greater than over the rest of the molecule, which might affect head-to-tail filament assembly on F-actin. Since the penultimate residue of striated muscle tropomyosin, Ser283, is a natural target of phosphorylating enzymes, we have assessed here if phosphorylation adjusts the mechanical properties of the tropomyosin overlap domain. MD simulations show that phosphorylation straightens the overlap to match the curvature of the remainder of tropomyosin while stiffening it to equal or exceed the rigidity of canonical coiled-coil regions. Corresponding EM data on phosphomimetic tropomyosin S283D corroborate these findings. The phosphorylation-induced change in mechanical properties of tropomyosin likely results from electrostatic interactions between C-terminal phosphoSer283 and N-terminal Lys12 in the four-chain overlap bundle, while promoting stronger interactions among surrounding residues and thus facilitating tropomyosin cable assembly. The stiffening effect of D283-tropomyosin noted correlates with previously observed enhanced actin-tropomyosin activation of myosin S1-ATPase, suggesting a role for the tropomyosin phosphorylation in potentiating muscle contraction.

Keywords: Actin; Cardiomyopathy; Molecular Dynamics; Muscle regulation; Phosphorylation; Tropomyosin.

Fig. 1

The effect of phosphorylation on tropomyosin structure. Ribbon representation of (a) isolated C-terminal domains, (b–d) N-C-terminal overlapping domains, all averaged from MD trajectories; (the coiled-coil rotation differs in (a) and (b,c,d) to best display the serine and phosphoserine 283 residues). (a) upper and middle rows: unphosphorylated (cyan) and phosphorylated (pink) C-terminal domains, bottom row, superposition of the two. Note that the last C-terminal residues are disordered (particularly at the ends one of the two component coiled-coil helices) beginning at residue 281 in unphosphorylated structure and at 279 in the phosphorylated one. The last respective helical residues (viz. 280 and 278) on one chain are labeled as are serine 283 (yellow) and phosphoserine 283 (magenta). (b) upper and middle rows: unphosphorylated and phosphorylated overlapping domains, bottom row superposition of the two (unphosphorylated C-terminal helices (cyan), N-terminal helices (green); phosphorylated C-terminal helices (pink), N-terminal helices (brown)). Average side-chain positions of serine 283 (yellow) and phosphoserine 283 (magenta); lysine 12 (dark blue), glutamine 9 (light blue), glutamate 280 (red) also indicated. Note that the phosphorylated overlapping domain is slightly straighter than unphosphorylated domain (see Table 1), and thus the superposition is not perfect. (c,d) Enlargement of the central region of overlapping domains shown in (b): unphosphorylated (left) and phosphorylated (right). Note the close interaction of phosphoserine 283 (magenta) and lysine 12 (dark blue) in the phosphorylated structure; and that its junctional region becomes more compact with corresponding shorter distances between lysine 12 and glutamate 280 and glutamine 9 and glutamate 280 (distances measured between the set of respective side chain pairs viewed face-on). Graphics and alignment of helices done with Chimera [39].

Fig. 2

Electron microscopy of isolated tropomyosin molecules. (a, b, c) Rotary shadowed wild-type, S283D and S283A tropomyosin molecules. Micrographs show a mixture of tropomyosin monomers, dimers and oligomers. Arrows indicate examples of dimers used to compare potential contributions of the tropomyosin head-to-tail overlap domains to the stiffness of the tropomyosin cable. For this purpose, thirty or more dimers were skeletonized and persistence lengths determined (see Table 2). Scale bars – 100 nm.