Role of Residues 311/312 in Actin-Tropomyosin Interaction

Abstract

The contraction of vertebrate striated muscle is regulated by the thin filament-associated proteins tropomyosin (Tm) 1 and troponin (Tn), which modulate the interaction of actin and myosin in a Ca 2ϩ -dependent fashion (2). Each Tm molecule associates with one Tn molecule and seven actin monomers. The amino acid sequence of Tm contains a pattern of charged and uncharged amino acids that repeats 14 times along its length (3). As each pair of repeats corresponds to an actin monomer along the actin filament, it has been inferred that the binding of Tm to actin is dominated by electrostatic interactions (1).Tn is composed of three subunits: TnT, TnI, and TnC. The TnC subunit binds Ca 2ϩ and confers calcium sensitivity to the actin-Tm-Tn system. According to the three-state model of thin filament regulation (4), the actin-Tm-Tn complex can assume three structural states: blocked, closed, and open. In the blocked state there is a very low incidence of myosin binding. When Ca 2ϩ binds to TnC, the Tm-Tn complex shifts to the closed state, uncovering additional myosin weak binding sites on the actin filament. The increase in weak binding and the initial strong binding of myosin induce Tm-Tn to shift from the closed (where it prevents myosin strong binding) to the open state. According to this model, the azimuthal shift of Tm-Tn around the axis of the actin filament sterically regulates myosin binding to actin.Despite the elegance of the three-state model, it cannot explain well the findings of earlier acto-S1 ATPase solution studies (5-7). Results from these studies describe the ability of Tm alone to both inhibit, at low S1 concentrations, actin-activated S1 ATPase rates and to potentiate the reaction at intermediate, nonsaturating concentrations of S1. Although inhibition can readily be explained using the steric block model, the potentiation suggests the presence of an allosteric component in actomyosin regulation.The Ca 2ϩ -induced Tm-Tn movement on actin was first indicated by x-ray diffraction studies (8 -10). Electron microscopy has also been used to directly visualize this shift in Tm-Tn position (11)(12)(13)(14)(15)(16), and the studies of Limulus muscle (17, 18) and vertebrate muscle (19) identified the positions for the Tm-Tn complex on actin in the presence and absence of Ca 2ϩ . A high resolution model of the Tm-F-actin complex was proposed by Lorenz et al. (1) on the basis of their x-ray fiber diffraction investigation. According to these authors, Tm alone and Tm-Tn in the presence of Ca 2ϩ reside in the same closedstate orientation on the actin filament. In this study Lorenz et al. (1) predict that 16 actin residues participate in the electrostatic interactions between F-actin and Tm and calculate their ⌬G contributions to this interaction. Our choice of residue 311 as a suitable starting point to test the predictions of this model and to gain additional insight into Tm regulatory function was based on two criteria. First, according to Lorenz et al. (1) residue 311 has a relatively high energy contrib...

Tropomyosin and Troponin Regulation of Wild Type and E93K Mutant Actin Filaments from Drosophila Flight Muscle

“…The putative strong and weak myosin binding sites on actin are exposed and the myosin cross-bridges may cycle unimpeded. Recent work using actin mutants supports this model: charge change mutations within the trough of actin such as E311A/R312A in yeast (13) and K238A/E241A/E360H in Dictyostelium (14) result in weaker tropomyosin binding and destabilization of the on state.…”

Mutations in Actin Subdomain 3 That Impair Thin Filament Regulation by Troponin and Tropomyosin

“…Mutation to Ala could then promote reversal of strong crossbridge binding. This would explain not only the current MgATPase data (reversal before product release), but also a preliminary report using in vitro motility to examine the yeast actin mutant E311A/R312A (49). These studies showed that motility rates were similar for E311A/R312A F-actin and wild type F-actin alone, but in the presence of the regulatory proteins the sliding speed was increased for the E311A/R312A thin filaments when compared with the wild type, implying faster cross-bridge detachment.…”

Role of Residues 230 and 236 of Actin in Myosin-ATPase Activation by Actin-Tropomyosin

“…The in vitro motility assays were performed according to previously described protocol (11,27). HMM was applied to the silicone-coated coverslips (Matsunami, Japan) at concentrations ranging from 15 to 300 g/ml.…”