Ca 2 +-Induced Conformational Change of Troponin C from the Japanese Pearl Oyster , Pinctada fucata

Troponin is a thin filament-associated regulator of vertebrate striated muscle contraction. Troponin changes its structure upon Ca binding to troponin C, one of the subunits of troponin, allowing myosin to interact with actin. We recently elucidated the molecular characteristics of the Japanese pearl oyster Pinctada fucata troponin C (Pifuc-TnC), revealing the possibilities that Pifuc-TnC and vertebrate muscle TnC play dissimilar roles in muscle contraction. Pifuc-TnC has four EF-hand motifs, but, unlike vertebrate TnC, only one (site IV) was predicted to bind Ca. To confirm the number of Ca-binding sites in Pifuc-TnC and whether Ca binding induces a conformational change, we purified the full-length protein and a variant, Pifuc-TnC-E142Q (that has a mutation in the predicted Ca-binding site of site IV), following their expression in laboratory E. coli. Isothermal titration calorimetry demonstrated Ca binding to Pifuc-TnC, whereas Pifuc-TnC-E142Q was unable to bind Ca, confirming that site IV is the only Ca-binding site in Pifuc-TnC. Pifuc-TnC eluted in a later fraction from a gel filtration column in the presence of Ca compared with the condition when Ca was absent. In contrast, the elution profiles of Pifuc-TnC-E142Q were equivalent in both the presence and absence of Ca, suggesting that Ca binding to Pifuc-TnC induces a conformational change that delays its elution from the column. UV-absorption spectral analysis revealed that binding of Ca to Pifuc-TnC caused an increase in absorption at a wavelength of approximately 250 nm, possibly because phenylalanine residues had been exposed on the surface of the molecule as a result of a conformational change. Differential scanning calorimetric analyses of Pifuc-TnC showed aggregation in the presence of Ca in accordance with an increase of temperature, but no aggregation was seen in the absence of Ca. In combination, these findings suggest that Ca binding to site IV induces a conformational change in Pifuc-TnC. How to cite this paper: Funabara, D., Ishikawa, D., Urakawa, Y. and Kanoh, S. (2018) Ca2+-Induced Conformational Change of Troponin C from the Japanese Pearl Oyster, Pinctada fucata. American Journal of Molecular Biology, 8, 205-214. https://doi.org/10.4236/ajmb.2018.84018 Received: September 26, 2018 Accepted: October 17, 2018 Published: October 20, 2018 Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/

fuc-TnC and vertebrate muscle TnC play dissimilar roles in muscle contraction.Pifuc-TnC has four EF-hand motifs, but, unlike vertebrate TnC, only one (site IV) was predicted to bind Ca 2+ .To confirm the number of Ca 2+ -binding sites in Pifuc-TnC and whether Ca 2+ binding induces a conformational change, we purified the full-length protein and a variant, Pifuc-TnC-E142Q (that has a mutation in the predicted Ca 2+ -binding site of site IV), following their expression in laboratory E. coli.Isothermal titration calorimetry demonstrated Ca 2+ binding to Pifuc-TnC, whereas Pifuc-TnC-E142Q was unable to bind Ca 2+ , confirming that site IV is the only Ca 2+ -binding site in Pifuc-TnC.Pifuc-TnC eluted in a later fraction from a gel filtration column in the presence of Ca 2+ compared with the condition when Ca 2+ was absent.In contrast, the elution profiles of Pifuc-TnC-E142Q were equivalent in both the presence and absence of Ca 2+ , suggesting that Ca 2+ binding to Pifuc-TnC induces a conformational change that delays its elution from the column.UV-absorption spectral analysis revealed that binding of Ca 2+ to Pifuc-TnC caused an increase in absorption at a wavelength of approximately 250 nm, possibly because phenylalanine residues had been exposed on the surface of the molecule as a result of a conformational change.Differential scanning calorimetric analyses of Pifuc-TnC showed aggregation in the presence of Ca 2+ in accordance with an increase of temperature, but no aggregation was seen in the absence of Ca 2+ .In combination, these findings suggest that Ca 2+ binding to site IV induces a conformational change in Pifuc-TnC.

Introduction
Troponin (Tn) is the sarcomeric Ca 2+ -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 Ca 2+ to TnC induces a conformational change in the troponin complex structure that enables myosin to interact with actin [1]- [6].
Both vertebrate and molluscan muscle contraction are regulated by intracellular Ca 2+ concentrations.However, in contrast to vertebrates, mollusks employ a thick filament-linked regulatory system where myosin binds Ca 2+ 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) [7].Recently, we revealed the molecular characteristics of troponin C from the Japanese pearl oyster Pinctada fucata (Pifuc-TnC) [8] [9].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 [10].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 Ca 2+ [8].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 Ca 2+ only at site IV, is likely to have distinct functions from vertebrates [11].

Protein Preparation
We designed Pifuc-TnC-E142Q, a mutant variant lacking the ability to bind Ca 2+ 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 [12].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).

Gel Filtration Analysis
To compare the gel filtration chromatography elution profiles of Pifuc-TnC in the presence and absence of Ca 2+ , Pifuc-TnC (2 mg) was injected onto a Hi-Prep16/60 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 CaCl 2 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 Ca 2+ .

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 Ca 2+ (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 Ca 2+ were then compared.Equivalent procedures were used to analyze the UV-absorption spectra of Pifuc-TnC-E142Q in the presence and absence of Ca 2+ .

Differential Scanning Calorimetric Analysis
The thermostability of Pifuc-TnC in the presence or absence of Ca 2+ 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 Ca 2+ (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.

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.

Isothermal Calorimetric Analysis
Ca 2+ binding to Pifuc-TnC was confirmed by ITC analysis (Kd = 16.7 mM), whereas no Ca 2+ binding was detected for Pifuc-TnC-E142Q.This confirms that only the Pifuc-TnC site IV EF-hand motif is able to bind to Ca 2+ (Figure 3).Each injection of Ca 2+ 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 Ca 2+ to Pifuc-TnC was driven by entropy changes.

Gel Filtration Chromatography
In the presence of Ca 2+ , Pifuc-TnC eluted in fraction 30 from a HiPrep16/60 Se-

UV-Absorption Spectra
UV-absorption spectral analysis revealed that the binding of Ca 2+ 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 Ca 2+ .

Differential Scanning Calorimetry
We also examined if Ca 2+ binding affects Pifuc-TnC stability by monitoring its thermal unfolding using differential scanning calorimetry (DSC).The DSC thermogram of the Ca 2+ -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 Ca 2+ -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 Ca 2+ -bound and -unbound forms is likely to be a result of changes in protein folding, owing to a conformational change upon Ca 2+ binding.Unfortunately, no thermodynamic parameters were obtained from the DSC data.

Discussion
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 Ca 2+ only at its site IV EF-hand motif (Figure 3  Pifuc-TnC is predominantly distributed in phasic adductor muscle [8].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 Ca 2+ to myosin, which leads to its activation and subsequent interaction with actin.The detachment of Ca 2+ from myosin reverses the process and relaxes the muscle [17].
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 Ca 2+ -induced conformational

Figure 1 .
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 Ca 2+ binding.Only the site IV EF-hand motifs in Pifuc-TnC is able to bind Ca 2+ (Pifuc-TnC-E142Q is unable to bind Ca 2+ ).

D.
Funabara et al.DOI: 10.4236/ajmb.2018.84018209 American Journal of Molecular Biology phacryl S-200 high resolution gel filtration column, whereas it eluted earlier, in fraction 27, in the absence of Ca 2+ (Figure4(a)).In contrast, the elution profiles of the mutant variant, Pifuc-TnC-E142Q, which is unable to bind Ca 2+ , were equivalent in both the presence and absence of Ca 2+ (Figure4(b)).This indicates that Ca 2+ binding to Pifuc-TnC induces a conformational change that delays its elution from the column.

Figure 3 .Figure 4 .
Figure 3. Isothermal titration calorimetric analysis of Ca 2+ binding to Pifuc-TnC and Pifuc-TnC-E142C.Titration curve of interactions between Pifuc-TnC and Ca 2+ (a) and Pifuc-TnC-E142C and Ca 2+ (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 Ca 2+ .