Localization of the two tropomyosin-binding sites of troponin T

Abstract

Troponin T (TnT) binds to tropomyosin (Tm) to anchor the troponin complex in the thin filament, and it thus serves as a vital link in the Ca(2+) regulation of striated muscle contraction. Pioneer work three decades ago determined that the T1 and T2 chymotryptic fragments of TnT each contains a Tm-binding site. A more precise localization of the two Tm-binding sites of TnT remains to be determined. In the present study, we tested serial deletion constructs of TnT and carried out monoclonal antibody competition experiments to show that the T1 region Tm-binding site involves mainly a 39 amino acids segment in the N-terminal portion of the conserved middle region of TnT. We further employed another set of TnT fragments to locate the T2 region Tm-binding site to a segment of 25 amino acids near the beginning of the T2 fragment. The localization of the two Tm-binding sites of TnT provided new information for the structure-function relationship of TnT and the anchoring of troponin complex on muscle thin filament.

Fig. 1. Structural and functional regions of TnT and TnT fragments studied

(A) The coding regions of the three muscle fiber type isoforms of TnT are aligned. The exon organizations are shown with alternatively spliced exons highlighted (the developmentally regulated fetal exons are in solid black). The structural differences among cardiac, fast and slow skeletal muscle TnT isoforms are mainly in the N-terminal variable region, which result in differences in exon and amino acid numbers in homologous regions of different TnT isoforms. The regions of T1, T2 and CB2 fragments are outlined along with the linear maps of the TnT isoforms and the epitope locations of three anti-TnT mAbs used as structural probes. Three N-terminal truncated cardiac TnTs with deletion of the N-terminal variable region alone (McTnT-ND₇₂) or together with portions of the conserved middle region encoded by exons 9 and 10 (McTnT-ND₉₂ and McTnT-ND₁₃₀) and the T2 fragment of cardiac TnT (McTnT-T2) were used in the mapping of the CB2 region Tm-binding site. Two C-terminal truncated slow TnT fragments (MsTnT_1–179, MsTnT_1–204,) were used in the mapping of the T2 region Tm-binding site. The corresponding regions of the TnT fragments are outlined with the dashed lines. (B) The epitope specificities of mAbs CT3, 2C8 and T12 were determined by Western blotting using the N-terminal truncated cardiac TnT fragments. (C) The two representative slow TnT fragments used in the mapping of the T2 region Tm-binding site were expressed in E. coli. The SDS-gel showed intact MsTnT and the MsTnT_1–179 and MsTnT_1–204 fragments prepared from bacterial culture.

Fig. 2. Binding of N-terminal truncated TnT fragments to Tm

N-terminal serially truncated cardiac TnT fragments (Fig. 1) were tested for Tm-binding affinity in ELISA protein binding experiments. The results showed that the deletion of the N-terminal variable region (McTnT-ND₇₂) alone did not significantly affect Tm-binding whereas further deletion of 20 amino acids in the conserved middle region (McTnT-ND₉₂) produced a detectable decrease in Tm-binding affinity. Extending the deletion to remove 39 more amino acids encoded by exon 10 (McTnT-ND₁₃₀) resulted in a major decrease in Tm-binding affinity.

Fig. 3. mAb competition/blocking mapping of the T1 region Tm-binding site of TnT

(A) The competition curves showed that mAb 2C8 significantly blocked the binding of TnT to Tm (P<0.01). In contrast, mAbs CT3 and T12 recognizing the flanking regions (Fig. 1) had only marginal effects. (B) The ELISA titration curves showed that mAb 2C8 raised against mouse cardiac TnT binds fast, slow and cardiac TnT isoforms with very similar affinities, indicating a highly conserved epitope structure with conserved function. Data are plotted as mean ± SD.

Fig. 4. Mapping of the T2 region Tm-binding site

Intact MsTnT, MsTnT_1–179, and MsTnT_1–204 fragments (Fig. 1) were examined for Tm-binding in ELISA protein binding experiments. The binding curves showed that whereas MsTnT_1–179, containing the T1 region Tm-binding site had rather weak binding to Tm (P<0.01), MsTnT_1–204 exhibited high affinity binding to Tm similar to that of intact MsTnT. Data are plotted as mean ± SD. In contrast to the normalized binding curves of the N-terminal truncated cardiac TnT fragments shown in Fig. 2 based on their similar maximum binding to Tm, the binding curves here were plotted from actual absorbance to demonstrate the significantly lower maximum Tm-binding of MsTnT_1–179 than that of intact MsTnT and MsTnT_1–204 in the solid phase binding assay.

Fig. 5. Evolutionarily conserved primary structure of the two Tm-binding sites of TnT

(A) Amino acid sequences of mammalian and avian fast, slow and cardiac TnT isoforms are aligned to demonstrate the highly conserved primary structure in the T1/CB2 region Tm-binding site (Site 1). The beginning of the CB2 region is indicated with an arrowhead. (B) Amino acid alignment demonstrates the highly conserved primary structure in the T2 region Tm-binding site (Site 2). The beginning of the T2 region and the truncation site of Amish nemaline myopathy slow TnT are indicated with arrowheads. The homologous exons encoding these segments are indicated in the background. Identical or conserved residues encoded by the two Tm-binding sites’ main coding exons are outlined with the grey bars above the sequences. The amino acid numbers of rabbit fast TnT match that in the previous Tm-binding studies [2]. The amino acid numbers for all other TnT isoforms were deduced from the longest splice forms [23]. The variable amino acid start positions and different exon numbers reflect the variation of the N-terminal region of the TnT isoforms (Fig. 1A). Met₁ was not counted in this figure to match the earlier studies using rabbit fast skeletal muscle TnT [2].

Fig. 6. The two Tm-binding sites of TnT in troponin-tropomyosin assembly

This proposed model illustrates that the CB2 region Tm-binding site of TnT (Site 1) binds the head-tail (N/C) junction of Tm (indicated by the gray box) in striated muscle thin filament, whereas the T2 region Tm-binding site of TnT (Site 2) binds Tm near Cys₁₉₀. The high resolution structure of partial troponin complex determined by crystallography is redrawn from published data [11,12]. The boundary between the T1 and T2 regions is indicated with a vertical line. The proteolysis site in cardiac TnT that selectively removes the N-terminal variable region [32], the position of the Amish nemaline myopathy (ANM) nonsense mutation [20], and the position of the MsTnT_1–204 truncation (Fig. 1A) are indicated with the arrows.