The Active State of the Thin Filament Is Destabilized by an Internal Deletion in Tropomyosin

Troponin (Tn) plays the key roles in the regulation of striated muscle contraction. Tn consists of three subunits (TnT, TnC, and TnI). In combination with the stopped-flow method, fluorescence resonance energy transfer between probes attached to Cys-60 or Cys-250 of TnT and Cys-374 of actin was measured to determine the rates of switching movement of the troponin tail domain (Cys-60) and of the TnT-TnI coiled-coil C terminus (Cys-250) between three states (relaxed, closed, and open) of the thin filament. When the free Ca 2؉ concentration was rapidly changed, these domains moved with rates of ϳ450 and ϳ85 s ؊1 at pH 7.0 on Ca 2؉ up and down, respectively. When myosin subfragment 1 (S1) was dissociated from thin filaments by rapid mixing with ATP, these domains moved with a single rate constant of ϳ400 s ؊1 in the presence and absence of Ca 2؉ . The light scattering measurements showed that ATP-induced S1 dissociation occurred with a rate constant >800 s ؊1 . When S1 was rapidly mixed with the thin filament, these domains moved with almost the same or slightly faster rates than those of S1 binding measured by light scattering. In most but not all aspects, the rates of movement of the troponin tail domain and of the TnT-TnI coiled-coil C terminus were very similar to those of certain TnI sites (N terminus, Cys-133, and C terminus) previously characterized (Shitaka, Y., Kimura, C., Iio, T., and Miki, M. (2004) Biochemistry 43, 10739 -10747), suggesting that a series of conformational changes in the Tn complex during switching on or off process occurs synchronously.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

The Rates of Switching Movement of Troponin T between Three States of Skeletal Muscle Thin Filaments Determined by Fluorescence Resonance Energy Transfer

Shitaka

Kimura

Miki

2005

“…The present work suggests that these functional defects can occur with preserved affinity of the regulatory proteins for actin and, at least in the present case, may best be understood as consequences of altered affinity of myosin for actin. Relief of the inhibitory effects of troponin-tropomyosin requires not just Ca 2ϩ binding to troponin but also strong myosin binding to the thin filament (11). Subdomain 2 may play an important role in myosin binding and myosin-induced activation, which is suppressed by the D56A/E57A mutation.…”

Section: Discussionmentioning

confidence: 99%

An Actin Subdomain 2 Mutation That Impairs Thin Filament Regulation by Troponin and Tropomyosin

Korman¹,

Hatch²,

Dixon³

et al. 2000

Self Cite

Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca 2؉ to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca 2؉ showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosinactin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex.Cardiac and skeletal muscle contraction is controlled by a complex allosteric system in which myosin-actin interactions are regulated by tropomyosin and troponin. Tropomyosin is an extended, dimeric coiled-coil and spans seven actin monomers on the thin filament. Troponin is composed of three subunits: troponin T connects the other two subunits and tropomyosin to the actin filament, troponin I inhibits myosin binding to actin, and troponin C serves as a Ca 2ϩ sensor for the system (for reviews see Refs. 1 and 2). Structural studies have shown that tropomyosin can bind to three different regions of the actin filament (3-7). In the absence of Ca 2ϩ , tropomyosin appears to bind to subdomain 1 of actin and to bridge over subdomain 2 to the next actin monomer (4). In the presence of Ca 2ϩ , tropomyosin moves toward subdomains 3 and 4 (3) and moves even further in the presence of myosin (8). Whereas these and other results support a three state model of muscle regulation involving tropomyosin movement (9 -12), information defining the position and interactions of troponin on the filament and actin residues that specifically interact with the regulatory proteins is limited.One strategy to examine muscle protein-protein interactions has been to exploit functionally altered mutants (13-15). For example, a Drosophila melanogaster actin subdomain 2 mutant, E93K, which inhibits tropomyosin-actin sliding in isolated protein preparations, has been utilized to investigate tropomyosin-based regulation (16). These and other data (17)(18)(19)(20) suggested that actin subdomain 2 may be ...

“…of Lys 238 by pentane dione (35)) or by mutations (E311A/R312A (13) and K238A/E241A/E360H (14)) lead to a reduction in tropomyosin binding affinity and modification of regulatory behavior. In addition mutations in tropomyosin can also modulate regulation (33,36) The likely position of tropomyosin when in the off state has only recently been visualized by electron microscopy and threedimensional reconstructions. In both vertebrates and arthropods tropomyosin is seen to have moved across the face of the actin monomer so that it is over the junction between domains 1 and 3 and over domain 2 at the top of the actin monomer, covering the putative myosin binding site (8,10) (Fig.…”

Section: Glu 93mentioning

confidence: 99%

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

Bing¹,

Razzaq²,

Sparrow³

et al. 1998

In the Drosophila flight muscle actin mutant E93K there is a charge reversal on the surface of actin close to the proposed position of tropomyosin when it is in the off state. Using a quantitative in vitro motility assay we have found that the wild type Drosophila ACT88F actin behaved like rabbit skeletal muscle actin when tropomyosin and troponin were added at pCa5 and pCa9. In contrast the effect of tropomyosin upon the E93K mutant actin filament movement was completely different from wild type and resembled the response of wild type with tropomyosin؉troponin at pCa9 (i.e. the filaments were switched off). Velocity of E93K actin did not increase, and the fraction of filaments motile was reduced to less than 15% by adding up to 30 nM tropomyosin. When myosin subfragment-1 modified by N-ethylmaleimide was mixed with mutant E93K actin-tropomyosin filaments we observed that it restored motility of the filaments to the level observed with E93K actin alone. We conclude that electrostatic charge on the surface of domain 2 of actin plays a critical role in determining the state of actin-tropomyosin that is a central feature of the steric blocking mechanism of actin filament regulation.