Roles for the Troponin Tail Domain in Thin Filament Assembly and Regulation

Abstract

Striated muscle contraction is regulated by Ca2؉ binding to troponin, which has a globular domain and an elongated tail attributable to the NH 2 -terminal portion of the bovine cardiac troponin T (TnT) subunit. Truncation of the bovine cardiac troponin tail was investigated using recombinant TnT fragments and subunits TnI and TnC. Progressive truncation of the troponin tail caused progressively weaker binding of troponin-tropomyosin to actin and of troponin to actin-tropomyosin. A sharp drop-off in affinity occurred with NH 2 -terminal deletion of 119 rather than 94 residues. Deletion of 94 residues had no effect on Ca 2؉ -activation of the myosin subfragment 1-thin filament MgATPase rate and did not eliminate cooperative effects of Ca 2؉ binding. Troponin tail peptide TnT1-153 strongly promoted tropomyosin binding to actin in the absence of TnI or TnC. The results show that the anchoring function of the troponin tail involves interactions with actin as well as with tropomyosin and has comparable importance in the presence or absence of Ca 2؉. Residues 95-153 are particularly important for anchoring, and residues 95-119 are crucial for function or local folding. Because striated muscle regulation involves switching among the conformational states of the thin filament, regulatory significance for the troponin tail may arise from its prominent contribution to the protein-protein interactions within these conformations.Troponin is a striated muscle regulatory protein (see reviews in Refs. 1-4) that is located at periodic, 38-nm spacing along muscle thin filaments. This spacing is due to the 1:1 complex formation of troponin with tropomyosin, an elongated coiledcoil protein that stretches along seven actin monomers. Ca 2ϩ binding to troponin triggers conformational changes in the thin filament, thereby allowing actin and myosin to interact to produce force and movement. Troponin contains two domains: a globular region, which is composed of subunits TnC, 1 TnI, and the COOH-terminal portion of TnT, and a highly extended region, or tail, containing the remainder of TnT (5, 6). The globular region has a central role in regulation, because it is the site of calcium binding. In contrast, the tail region of troponin, which is the subject of this report, has an uncertain role in conformational changes of the thin filament. One possibility is that it has little direct effect on regulation, acting instead as a calcium-insensitive anchor that holds troponin onto tropomyosin (7,8). However, the details of the interactions of troponin with actin and tropomyosin are unknown, in any of the conformations of the thin filament. Moreover, there is increasing evidence that the structure of the troponin tail can alter thin filament function in a complex manner (9 -16). To better understand the troponin tail region, the present study reports the properties of a series of troponin complexes containing progressively less of this region. Cardiac TnC plus TnI was reconstituted with either cardiac TnT or a series of recombinant NH 2 -termina...

Cardiac Troponin T, a Sarcomeric AKAP, Tethers Protein Kinase A at the Myofilaments

“…These fragments of cTnT (Fig. 5A) were previously confirmed as good functional representations of that particular fragment of the protein (27). Interactions between cTnT constructs and PKA-RI, or -RII, were assessed by two different colorimetric assays, using either ␣-gal (on selective medium plates) or ␤-gal (in a colony lift assay) (see Fig.…”

Novel Cardiac Troponin T Mutation as a Cause of Familial Dilated Cardiomyopathy

“…In vitro site-directed mutagenesis of the tail of cTnT showed that increasing elimination of residues 1 to 153 correlated with increasing to virtually complete elimination of the tropomyosin binding to actin. 28 It appears that the amino-terminal tail of cTnT is essential for assembly and anchoring of the troponin-tropomyosin complex onto the thin filament. Thus, despite mitotic and trophic simulation, the autosomal dominant inherited cTnT (Arg141Trp) acts as a poison peptide to inhibit filament assembly, which stymies the growth response.…”

“…It is expected that cardiac contractility will be impaired, because residue 141 is required for the troponin-tropomyosin complex to bind to actin. 27,28 The growth factors will most likely be upregulated, but why no growth? A likely reason stems from recent studies showing that the amino terminus (residues 1 to 153) of cTnT is essential for filament and sarcomere assembly.…”

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization

“…Gel filtration chromatography-The standards used in calibration of the Superdex G200 HR 16/60 (GE Healthcare) column and their corresponding Stokes radii were: ovoalbumin (30.5 Å); bovine serum albumin (35.5 Å); aldolase (48.1 Å); ferritin (61 Å) and thyroglobulin (85 Å) [17,18].…”