Structural and functional effects of two stabilizing substitutions, D137L and G126R, in the middle part of α-tropomyosin molecule (original) (raw)
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Acta naturae, 2013
We show that the mutations D137L and G126R, which stabilize the central part of the tropomyosin (Tm) molecule, increase both the maximal sliding velocity of the regulated actin filaments in the in vitro motility assay at high Са(2+) concentrations and the Са(2+)-sensitivity of the actin-myosin interaction underlying this sliding. Based on an analysis of the recently published data on the structure of the actin-Tm-myosin complex, we suppose that the physiological effects of these mutations in Tm can be accounted for by their influence on the interactions between the central part of Tm and certain sites of the myosin head.
The Role of Tropomyosin Domains in Cooperative Activation of the Actin–Myosin Interaction
Journal of Molecular Biology, 2011
To establish α-tropomyosin (Tm)'s structure-function relationships in cooperative regulation of muscle contraction, thin filaments were reconstituted with a variety of Tm mutants (Δ2Tm, Δ3Tm, Δ6Tm, P2sTm, P3sTm, P2P3sTm, P1P5Tm, wtTm), and force and sliding velocity of the thin filament were studied using an in vitro motility assay. In the case of deletion mutants, Δ indicates which of the quasi-equivalent repeats in Tm was deleted. In the case of period (P) mutants, an Ala cluster was introduced into the indicated period to strengthen the Tm-actin interaction. In P1P5Tm, the N-terminal half of period 5 was substituted with that of period 1 to test the quasiequivalence of these two Tm periods. The reconstitution included bovine cardiac troponin. Deletion studies revealed that period 3 is important for the positive cooperative effect of Tm on actin filament regulation, and that period 2 also contributes to this effect at low ionic strength, but to a lesser degree. Furthermore, Tm with one extra Ala cluster at period 2 (P2s) or period 3 (P3s) did not increase force or velocity, whereas Tm with two extra Ala clusters (P2P3s) increased both force and velocity, demonstrating interaction between these periods. Most mutants did not move in the absence of Ca 2+. Notable exceptions were Δ6Tm and P1P5Tm, which moved near at the full velocity, but with reduced force, which indicate impaired relaxation. These results are consistent with the mechanism that the Tm-actin interaction cooperatively affects actin to result in generation of greater force and velocity.
European Journal of Biochemistry, 1983
The interaction of regulated and unregulated actin of myosin-free ghost single fibre with myosin subfragment-1 free of 5,5'-dithiobis(2-nitrobenzoic acid) light chains was investigated by polarized microphotometry. The anisotropy of intrinsic tryptophan fluorescence of regulated actin is Ca2 +-dependent and has a maximal value at low (pCa2 7) and a minimal value at high ( p C a I 6) concentrations of Ca2+. The interaction of myosin subfragment-1 with actin induces cooperative changes in actin structure, which manifest themselves in a decrease in the anisotropy of tryptophan fluorescence. The cooperativity of conformational changes in actin, induced by myosin subfragment-l , is high for regulated actin in the absence of Ca2+ and low for unregulated and regulated actin in the presence of Ca2+. The data obtained suggest that the decrease of the flexibility of actin filaments, induced by tropomyosin or by Caunsaturated troponin-tropomyosin complex, results in increased cooperativity of conformational changes of actin induced by myosin subfragment-1.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2001
In order to help understand the spatial rearrangements of thin filament proteins during the regulation of muscle contraction, we used fluorescence resonance energy transfer (FRET) to measure Ca 2 -dependent, myosin-induced changes in distances and fluorescence energy transfer efficiencies between actin and the inhibitory region of troponin I (TnI). We labeled the single Cys-117 of a mutant TnI with N-(iodoacetyl)-NP-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS) and Cys-374 of actin with 4-dimethylaminophenylazophenyl-4P-maleimide (DABmal). These fluorescent probes were used as donor and acceptor, respectively, for the FRET measurements. We reconstituted a troponin^tropomyosin (Tn^Tm) complex which contained the AEDANS-labeled mutant TnI, together with natural troponin T (TnT), troponin C (TnC) and tropomyosin (Tm) from rabbit fast skeletal muscle. Fluorescence titration of the AEDANS-labeled Tn^Tm complex with DABmal-labeled actin, in the presence and absence of Ca 2 , resulted in proportional, linear increases in energy transfer efficiency up to a 7:1 molar excess of actin over Tn^Tm. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased from 37.9 A î to 44.1 A î when Ca 2 bound to the regulatory sites of TnC. Titration of reconstituted thin filaments, containing AEDANS-labeled Tn^Tm and DABmal-labeled actin, with myosin subfragment 1 (S1) decreased the energy transfer efficiency, in both the presence and absence of Ca 2 . The maximum decrease occurred at well below stoichiometric levels of S1 binding to actin, showing a cooperative effect of S1 on the state of the thin filaments. S1:actin molar ratios of V0.1 in the presence of Ca 2 , and V0.3 in the absence of Ca 2 , were sufficient to cause a 50% reduction in normalized transfer efficiency. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased by V7 A î in the presence of Ca 2 and by V2 A î in the absence of Ca 2 when S1 bound to actin. Our results suggest that TnI's interaction with actin inhibits actomyosin ATPase activity by modulating the equilibria among active and inactive states of the thin filament. Structural rearrangements caused by myosin S1 binding to the thin filament, as detected by FRET measurements, are consistent with the cooperative behavior of the thin filament proteins. ß
Regulation of actin-myosin interaction by conserved periodic sites of tropomyosin
Proceedings of the National Academy of Sciences, 2012
Cooperative activation of actin-myosin interaction by tropomyosin (Tm) is central to regulation of contraction in muscle cells and cellular and intracellular movements in nonmuscle cells. The steric blocking model of muscle regulation proposed 40 y ago has been substantiated at both the kinetic and structural levels. Even with atomic resolution structures of the major players, how Tm binds and is designed for regulatory function has remained a mystery. Here we show that a set of periodically distributed evolutionarily conserved surface residues of Tm is required for cooperative regulation of actomyosin. Based on our results, we propose a model of Tm on a structure of actin-Tm-myosin in the “open” (on) state showing potential electrostatic interactions of the residues with both actin and myosin. The sites alternate with a second set of conserved surface residues that are important for actin binding in the inhibitory state in the absence of myosin. The transition from the closed to op...
Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments
Journal of Molecular Biology, 2000
Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin ®lament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca 2 binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin ®laments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and nonmuscle sources, was investigated. Tropomyosin position on actin appeared to be de®ned by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the speci®c actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin ®lament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of signi®cant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.
Journal of Biological Chemistry, 2018
The metazoan actin cytoskeleton supports a wide range of contractile and transport processes. Recent studies have shown how the dynamic association with specific tropomyosin isoforms generates actin filament populations with distinct functional properties. However, critical details of the associated molecular interactions remain unclear. Here, we report the properties of actomyosintropomyosin complexes containing filamentous -actin, nonmuscle myosin-2B (NM-2B) constructs, and either tropomyosin isoform Tpm1.8cy (b.-.b.d), Tpm1.12br (b.-.b.c), or Tpm3.1cy (b.-.a.d). Our results show the extent to which the association of filamentous-actin with these different tropomyosin cofilaments affects the actin-mediated activation of NM-2B and the release of the ATP hydrolysis products ADP and phosphate from the active site. Phosphate release gates a transition from weak to strong F-actin-binding states. The release of ADP has the opposite effect. These changes in dominant rate-limiting steps have a direct effect on the duty ratio, the fraction of time that NM-2B spends in strongly F-actin-bound states during ATP turnover. The duty ratio is increased ϳ3-fold in the presence of Tpm1.12 and 5-fold for both Tpm1.8 and Tpm3.1. The presence of Tpm1.12 extends the time required per ATP hydrolysis cycle 3.7-fold, whereas it is shortened by 27 and 63% in the presence of Tpm1.8 and Tpm3.1, respectively. The resulting Tpm isoform-specific changes in the frequency, duration, and efficiency of actomyosin interactions establish a molecular basis for the ability of these complexes to support cellular processes with widely divergent demands in regard to force production, capacity to move processively, and speed of movement.
Role of Residues 230 and 236 of Actin in Myosin-ATPase Activation by Actin-Tropomyosin
Biochemical and Biophysical Research Communications, 2000
The Dictyostelium/Tetrahymena-chimeric actin (Q228K/T229A/A230Y) showed higher Ca(2+)-activation of myosin S1 ATPase in the presence of tropomyosin-troponin. The crystal structure of the chimeric actin is almost the same as that of wild-type except the conformation of the side chain of Leu236. Here, we introduced an additional mutation (L236A), in which the side chain of Leu236 was truncated, into the chimeric actin (Q228K/T229A/A230Y/L236A). Without regulatory proteins, the new mutant actin showed normal myosin S1 activation and normal sliding velocity. However, in the presence of tropomyosin, the new mutant actin activated myosin S1 ATPase higher than the wild-type actin and showed higher velocities in in vitro motility assay at low HMM concentrations. These results suggest that the mutations of A230Y and L236A in the actin subdomain-4 facilitate the transition of thin filaments from a "closed" state to an "open" state.