Structural and functional effects of two stabilizing substitutions, D137L and G126R, in the middle part of α‐tropomyosin molecule

Abstract

Tropomyosin (Tm) is an a-helical coiled-coil protein that binds along the length of actin filament and plays an essential role in the regulation of muscle contraction. There are two highly conserved non-canonical residues in the middle part of the Tm molecule, Asp137 and Gly126, which are thought to impart conformational instability (flexibility) to this region of Tm which is considered crucial for its regulatory functions. It was shown previously that replacement of these residues by canonical ones (Leu substitution for Asp137 and Arg substitution for Gly126) results in stabilization of the coiled-coil in the middle of Tm and affects its regulatory function. Here we employed various methods to compare structural and functional features of Tm mutants carrying stabilizing substitutions Arg137Leu and Gly126Arg. Moreover, we for the first time analyzed the properties of Tm carrying both these substitutions within the same molecule. The results show that both substitutions similarly stabilize the Tm coiled-coil structure, and their combined action leads to further significant stabilization of the Tm molecule. This stabilization not only enhances maximal sliding velocity of regulated actin filaments in the in vitro motility assay at high Ca 2+ concentrations but also increases Ca 2+ sensitivity of the actin-myosin interaction underlying this sliding. We propose that the effects of these substitutions on the Ca 2+ -regulated actin-myosin interaction can be accounted for not only by decreased flexibility of actin-bound Tm but also by their influence on the interactions between the middle part of Tm and certain sites of the myosin head.

The Relaxation Properties of Myofibrils Are Compromised by Amino Acids that Stabilize α-Tropomyosin

“…Both sets of data can be explained by the increased contribution of an active open state in the absence of Ca 2þ (M À (20)) according to the three-state muscle regulation theory (21). Furthermore, our results support the hypotheses that the local and long-range flexibility of the Tm coiled-coil structure, as well as some interactions of the Tm central part with TnI (2,22) and/or myosin (16), may be crucial to maintain the full inhibition of acto-myosin interactions of the resting state. These results confirm previous preliminary observations in skeletal myofibrils replaced with D137LTm-Tn (23) or for a C-terminal truncated form of cTnI (24).…”

“…Previous results have shown that these stabilizing mutants do not alter the Tm-actin binding capability (9,16), likely preserving the preshaped Tm configuration (Gestalt-binding (49)) despite the presence of some long-range overall molecular straightening and local stiffening (11). Myosin has been shown to dissociate TnI from actin 7 TmTnI (22), indicating a direct shift in the actin-Tm state from blocked to open.…”

“…Also, the three recombinants (control, D137L, and D137L/G126R) included the mutation C190A to prevent air oxidation. As described previously (16), all of these Tm species were overexpressed in BL21(DE3) according to standard methods, extracted by heating, fractionated by reducing the pH to 4.8, and purified by anion exchange chromatography using a HiTrap QXL column. The C190A mutation mimics the reduced state of cysteine in the Tm molecule, which is typical of living muscle (27), and had no appreciable effect on the tryptic digestion of Tm (9) or on its thermal unfolding and domain structure (12).…”

“…Interestingly, Ca 2þ regulation dynamics in the intact thin filament are also affected, as shown by measurements of thin-filament-induced activation of S1 ATPase activity in the presence of D137L or G126R aaTm (9,10), and by the maximal sliding velocity of regulated actin filaments in in vitro motility assays (15,16). All of these studies reported a clear functional change in the presence of one or both stabilizing substitutions and a generalized increase in apparent Ca 2þ sensitivity.…”

Tropomyosin as a Regulator of Actin Dynamics

“…The figure is from Minakata et al (2008) and Dominguez (2011). residues Asp137 and Gly126 (Pato et al, 1981;Ueno, 1984;Brown et al, 2005;Sumida et al, 2008;. A separate and concerted action of these residues contributes to the tropomyosin flexibility and seems to modulate tropomyosin functions (Sumida et al, 2008;Matyushenko et al, 2014). The evidence on the colocalization of six Ala clusters with regions of tropomyosin's high local flexibility as well as on the existence of numerous other sites of local flexibility was obtained by molecular dynamics simulation (Zheng et al, 2013).…”