Tropomyosin ends determine the stability and functionality of overlap and troponin T complexes

Abstract

Tropomyosin binds end to end along the actin filament. Tropomyosin ends, and the complex they form, are required for actin binding, cooperative regulation of actin filaments by myosin, and binding to the regulatory protein, troponin T. The aim of the work was to understand the isoform and structural specificity of the end-to-end association of tropomyosin. The ability of N-terminal and C-terminal model peptides with sequences of alternate alpha-tropomyosin isoforms, and a troponin T fragment that binds to the tropomyosin overlap, to form complexes was analyzed using circular dichroism spectroscopy. Analysis of N-terminal extensions (N-acetylation, Gly, AlaSer) showed that to form an overlap complex between the N-terminus and the C-terminus requires that the N-terminus be able to form a coiled coil. Formation of a ternary complex with the troponin T fragment, however, effectively takes place only when the overlap complex sequences are those found in striated muscle tropomyosins. Striated muscle tropomyosins with N-terminal modifications formed ternary complexes with troponin T that varied in affinity in the order: N-acetylated > Gly > AlaSer > unacetylated. The circular dichroism results were corroborated by native gel electrophoresis, and the ability of the troponin T fragment to promote binding of full-length tropomyosins to filamentous actin.

FIGURE 1

Model of the troponin-tropomyosin complex on actin (modified from Heeley et al., 1987). The dark shaded area represents residues 70–170 of TnT. The dotted area is the part of tropomyosin that corresponds to the N-terminal model peptides used in this study and the striped area corresponds to the C-terminal model peptides.

FIGURE 2

Thermal transitions of the N-terminal tropomyosin model peptides AcTM1aZip (A), GlyTM1aZip (B), AlaSerTM1aZip (C), AcTM1bZip (D), and unacetylated TM1aZip (E) alone (□), in complex with TM9a_251–284 (○), and in complex with TM9a_251–284 and hcTnT_70–170 (◊). Solid lines represent fits to the mixtures, and dotted lines represent fits to the sum of the components. Thermal transitions of TM9a_251–284 (▵) and hcTnT_70–170 (▿) are shown in panel F.

FIGURE 3

Ternary complexes of hcTnT_70–170, TM9a_251–284, and different N-terminal tropomyosin model peptides run at nondenaturing conditions on a 10% glycerol, 10% acrylamide gel at pH = 8.8, 4°C.

FIGURE 4

Effect of hcTnT_70–170 on the binding of the tropomyosin isoforms to actin measured at 300 mM NaCl (A) and 100 mM NaCl (B), respectively: AcTM1a9a (□), AlaSerTM1a9a (○), and TM1b9a (▵), unacetylated TM1a9a (◊), and TM1a9d (▿) (open symbols and solid line, + hcTnT_70–170; filled symbols and dotted line, no TnT). Terminology: full length rat α-tropomyosins are designated by the codons that express their N- and C-terminal ends; i.e. TM1a9a is full length rat α-TM with an N-terminus encoded by exon 1a and a C-terminus encoded by exon 9a. Prefixes Ac and AlaSer stand for the respective N-terminal modifications. Concentrations are in μM.