Troponin (Tn) is the sarcomeric Ca2+-dependent regulator for striated muscle contraction in vertebrates. It is distributed on thin filaments and inhibits the interaction between actin and myosin. Troponin consists of three subunits: troponin C (TnC), troponin I (TnI), and troponin T (TnT). The binding of Ca2+ to TnC induces a conformational change in the troponin complex structure that enables myosin to interact with actin  -  .
Both vertebrate and molluscan muscle contraction are regulated by intracellular Ca2+ concentrations. However, in contrast to vertebrates, mollusks employ a thick filament-linked regulatory system where myosin binds Ca2+ directly, leading to its activation and subsequent interaction with actin. Although Tn is also located in molluscan muscles, it is currently unclear whether it is involved in a similar thin filament-linked regulatory system to that in vertebrates.
Bivalve adductor muscle is composed of two types of muscles, phasic and catch. Phasic muscle is used for quick shell closures and catch muscle for the prolonged closure of shells, a process that utilizes little energy (catch contraction)  . Recently, we revealed the molecular characteristics of troponin C from the Japanese pearl oyster Pinctada fucata (Pifuc-TnC)   . The Pifuc-TnC gene is predominantly expressed in phasic muscle, which leads us to suggest that Pifuc-TnC is involved in phasic muscle contraction. Our prediction is also supported by a recent comprehensive survey of gene expression patterns in scallop adductor muscle using proteomic and transcriptomic analyses that showed that the genes of all the troponin subunits are more highly expressed in phasic muscle compared with catch muscle  . However, to evaluate our prediction, elucidation of the function(s) of Tn in molluscan phasic muscle is required.
Pifuc-TnC possesses four EF-hand motifs (site I - IV) and, analogous to other molluscan TnCs, primary structure analyses indicate that only site IV is able to bind to Ca2+  . A predicted three-dimensional model of Pifuc-TnC resembled that of chicken fast skeletal TnC. However, there was an additional loop structure in the α-helix connecting its N- and C-terminal lobes, suggesting that Pifuc-TnC might work differently in molluscan muscles compared with TnC in vertebrate muscles. Moreover, it has been reported that scallop TnC, which binds to Ca2+ only at site IV, is likely to have distinct functions from vertebrates  .
Here, we studied Pifuc-TnC and Pifuc-TnC-E142Q, a Pifuc-TnC mutant variant lacking Ca2+-binding ability, and investigated whether the Pifuc-TnC structure changes upon binding of Ca2+.
2. Materials and Methods
2.1. Protein Preparation
We designed Pifuc-TnC-E142Q, a mutant variant lacking the ability to bind Ca2+ due to the substitution of a glutamic acid (E142) residue located in the site IV EF-hand motif to glutamine (Q) (Figure 1). DNA fragments encoding Pifuc-TnC or Pifuc-TnC-E142Q, with codon usage optimized for expression in Escherichia coli, were commercially synthesized by GenScript Gene Synthesis Service (GenScript, Piscataway, NJ, USA) and inserted into the T7 expression vector pET15b (Novagen Darmstadt, Germany), creating an in-frame N-terminal fusion of six histidine residues. E. coli BL21(DE3) transformed with pET-Pifuc-TnC or pET-Pifuc-TnC-E142Q were cultured in auto-induction media at 37˚C for 24 hours  . The cultured E. coli collected by centrifugation was suspended in a lysis buffer included in a kit EzBactYeast Crusher (ATTO, Tokyo, Japan). The supernatant of the lysate obtained by centrifugation containing Pifuc-TnC or Pifuc-TnC-E142Q was subjected to affinity chromatography with a Bio-Scale Mini Profinity IMAC cartridge (Bio-Rad, Hercules, CA, USA) under native conditions according to the manufacturer’s instructions. The purity of the eluted proteins was confirmed using SDS-PAGE and Coomassie blue staining. Protein concentrations were measured by the Bradford method using bovine serum albumin as a standard. Purified protein samples were freeze-dried following dialysis against 10 mM ammonium bicarbonate (pH 8.0).
2.2. Isothermal Titration Calorimetric Analysis
Ca2+ binding to Pifuc-TnC or Pifuc-TnC-E142Q was measured by isothermal
Figure 1. Sequences of Pinctada fucata troponin C (Pifuc-TnC) and Pifuc-TnC-E142Q. (a) Primary structures of Pifuc-TnC and its mutant variant, Pifuc-TnC-E142Q, which was constructed in this study. Additional sequences, including a histidine tag and a thrombin recognition site are shaded. Each of the four EF-hand motifs is underlined. Dots represent identical residues in Pifuc-TnC and Pifuc-TnC-E142Q; (b) Schematic representation of Pifuc-TnC and Pifuc-TnC-E142Q. The four EF-hand motifs are represented by semicircles. The black circle represents Ca2+ binding. Only the site IV EF-hand motifs in Pifuc-TnC is able to bind Ca2+ (Pifuc-TnC-E142Q is unable to bind Ca2+).
titration calorimetry (ITC) using a MicroCal iTC200 calorimeter (Malvern Panalytical Ltd., Malvern, UK). Freeze-dried protein samples were resuspended and dialyzed overnight against 10 mM PIPES-KOH (pH 6.8) containing 0.15 M NaCl and 1 mM 2-mercaptoethanol at 4˚C. The protein samples and CaCl2 solution were adjusted to final concentrations of 2.4 mM and 120 mM, respectively, using the external dialysis buffer. Experimental parameters were: total injections, 18 times; cell temperature, 25˚C; reference power, 10 μcal/s; syringe concentration, 120 mM; cell concentration, 2.4 mM; stirring speed, 1000 rpm. Injection parameters were: volume, 2.0 μL; spacing, 200 s. Titration was performed at 25˚C by injecting 2.0 μL of 120 mM CaCl2 into the ITC cell containing 300 μL of 2.4 mM Pifuc-TnC or Pifuc-TnC-E142Q. The data thus obtained were corrected for the heat of dilution and analyzed using MicroCal Analysis Launch software (Malvern Panalytical Ltd.).
2.3. Gel Filtration Analysis
To compare the gel filtration chromatography elution profiles of Pifuc-TnC in the presence and absence of Ca2+, Pifuc-TnC (2 mg) was injected onto a HiPrep16/60 Sephacryl S-200 high resolution column (GE Healthcare Ltd., Buckinghamshire, UK) equilibrated with 10 mM PIPES-KOH (pH 6.8) containing 0.15 M NaCl, 1 mM 2-mercaptoethanol, and 1 mM CaCl2 or 0.5 mM EDTA at 4˚C. Elution was performed at the flow rate of 0.5 mL/min and fraction volumes were 3.0 mL. The column effluent was monitored at 280 nm. Equivalent procedures were used to analyze the elution profiles of Pifuc-TnC-E142Q in the presence and absence of Ca2+.
2.4. UV-Absorption Spectral Analysis
Pifuc-TnC was dialyzed against phosphate buffered saline (PBS) and adjusted to a final concentration of 2.0 mg/mL. The absorption spectrum of Pifuc-TnC (2.0 mg/mL) in the presence or absence of 1 mM Ca2+ (where appropriate), was measured between 240 nm and 320 nm using an Agilent Cary 60 UV-Vis scanning spectrometer (Agilent Technologies, Santa Clara, CA) at room temperature. The UV-absorption spectra of Pifuc-TnC in the presence and absence of Ca2+ were then compared. Equivalent procedures were used to analyze the UV-absorption spectra of Pifuc-TnC-E142Q in the presence and absence of Ca2+.
2.5. Differential Scanning Calorimetric Analysis
The thermostability of Pifuc-TnC in the presence or absence of Ca2+ was analyzed by differential scanning calorimetry (DSC). Pifuc-TnC was dialyzed against PBS and adjusted to a final concentration of 2.0 mg/mL. Pifuc-TnC was analyzed by DSC in the presence or absence of 1 mM Ca2+ (where appropriate) using a Microcal VP-DSC (Malvern Panalytical Ltd.). Thermal scanning was performed between 10˚C and 80˚C with a scanning rate of 1˚C/min.
3.1. Protein Preparation
Histidine-tagged Pifuc-TnC and Pifuc-TnC-E142Q were successfully expressed in laboratory E. coli and purified to near-homogeneity using affinity chromatography as shown in Figure 2. The purity was sufficient for use in all experiments carried out in this study. Following freeze-drying, both proteins were able to be resuspended in the buffers required for all analyses.
3.2. Isothermal Calorimetric Analysis
Ca2+binding to Pifuc-TnC was confirmed by ITC analysis (Kd = 16.7 mM), whereas no Ca2+ binding was detected for Pifuc-TnC-E142Q. This confirms that only the Pifuc-TnC site IV EF-hand motif is able to bind to Ca2+ (Figure 3). Each injection of Ca2+ into the calorimetry cell containing Pifuc-TnC produced an endothermic heat of reaction that decreased in magnitude with subsequent injections. The results indicated that the binding of Ca2+ to Pifuc-TnC was driven by entropy changes.
3.3. Gel Filtration Chromatography
In the presence of Ca2+, Pifuc-TnC eluted in fraction 30 from a HiPrep16/60 Sephacryl S-200 high resolution gel filtration column, whereas it eluted earlier, in fraction 27, in the absence of Ca2+ (Figure 4(a)). In contrast, the elution profiles of the mutant variant, Pifuc-TnC-E142Q, which is unable to bind Ca2+, were equivalent in both the presence and absence of Ca2+ (Figure 4(b)). This indicates that Ca2+ binding to Pifuc-TnC induces a conformational change that delays its elution from the column.
Figure 2. Expression and purification of Pifuc-TnC and Pifuc-TnC-E142Q. (a) Coomassie blue-stained SDS-PAGE gel showing Pifuc-TnC upon expression in laboratory E. coli (lane 1) and following affinity purification (lane 2); (b) SDS-PAGE gel showing Pifuc-TnC-E142Q upon expression (lane 1) and following affinity purification (lane 2). M: molecular weight markers. Arrowheads indicate the purified protein bands.
Figure 3. Isothermal titration calorimetric analysis of Ca2+ binding to Pifuc-TnC and Pifuc-TnC-E142C. Titration curve of interactions between Pifuc-TnC and Ca2+ (a) and Pifuc-TnC-E142C and Ca2+ (b). In each case, the upper panel shows raw energy changes during the titration (time), while the lower panel presents the derived integrated total energy changes as a function of the molar ratio of the interactants. An endothermal reaction was detected upon interactions between Pifuc-TnC and Ca2+.
Figure 4. Isothermal titration calorimetric analysis of Ca2+ binding to Pifuc-TnC and Pifuc-TnC-E142C. Titration curve of interactions between Pifuc-TnC and Ca2+ (a) and Pifuc-TnC-E142C and Ca2+ (b). In each case, the upper panel shows raw energy changes during the titration (time), while the lower panel presents the derived integrated total energy changes as a function of the molar ratio of the interactants. An endothermal reaction was detected upon interactions between Pifuc-TnC and Ca2+.
3.4. UV-Absorption Spectra
UV-absorption spectral analysis revealed that the binding of Ca2+ to Pifuc-TnC caused an increase in absorption at a wavelength of approximately 250 nm (Figure 5). In contrast, no significant difference was observed in UV absorption spectra of the Pifuc-TnC-E142Q mutant variant in the presence and absence of Ca2+.
3.5. Differential Scanning Calorimetry
We also examined if Ca2+ binding affects Pifuc-TnC stability by monitoring its thermal unfolding using differential scanning calorimetry (DSC). The DSC thermogram of the Ca2+-unbound Pifuc-TnC did not show a clear thermal transition, which may be due to gradual unfolding as the temperature increases. However, a very different spectrum was observed for the Ca2+-bound Pifuc-TnC: a possible structural transition was observed at 50˚C followed by a number of smaller peaks that are probably the result of aggregation at higher temperatures (Figure 6). The difference between the Ca2+-bound and -unbound forms is likely to be a result of changes in protein folding, owing to a conformational change upon Ca2+ binding. Unfortunately, no thermodynamic parameters were obtained from the DSC data.
Here, we successfully constructed, expressed, and purified recombinant Pifuc-TnC and its mutant variant Pifuc-TnC-E142Q (Figure 1 and Figure 2). ITC analyses confirmed that Pifuc-TnC, as predicted from its primary structure, is able to bind to Ca2+ only at its site IV EF-hand motif (Figure 3), analogous to other molluscan TnCs. Gel-filtration chromatographic studies of Pifuc-TnC in the presence and absence of Ca2+ ions strongly suggested that Ca2+ binding
Figure 5. UV-absorption spectral analysis. (a) UV-absorption spectrum of Pifuc-TnC in the presence (solid line) and absence (dotted line) of Ca2+; (b) UV-absorption spectrum of Pifuc-TnC-E142Q in the presence (solid line) and absence (dotted line) of Ca2+.
Figure 6. Differential scanning calorimetric analysis. DSC thermograms of Pifuc-TnC in the absence (a) and presence (b) of Ca2+.
induces a conformational change, an observation that was supported by UV-absorption spectra and DSC analyses (Figures 4-6). It is possible that this increase in absorption at 250 nm of the UV-absorption spectra was caused by the exposure of phenylalanine residue at the surface of Pifuc-TnC as a result of a conformational change induced upon Ca2+ binding. These findings agree with reports that revealed that troponin C from Akazara scallop adductor muscle also has a single Ca2+ binding site at site IV, and, like Pifuc-TnC, alters its steric structure upon Ca2+ binding    .
In our previous study, we predicted a three-dimensional model of Pifuc-TnC using SWISS-MODEL. The predicted structure of Pifuc-TnC was very similar to TnCs from chicken fast skeletal muscle and American lobster  . The only notable difference was the presence of a short loop (four amino acids) within the α-helix connecting the N- and C-lobes, suggesting functional differences between molluscan and vertebrate TnCs. In contrast to molluscan TnCs, Ca2+ binding of vertebrate skeletal TnC involves all four EF-hand motifs. Ca2+ binding to sites I and II in the N-terminal lobe of skeletal TnC triggers actin-myosin activation, whereas sites III and IV in the C-terminal lobe are thought to contribute to the stabilization of the molecular structure. In contrast, Ca2+ binding to site IV of scallop TnC confers Ca2+ sensitivity to actomyosin, which suggests that the conformational change induced by Ca2+ binding of molluscan TnC may control the interaction between actin and myosin filaments   .
Pifuc-TnC is predominantly distributed in phasic adductor muscle  . Our findings in this study support previous reports that suggest molluscan TnC plays a role in the regulation of phasic muscle contraction. In mollusks, it is thought that muscle contraction begins following the direct binding of Ca2+ to myosin, which leads to its activation and subsequent interaction with actin. The detachment of Ca2+ from myosin reverses the process and relaxes the muscle  . Troponin is distributed on thin filaments and it is currently unclear how thin-filament related proteins contribute to the regulation of muscle contraction in mollusks. However, it is highly likely that the Ca2+-induced conformational change of TnC is involved in the regulation of molluscan muscle contraction.
Troponin is a complex of three regulatory proteins, TnC, TnI and TnT. Previous studies have revealed that Ca2+-bound scallop TnC binds to a different site in TnI in the troponin complex compared with vertebrate TnCs  . Further studies on the molecular interaction of Ca2+-bound TnC with the other subunits of the troponin complex are required to clarify the function of troponin in molluscan muscle contraction.
This study was supported by JSPS KAKENHI Grant Number JP16K07872. We thank Emma Andrew, PhD, from Edanz Group (http://www.edanzediting.com/ac) for editing a draft of this manuscript.
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