The Second Half of the Fourth Period of Tropomyosin Is a Key Region for Ca²⁺-Dependent Regulation of Striated Muscle Thin Filaments

Abstract

Rabbit skeletal muscle alpha-tropomyosin (Tm), a 284-residue dimeric coiled-coil protein, spans seven actin monomers and contains seven quasiequivalent periods. X-ray analysis of cocrystals of Tm and troponin (Tn) placed the Tn core domain near residues 150-180 of Tm. To identify the Ca(2+)-sensitive Tn interaction site on Tm, we generated three Tm mutants to compare the consequences of sequence substitution inside and outside of the Tn core domain-binding region. Residues 152-165 and 156-162 in the second half of period 4 were replaced by corresponding residues 33-46 and 37-43 in the second half of period 1, respectively (termed mTm152-165 and mTm156-162, respectively), and residues 134-147 in the first half of period 4 were replaced with residues 15-28 in the first half of period 1 (mTm134-147). Recombinant Tms designed with an additional tripeptide, Ala-Ala-Ser, at the N-terminus were expressed in Escherichia coli. Both mTm152-165 and mTm156-162 suppressed the actin-activated myosin subfragment-1 Mg(2+)-ATPase rate regardless of whether Ca(2+) and Tn were present. On the other hand, mTm134-147 retained the normal Ca(2+)-sensitive regulation, although the actin binding of mTm alone was significantly impaired. Differential scanning calorimetry showed that the sequence substitution in the second half of period 4 affected the thermal stability of the complete Tm molecule and also the actin-induced stabilization. These results suggest that the second half of period 4 of Tm is a key region for inducing conformational changes of the regulated thin filament required for its fully activated state.

Tropomyosin Period 3 Is Essential for Enhancement of Isometric Tension in Thin Filament-Reconstituted Bovine Myocardium

“…However, these analyses were carried out in the absence of regulatory proteins. Our results imply that the number of hydrophobic amino acid residues at the actin-myosin interface increases as the regulatory proteins are added and as hypothesized earlier [11, 56]. This is because the allosteric interaction between Tm and actin may modify the actin molecule for better stereoscopic match with the myosin molecule, and that this increased actin-myosin interaction may be further responsible for the increased slope of the temperature-tension plot.…”

“…While our investigation demonstrates the significance of period 3 for the positive allosteric effect of Tm on actin and excludes period 2 for this effect in the muscle fiber system, it does not rule out the possibility that other periods are also involved in eliciting this positive allosteric effect. Indeed, solution studies have suggested that periods 4 and 5 are involved in this fundamental regulatory function [17, 19, 56], and this possibility needs to be investigated in the muscle fiber system in future studies. It is likely that Tn also plays a role for the allosteric effect and as reported by us [54] as well as others [55].…”

Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T

“…The proposed azimuthal shift of tropomyosin between blocks IV and V is supported by tropomyosin crystal structures (29), which showed that the coiled coil is disturbed around residues 151-162, located in the C-terminal half of block IV. The flexibility generated by an alanine cluster (Ala-151, 155, and 158) and nearby Tyr-162 in the coiled-coil core would also help accommodate the difference in the azimuthal position between blocks IV and V. When the alanine cluster (156-162) is replaced, the actin-activated myosin ATPase is suppressed (30). In block I, the EM density corresponding to tropomyosin was too weak to determine the azimuthal position of tropomyosin unambiguously (6).…”

The Role of Tropomyosin Domains in Cooperative Activation of the Actin–Myosin Interaction

“…Although these repeats were originally proposed to each serve as a binding partner for the seven actin monomers along its length 7; 9; 10 , and they have clear sequence homologies, each contributes to actin affinity and regulatory function in a different way. Specifically, the two ends of Tm (periods 1 and 7) and the first half of period 5 are essential for actin binding 11; 12; 13 ; the middle of the molecule, in particular period 3, is involved in positive cooperative regulation of actin 5; 12; 14; 15; 16; 17 ; and the second half of period 4, the entire length of period 5, and the first half of period 6 are involved in Ca 2+ -dependent regulatory function 12; 18; 19 .…”