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X-ray fiber diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles

View Article: PubMed Central - PubMed

ABSTRACT

In order to clarify the structural changes of the thin filaments related to the regulation mechanism in skeletal muscle contraction, the intensities of thin filament-based reflections in the X-ray fiber diffraction patterns from live frog skeletal muscles at non-filament overlap length were investigated in the relaxed state and upon activation. Modeling the structural changes of the whole thin filament due to Ca2+-activation was systematically performed using the crystallographic data of constituent molecules (actin, tropomyosin and troponin core domain) as starting points in order to determine the structural changes of the regulatory proteins and actin. The results showed that the globular core domain of troponin moved toward the filament axis by ∼6 Å and rotated by ∼16° anticlockwise (viewed from the pointed end) around the filament axis by Ca2+-binding to troponin C, and that tropomyosin together with the tail of troponin T moved azimuthally toward the inner domains of actin by ∼12° and radially by ∼7 Å from the relaxed position possibly to partially open the myosin binding region of actin. The domain structure of the actin molecule in F-actin we obtained for frog muscle thin filament was slightly different from that of the Holmes F-actin model in the relaxed state, and upon activation, all subdomains of actin moved in the direction to closing the nucleotide-binding pocket, making the actin molecule more compact. We suggest that the troponin movements and the structural changes within actin molecule upon activation are also crucial components of the regulation mechanism in addition to the steric blocking movement of tropomyosin.

No MeSH data available.


Modeling thin filaments with an unchanging Holmes F-actin. (A) A model with the V-shaped troponin core domain is oriented toward the pointed/M-line end of the actin filament. (B) A model with the troponin core domain is oriented toward the barbed/Z-band end of the actin filament. The upper side is toward the pointed/M-line end. The color assignment for the constituent molecules is the same as in Fig. 3. The calculated and observed layer line intensities are compared in the relaxed state (C) and in the activated state (D).
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f4-6_13: Modeling thin filaments with an unchanging Holmes F-actin. (A) A model with the V-shaped troponin core domain is oriented toward the pointed/M-line end of the actin filament. (B) A model with the troponin core domain is oriented toward the barbed/Z-band end of the actin filament. The upper side is toward the pointed/M-line end. The color assignment for the constituent molecules is the same as in Fig. 3. The calculated and observed layer line intensities are compared in the relaxed state (C) and in the activated state (D).

Mentions: In the second step, the troponin core domain was introduced based upon observation in the immuno-electron micrographs17 and by connecting the N-terminus of TNT2 part to the C-terminus of TNT1 part on tropomyosin molecules. The orientation of the core domain was varied around the N-terminus of TNT2 so as not to break the connecting portion between the C-terminus of TNT1 and the N-terminus of TNT2 where tropomyosin is adjacent. Its optimal disposition of the troponin core domain on tropomyosin including the TNT1 part was searched by varying the rotation angles (α, β, γ) around three axes of the Cartesian coordinate, the radial (r), angular (θ) and axial (z) coordinates of the core domain (Fig. 3B) without changes of the subunit arrangement within the core domain to obtain a better fit of the calculated intensities to the observed ones of the first and the second layer lines including the 51 Å and the 59 Å layer lines. The variable ranges and the step sizes for these parameters are given in Table 1B. The position r = 0 corresponds to that where the TNT2 N-terminus contacts with tropomyosin, and the position θ = 0 corresponds to that where the line connecting the N- and the C-termini of TNT2 in the core domain is parallel to the tropomyosin strand. Four optimal models for the disposition of the TN core domain were obtained with similar values of Rf. The intensities of the 51 Å and the 59 Å layer lines are not in fact affected by the movement of the tropomyosin + TNT1 tail. The orientation of the TN core domain in the two representative models depicted in Fig. 4, which yielded similar intensity fits, was in the opposite direction with each other. Unfortunately, we could not determine the correct orientation of the TN core domain with respect to the F-actin and the tropomyosin polarity by X-ray fiber diffraction, and therefore two types of models with either orientation remain possible. However, the orientation of the troponin core domain in the model of Fig. 4A, in which the apex (taper end) of the V-shaped core domain orients toward the barbed end of F-actin, was consistent with that shown by the recent EM single-particle image analysis18. The troponin core domain placed over subdomain 1 of actin and lay at a radius of 54 Å. This model was also consistent with the result suggested from distance measurements between the amino acid residues in reconstituted thin filaments obtained by fluorescence resonance energy transfer (FRET) method19. In addition, the D/E helix linker of TNC was oriented almost perpendicularly to the filament axis. This orientation was consistent with the data from fluorescence polarization measurement from the TN core domain20,21. Considering these features, we adopted the model depicted in Fig. 4A and refined it further, although firm evidence for the correct orientation is still under investigation (see below). As seen in Fig. 4, the fit to the observed data, however, was poor for the low-angle layer lines, suggesting that the F-actin model used here may not be compatible with the structure of F-actin in frog skeletal muscle thin filament. We therefore refined the initial F-actin model against the observed diffraction data by changing the conformation of actin molecule consisting of four subdomains in the crystal structure15.


X-ray fiber diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles
Modeling thin filaments with an unchanging Holmes F-actin. (A) A model with the V-shaped troponin core domain is oriented toward the pointed/M-line end of the actin filament. (B) A model with the troponin core domain is oriented toward the barbed/Z-band end of the actin filament. The upper side is toward the pointed/M-line end. The color assignment for the constituent molecules is the same as in Fig. 3. The calculated and observed layer line intensities are compared in the relaxed state (C) and in the activated state (D).
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Related In: Results  -  Collection

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f4-6_13: Modeling thin filaments with an unchanging Holmes F-actin. (A) A model with the V-shaped troponin core domain is oriented toward the pointed/M-line end of the actin filament. (B) A model with the troponin core domain is oriented toward the barbed/Z-band end of the actin filament. The upper side is toward the pointed/M-line end. The color assignment for the constituent molecules is the same as in Fig. 3. The calculated and observed layer line intensities are compared in the relaxed state (C) and in the activated state (D).
Mentions: In the second step, the troponin core domain was introduced based upon observation in the immuno-electron micrographs17 and by connecting the N-terminus of TNT2 part to the C-terminus of TNT1 part on tropomyosin molecules. The orientation of the core domain was varied around the N-terminus of TNT2 so as not to break the connecting portion between the C-terminus of TNT1 and the N-terminus of TNT2 where tropomyosin is adjacent. Its optimal disposition of the troponin core domain on tropomyosin including the TNT1 part was searched by varying the rotation angles (α, β, γ) around three axes of the Cartesian coordinate, the radial (r), angular (θ) and axial (z) coordinates of the core domain (Fig. 3B) without changes of the subunit arrangement within the core domain to obtain a better fit of the calculated intensities to the observed ones of the first and the second layer lines including the 51 Å and the 59 Å layer lines. The variable ranges and the step sizes for these parameters are given in Table 1B. The position r = 0 corresponds to that where the TNT2 N-terminus contacts with tropomyosin, and the position θ = 0 corresponds to that where the line connecting the N- and the C-termini of TNT2 in the core domain is parallel to the tropomyosin strand. Four optimal models for the disposition of the TN core domain were obtained with similar values of Rf. The intensities of the 51 Å and the 59 Å layer lines are not in fact affected by the movement of the tropomyosin + TNT1 tail. The orientation of the TN core domain in the two representative models depicted in Fig. 4, which yielded similar intensity fits, was in the opposite direction with each other. Unfortunately, we could not determine the correct orientation of the TN core domain with respect to the F-actin and the tropomyosin polarity by X-ray fiber diffraction, and therefore two types of models with either orientation remain possible. However, the orientation of the troponin core domain in the model of Fig. 4A, in which the apex (taper end) of the V-shaped core domain orients toward the barbed end of F-actin, was consistent with that shown by the recent EM single-particle image analysis18. The troponin core domain placed over subdomain 1 of actin and lay at a radius of 54 Å. This model was also consistent with the result suggested from distance measurements between the amino acid residues in reconstituted thin filaments obtained by fluorescence resonance energy transfer (FRET) method19. In addition, the D/E helix linker of TNC was oriented almost perpendicularly to the filament axis. This orientation was consistent with the data from fluorescence polarization measurement from the TN core domain20,21. Considering these features, we adopted the model depicted in Fig. 4A and refined it further, although firm evidence for the correct orientation is still under investigation (see below). As seen in Fig. 4, the fit to the observed data, however, was poor for the low-angle layer lines, suggesting that the F-actin model used here may not be compatible with the structure of F-actin in frog skeletal muscle thin filament. We therefore refined the initial F-actin model against the observed diffraction data by changing the conformation of actin molecule consisting of four subdomains in the crystal structure15.

View Article: PubMed Central - PubMed

ABSTRACT

In order to clarify the structural changes of the thin filaments related to the regulation mechanism in skeletal muscle contraction, the intensities of thin filament-based reflections in the X-ray fiber diffraction patterns from live frog skeletal muscles at non-filament overlap length were investigated in the relaxed state and upon activation. Modeling the structural changes of the whole thin filament due to Ca2+-activation was systematically performed using the crystallographic data of constituent molecules (actin, tropomyosin and troponin core domain) as starting points in order to determine the structural changes of the regulatory proteins and actin. The results showed that the globular core domain of troponin moved toward the filament axis by ∼6 Å and rotated by ∼16° anticlockwise (viewed from the pointed end) around the filament axis by Ca2+-binding to troponin C, and that tropomyosin together with the tail of troponin T moved azimuthally toward the inner domains of actin by ∼12° and radially by ∼7 Å from the relaxed position possibly to partially open the myosin binding region of actin. The domain structure of the actin molecule in F-actin we obtained for frog muscle thin filament was slightly different from that of the Holmes F-actin model in the relaxed state, and upon activation, all subdomains of actin moved in the direction to closing the nucleotide-binding pocket, making the actin molecule more compact. We suggest that the troponin movements and the structural changes within actin molecule upon activation are also crucial components of the regulation mechanism in addition to the steric blocking movement of tropomyosin.

No MeSH data available.