Terence Tao

Molecular mechanism of troponin-C function

ConclusionsThere is now a large body of evidence in support of the view that Ca2+ binding to the low affinity sites of TnC induces a movement of helices B and C away from helices A and D, thus opening a hydrophobic cavity, the site of interaction with TnI. Another site of similar structure is formed by the helical segments in the C-terminal domain. Both sites appear to interact with the inhibitory segment of TnI. Whereas the interactions at both sites are necessary for the full regulatory activity of TnC, the interaction at the C-terminal domain stabilizes the complex and that involving the N-terminal domain is directly linked to the release of inhibition. In the absence of Ca2+ the inhibitory region of TnI would preferentially bind to actin and on Ca2+ binding to sites I and II it would switch to the site in the N-terminal domain of TnC. Detachment of TnI from actin gives rise to further events in thin filament regulation.

IMMUNOCYTOCHEMICAL LOCALIZATION OF CALDESMON AND CALPONIN IN CHICKEN GIZZARD SMOOTH MUSCLE

SummaryThe distribution of caldesmon and calponin in chicken gizzard smooth muscle was investigated with immunofluorescence and immunogold electron microscopy. Immunofluorescence microscopy showed that in verapamil treated (relaxed) muscles the distributions of caldesmon and myosin appeared to be uniform throughout the cytoplasm, but clearly more textured than that of actin filaments as revealed by the distribution of tropomyosin. In shortened muscles both caldesmon and myosin became segregated, in contrast to the distribution of actin, which remained uniform. The distribution of calponin was even more textured, with no similarity to those of caldesmon or myosin. Instead, considerable overlap was observed between calponin and the cytoskeletal protein desmin and, to a lesser extent, β-actin. By immunogold electron microscopy caldesmon appeared mostly near and around myosin filaments in both relaxed and shortened muscle. Calponin, on the other hand, was found primarily at the periphery of cytoskeletal structures in the same general region as desmin, and very often adjacent to β-actin, which is mainly in the core. These observations indicated that caldesmon and calponin are associated with different subsets of actin filaments, caldesmon with contractile actin, while calponin with cytoskeletal actin. Thus the in situ localization of caldesmon is consistent with its proposed regulatory function. Calponin, on the other hand, is unlikely to directly regulate actomyosin interactions in these cells; instead, it may function as a bridging protein between the actin and the intermediate filament networks.

Ca2+-Dependent Photocrosslinking of Tropomyosin Residue 146 to Residues 157―163 in the C-Terminal Domain of Troponin I in Reconstituted Skeletal Muscle Thin Filaments

The Ca(2+)-dependent interaction of troponin I (TnI) with actin.tropomyosin (Tm) in muscle thin filaments is a critical step in the regulation of muscle contraction. Previous studies have suggested that, in the absence of Ca(2+), TnI interacts with Tm and actin in reconstituted muscle thin filaments, maintaining Tm at the outer domain of actin and blocking myosin-actin interaction. To obtain direct evidence for this Tm-TnI interaction, we performed photochemical crosslinking studies using Tm labeled with 4-maleimidobenzophenone at position 146 or 174 (Tm*146 or Tm*174, respectively), reconstituted with actin and troponin [composed of TnI, troponin T (TnT), and troponin C] or with actin and TnI. After near-UV irradiation, SDS gels of the Tm*146-containing thin filament showed three new high-molecular-weight bands determined to be crosslinked products Tm*146-TnI, Tm*146-troponin C, and Tm*146-TnT using fluorescence-labeled TnI, mass spectrometry, and Western blot analysis. While Tm*146-TnI was produced only in the absence of Ca(2+), the production of other crosslinked species did not show Ca(2+) dependence. Tm*174 mainly crosslinked to TnT. In the absence of actin, a similar crosslinking pattern was obtained with a much lower yield. A tryptic peptide from Tm*146-TnI with a molecular mass of 2601.2 Da that was not present in the tryptic peptides of Tm*146 or TnI was identified using HPLC and matrix-assisted laser desorption/ionization time-of-flight. This was shown, using absorption and fluorescence spectroscopy, to be the 4-maleimidobenzophenone-labeled peptide from Tm crosslinked to TnI peptide 157-163. These data, which show that a region in the C-terminal domain of TnI interacts with Tm in the absence of Ca(2+), support the hypothesis that a TnI-Tm interaction maintains Tm at the outer domain of actin and will help efforts to localize troponin in actin.Tm muscle thin filaments.

Localization of Protein Regions Involved in the Interaction between Calponin and Myosin

Calponin is a 33-kDa smooth muscle-specific protein that has been suggested to play a role in muscle contractility. It has previously been shown to interact with actin, tropomyosin, and calmodulin. More recently we showed that calponin also interacts with myosin (Szymanski, P. T., and Tao, T. (1993) FEBS Lett. 331, 256-259). In the present study we used a combination of co-sedimentation and fluorescence assays to localize the regions in myosin and calponin that are involved in the interaction between these two proteins. We found that recombinant chicken gizzard α-calponin co-sediments with myosin rod and, to a lesser extent, with light meromyosin. Fluorescently labeled recombinant calponin shows interaction with heavy meromyosin and myosin subfragment 2 but not subfragment 1. A fragment comprising residues 7-182 and a synthetic peptide spanning residues 146-176 of calponin co-sediment with myosin, but fragments comprising residues 7-144 and 183-292 do not. Our results indicate that there are calponin binding sites in the subfragment 2 and light meromyosin regions of myosin, and that the region comprising residues 145-182 of calponin mediates its interaction with myosin.

LOCALIZATION OF CYS133 OF RABBIT SKELETAL TROPONIN-I WITH RESPECT TO TROPONIN-C BY RESONANCE ENERGY TRANSFER

We have used the technique of resonance energy transfer in conjunction with distance geometry analysis to localize Cys133 of troponin-I (TnI) with respect to troponin-C (TnC) in the ternary troponin complex and the binary TnC.TnI complex in the presence and absence of Ca2+. Cys133 of TnI was chosen because our previous work has shown that the region of TnI containing this residue undergoes Ca2+-dependent movements between actin and TnC, and may play an important role in the regulatory function of troponin. For this purpose, a TnI mutant with a single Cys at position 133, and TnC mutants, each with a single Cys at positions 5, 12, 21, 41, 49, 89, 98, 133, and 158, were constructed by site-directed mutagenesis. The distances between TnI Cys133 and each of the nine residues in TnC were then measured. Using a least-squares minimization procedure, we determined the position of TnI Cys133 in the coordinate system of the crystal structure of TnC. Our results show that in the presence of Ca2+, TnI Cys133 is located near residue 12 beneath the N-terminal lobe of TnC, and moves away by 12.6 A upon the removal of Ca2+. TnI Cys133 and the region of TnC that undergoes major change in conformation in response to Ca2+ are located roughly on opposite sides of TnCs central helix. This suggests that the region in TnI that undergoes Ca2+-dependent interaction with TnC is distinct from that interacting with actin.

Proximity Relationships between Residue 117 of Rabbit Skeletal Troponin-I and Residues in Troponin-C and Actin

We used resonance energy transfer and site-directed photo-cross-linking to probe the Ca(2+)-dependent proximity relationships between residue 117 next to the C-terminus of the inhibitory region in rabbit skeletal troponin-I (TnI) and residues in troponin-C (TnC) and in actin. A mutant TnI that contains a single cysteine at position 117 (I117) was constructed, and the distance between TnI residue 117 and TnC residue 98 was measured with the following results: for both the binary TnC-TnI complex and the ternary troponin complex, this distance was 30 and 41 A in the presence and absence of Ca(2+), respectively. The distance between TnI residue 117 and Cys374 of actin was 48 and 41 A in the presence and absence of Ca(2+), respectively. Six additional distances from this TnI residue to cysteines in TnC mutants were measured and used to localize this residue with respect to the crystal structure of TnC. The results show that in the presence of Ca(2+) it is localized near the B and C helices of TnCs N-terminal domain. In the absence of Ca(2+) this residue moves away from this location by approximately 8 A. Photo-cross-linking experiments show that I117 labeled with 4-maleimidobenzophenone photo-cross-linked to TnC but not to actin in both the presence and absence of Ca(2+). Taken together these results provide independent experimental support for the proposal (Y. Luo, J. L. Wu, B. Li, K. Langsetmo, J. Gergely, and T. Tao, 2000, J. Mol. Biol. 296:899-910) that upon Ca(2+) removal the region comprising TnI residues 114-125 triggers the movements of residues 89-113 and 130-150 toward actin, but does not itself interact with actin.

Identification of the photocrosslinking sites in troponin-I with 4-maleimidobenzophenone labelled mutant troponin-Cs having single cysteines at positions 158 and 21

Our previous studies have shown that 4-maleimidobenzophenone (BP-Mal) attached to troponin-C (TnC) mutants with single cysteines at positions 12, 57, 89 and 98 forms crosslinks to troponin-I (TnI), and the identified crosslinking regions indicate an antiparallel course of the two interacting polypeptide chains, in agreement with other studies using fragments of TnC and TnI. In this work we extended the mapping of the TnC–TnI interface by analysing photocrosslinking between TnI and BP-Mal labelled TnC mutants with single Cys residues at positions 21 (TnC21) and 158 (TnC158). We determined the sites of these photocrosslinks in TnI by progressive proteolysis of the crosslinked product, followed by N-terminal sequencing and mass spectrophotometric analyses. The results show that whereas TnC158 forms a specific crosslink with Met-21, TnC21 forms multiple crosslinks in the range of residues 96 to 134 of TnI. The results are discussed in light of the antiparallel model of the TnI–TnC complex and a structural model derived from low-angle X-ray and neutron scattering studies.

Conformational changes induced in troponin I by interaction with troponin T and actin/tropomyosin

Troponin I (TnI) is the inhibitory component of the striated muscle Ca2+ regulatory protein troponin (Tn). The other two components of Tn are troponin C (TnC), the Ca2+-binding component, and troponin T (TnT), the tropomyosin-binding component. We have used limited chymotryptic digestion to probe the local conformation of TnI in the free state, the binary TnC*TnI complex, the ternary TnC*. TnI*TnT (Tn) complex, and in the reconstituted Tn*tropomyosin*F-actin filament. The digestion of TnI alone or in the TnC*TnI complex produced initially two major fragments via a cleavage of the peptide bond between Phe100 and Asp101 in the so-called inhibitory region. In the ternary Tn complex cleavage occurred at a new site between Leu140 and Lys141. In the absence of Ca2+ this was followed by digestion of the 1-140 fragment at Leu122 and Met116. In the reconstituted thin filament the same fragments as in the case of the ternary complex were produced, but the rate of digestion was slower in the absence than in the presence of Ca2+. These results indicate firstly that in both free TnI and TnI complexed with TnC there is an exposed and flexible site in the inhibitory region. Secondly, TnT affects the conformation of TnI in the inhibitory region and also in the region that contains the 140-141 bond. Thirdly, the 140-141 region of TnI is likely to interact with actin in the reconstituted thin filament when Ca2+ is absent. These findings are discussed in terms of the role of TnI in the mechanism of thin filament regulation, and in light of our previous results [Y. Luo, J.-L. Wu, J. Gergely, T. Tao, Biochemistry 36 (1997) 13449-13454] on the global conformation of TnI.

The Molecular Switch in Troponin C

Conformational changes in troponin C (TnC) associated with Ca(2+)-induced triggering of muscle contraction are discussed in light of the model proposed by Herzberg, Moult and James (J. Biol. Chem. 261, 2638, 1986) and of our recent work on mutants of troponin C. The model involves a Ca(2+)-induced angular movement of one pair of alpha-helical segments relative to another pair of helices in the N-terminal domain. A disulfide bridge introduced into the N-terminal domain reversibly blocks the key conformational transition and the Ca(2+)-regulatory activity. Binding of troponin I (TnI) to the disulfide form of TnC is weakened owing to the blocking of its interaction with the N-terminal domain; however incorporation of the mutant into TnC-extracted myofibrils is not abolished. Introduction of a Cys residue in the C-terminal domain of TnC leads to disulfide formation between it and the indigenous Cys-98, with accompanying inhibition of regulatory activity attributable to interference with binding to TnI and, consequently, incorporation into the thin filaments. Evidence for movement of helical segments upon Ca(2+)-binding to TnC was obtained by measurements of excimer fluorescence and of resonance energy transfer with probes attached to Cys residues introduced by site-directed mutagenesis at suitable locations. Introduction of a disulfide bridge into calmodulin, another member of the super-family of Ca(2+)-binding proteins to which TnC belongs, abolishes its interaction with target enzymes. This suggests that the type of conformational change involving angular movement of helical segments that takes place in TnC is also involved in signal transmission in other Ca(2+)-dependent regulatory proteins.

pH-DEPENDENT CHANGES IN THE SPATIAL RELATIONSHIP BETWEEN THE DOMAINS OF TROPONIN C

Publisher Summary This chapter analyzes pH-dependent changes in the spatial relationship between the domains of Troponin C. The pH dependence of the TnC conformation in solution was studied by measuring the distance from the N-terminal domain to Cys-98 in the C-terminal domain using the technique of resonance energy transfer. Energy transfer measurements were carried out using as the donor either the luminescent rare earth ion Tb3+ bound to TnC or a fluorophor (IAEDANS) attached to Met-25 and, as the acceptor, the chromophoric maleimide derivative DAB attached to Cys-98. The findings show that the distance from the low affinity metal binding sites in the N-terminal domain to Cys-98 in the C-terminal domain of TnC changes from >5.2 nm at pH 5 to ∼2.7 nm at pH 7. Under similar conditions, the distance between probes at Met-25 and Cys-98 change only slightly from 4.6 to 4.3 nm. These results indicate that whereas the solution structure of TnC at pH 5 is compatible with the elongated crystal structure, it is more compactly folded at pH 7.