Introduction

Plastins are known to be a family of actin-binding proteins that are evolutionarily conserved from yeast to mammalian cells1. These proteins contain two Ca2+ binding sites, one calmodulin (CaM) binding domain, and two actin-binding domains (Fig. 1a). The two calmodulin-like Ca2+-binding domains in plastins, so-called EF-hand motifs2,3, suggested that Ca2+ could regulate actin-binding or other functions of plastins1. In human, three isoforms have been characterized: L-plastin, T-plastin and I-plasin1. Human L-plastin is the only isoform that possesses all the conserved amino acids essential for Ca2+-binding and bundles actin filaments in a strictly Ca2+-regulated manner. The bundling activity of I-plastin is inhibited by Ca2+ (ref.4). The sensitivity of T-plastin for Ca2+ is lower than that of L-plastin. In mice, two isoforms, L-plastin and T-plastin, have so far been identified5,6,7,8. L-plastin isoform is expressed in leukocytes of normal cells and in many types of cancer cells, whereas the T-plastin isoform is constitutively expressed in epithelial and mesenchymal cells of solid tissues. The two isoforms differ in 21% of amino acid sequences. The actin-bundling activity of plastins was demonstrated to be regulated by Ca2+ through N-terminal EF-hand Ca2+-binding domains. In our previous study, it was indicated that mice L- and T-plastin headpieces changed their structures in response to Ca2+, but that the sensitivity to Ca2+ was higher in the L-plastin headpiece compared to the T-plastin headpiece in analyses carried out using various spectroscopic methods, gel-filtration chromatography, and isothermal titration calorimetry9. These results suggest that L-plastin is suitable for dynamic rearrangement of cytoskeletons, while T-plastin is suitable for maintaining static cytoskeletons9.

Figure 1
figure 1

(a) Schematic structures of L- and T-plastins and (b) amino acid sequences for the two Ca2+-binding sites in the L- and T-plastin headpieces.

In the present study, Fourier transform infrared (FTIR) spectroscopy was employed to study the coordination structures of the divalent cation (M2+ = Mg2+ or Ca2+) bound in mice L- and T-plastin headpieces, each of which contains two EF-hand Ca2+-binding sites (Fig. 1b)9. The regions of COO antisymmetric and symmetric stretches provide information regarding the modes of coordination of a COO group to a metal ion10,11,12,13,14,15,16. The results showed that these headpieces have Ca2+ affinity in common with EF-hand calcium binding sites and a lower affinity for Mg2+. The amide-I profiles suggested that the T-plastin headpiece was more aggregable than the L-plastin headpiece. The implications of the FTIR spectral data for these plastin headpieces are discussed on the basis of the data obtained for the synthetic peptide analogs corresponding to a Ca2+-binding site.

Results and Discussion

FTIR spectra of L-plastin headpiece

Figure 2a shows the FTIR spectra for an L-plastin headpiece in the apo (M2+-free) and Mg2+ and Ca2+-loaded states in D2O solution in the wavenumber range of 1800–1300 cm−1. We observed the bands due to amide-I’, COO antisymmetric stretch, amide-II’, and COO symmetric stretch from the higher wavenumber side in Fig. 2a(A–C). A slight difference was detected in the region of COO antisymmetric stretching: a shoulder at approximately 1551 cm−1 appeared in the Ca2+-loaded state, although such a shoulder was not observed in the apo and Mg2+-loaded states.

Figure 2
figure 2

ATR-FTIR spectra for the L-plastin headpiece in the (A) apo, (B) Mg2+-loaded and (C) Ca2+-loaded states (a) in the D2O solutions and (b) in the H2O solutions.

Figure 2b also shows the FTIR spectra for the samples in H2O solution, where the contribution of the buffer has already been eliminated by subtracting the spectrum of the buffer, as mentioned in the Experimental section. The spectra indicated bands due to amide-I, amide-II, CH2 bending, and COO symmetric stretching from the higher wavenumber side. For the spectra obtained in H2O solution, the COO antisymmetric stretching mode is overlapped with the amide II mode, and therefore, we cannot extract the information about the COO antisymmetric stretch. However, the band at 1423 cm−1 was clearly observed only in the Ca2+-loaded state, which was thought to reflect the interaction of Ca2+ with the side-chain COO groups. The second-derivative spectra provide information in more detail regarding the spectral differences.

In Fig. 3, the second-derivative spectra corresponding to the data shown in Fig. 2 are shown. From the region of the COO antisymmetric stretch, information regarding the coordination modes of the COO groups to the metal ions such as bidentate or pseudo-bridging modes is obtained10,11,12,13,14,15,16,17. The bands at 1583 cm−1 and 1564 cm−1 in the apo as well as Mg2+-loaded states (Fig. 3a(A,B)) were very close to the band at 1585 cm−1 due to β-COO of Asp and the band at 1565 cm−1 due to γ-COO of Glu, respectively14,15,16. The band at 1612 cm−1 was slightly stronger in the Mg2+-loaded state in comparison with that in the apo state. We observed the bands at 1604, 1580, and 1551 cm−1 in the Ca2+-loaded state where the band at 1551 cm−1 was undoubtedly due to the sidechain COO group binding to Ca2+ in the bidentate coordination mode15,16. In the COO symmetric stretch region, two bands at 1423 and 1403 cm−1 were detected, although one band at 1402 cm−1 was found only for the apo state.

Figure 3
figure 3

Second-derivative spectra for the L-plastin headpiece in the (A) apo, (B) Mg2+-loaded and (C) Ca2+-loaded states (a) in the D2O solutions and (b) in the H2O solutions.

The COO symmetric stretching region for the L-plastin headpiece in H2O solution also provides information regarding the coordination modes of the COO groups to the metal ions despite other vibrational modes such as the CH2 bending mode contributing to this region15,16. The amplitude at 1425 cm−1, which corresponds to the band at 1423 cm−1 in Fig. 2b(C), was stronger in the Ca2+-loaded state than in the apo and Mg2+-loaded states, which undoubtedly reflects the coordination modes of the COO groups to the metal ions as pseudo-bridging and/or bidentate modes. It is noted that the band at 1587 cm−1 for the L-plastin headpiece in the Ca2+-loaded state in H2O solution may reflect the coordination modes of COO groups to Ca2+ in the pseudo-bridging mode because the corresponding band in D2O solution was stronger in the Ca2+-loaded state than in the apo state18.

We also refer to the amide-I region because the spectral profiles for the Ca2+-loaded state in D2O and H2O solution were, respectively, different from those obtained for the apo and Mg2+-loaded states. The main peak position for amide-I and amide-I’ was the same among the apo, Mg2+-loaded and Ca2+-loaded states, but the bandwidth at the 1652 cm−1 band for the Ca2+-loaded state was clearly narrower than for the apo and Mg2+-loaded states (Fig. 3b). This spectral difference may reflect a conformational difference such as α-helix formation induced by Ca2+ binding. The bands at approximately 1682 cm−1 and 1633 cm−1 are assigned to a β-sheet conformation according to the empirical assignment of proteins19,20. The band at 1633 cm−1 was thought to not be induced by Ca2+ binding because a shoulder at 1633 cm−1 was also observed in the apo and Mg2+-loaded states and because the amplitude at approximately 1682 cm−1 was observed to be constant for these states.

FTIR spectra for the T-plastin headpiece

Figure 4 depicts the second-derivative spectra for a T-plastin headpiece in the apo, Mg2+-loaded and Ca2+-loaded states in D2O and H2O solutions. Here, we left out the Attenuated total reflection (ATR) spectra for the T-plastin headpiece since the second-derivative spectra provide information regarding the coordination structure, as well as the conformational changes induced by M2+ in detail, as described in the FTIR section for the L-plastin headpiece. In the COO antisymmetric region, two bands at 1580 and 1564 cm−1 in the apo state, two bands at 1583 and 1563 cm−1 in the Mg2+-loaded state and three bands at 1582, 1564 and 1555 cm−1 in the Ca2+-loaded state were observed (Fig. 4a). The band at 1580 cm−1 in the apo state (Fig. 4a(A)) showed a 5 cm−1 downshift from the ionic Asp (1585 cm−1)14,15,16. The band at 1555 cm−1 in the Ca2+-loaded state (Fig. 4a(C)) was due to the side-chain COO groups binding to Ca2+ in the bidentate coordination mode15,16 but was 4-cm−1 higher than the corresponding band (1551 cm−1) for the L-plastin headpiece. In the region of the COO symmetric stretch, the bands at 1421 and 1400 cm−1 in D2O solution and the bands at approximately 1422 and 1401 cm−1 in H2O solution were observed.

Figure 4
figure 4

Second-derivative spectra for the T-plastin headpiece in the (A) apo, (B) Mg2+-loaded and (C) Ca2+-loaded states (a) in the D2O solutions and (b) in the H2O solutions.

The band at 1614 cm−1 was due to amide-I’ rather than the COO antisymmetric stretch (Fig. 4a) because the corresponding bands were observed at 1620 cm−1 in H2O solution (Fig. 4b), which moved together with the band at 1693 cm−1. The bands at 1693 and 1620 cm−1 were probably due to intermolecular interactions such as intermolecular β-strand since this protein seemed to easily aggregate with a spectral profile similar to that observed for a denatured protein19,20.

CD spectra for plastin headpieces

The effects of Mg2+- and Ca2+-binding in the headpieces were analyzed also by CD spectroscopy. The CD spectrum in each state showed two troughs around 208 and 222 nm (Fig. 5). For the L-plastin headpiece (Fig. 5(A)), both peaks at 208 nm and 222 nm in the CD spectra were shifted toward more negative values due to Ca2+-binding from apo and Mg2+-loaded states. On the other hand, the T-plastin headpiece showed a spectral change with an increasingly negative value only around the peak at 222 nm (Fig. 5(B)). These results suggest that the two headpieces are folded as a single polypeptide and are rich in α-helices. No change occurred due to the presence of Mg2+ on either of the headpieces, whereas some changes occurred due to Ca2+ -binding on both the peptides9. The secondary structural change induced by Ca2+-binding was greater in the L-plastin headpiece than in the T-plastin headpiece. Therefore, the increase in the α-helix content in the L-plastin headpiece due to Ca2+-binding should be larger than that in the T-plastin headpiece. This result was consistent with that data obtained using FTIR spectroscopy because these headpieces had a Ca2+ affinity in common with EF-hand calcium binding sites and less affinity for Mg2+; therefore, Mg2+ does not induce a conformational change in the headpieces.

Figure 5
figure 5

CD spectra for the headpiece from (A) L-plastin or (B) T-plastin in the apo (−), Mg2+(−), and Ca2+(······) states.

FTIR spectra for the synthetic peptide analogs of the Ca2+-binding sites

Figure 6 depicts the second-derivative spectra for 17-residue synthetic peptide analogs of the Ca2+-binding sites I and II of the L- and T-plastins in the wet film under D2O vapor atmosphere because the absorbance of the peptide in solution was too weak to analyze the amide I’ and COO stretching bands in detail. The bands at 1549 and 1553 cm−1 were detected for the peptide analogs corresponding to the site I and II of L-plastin in the Ca2+-loaded state, respectively (Fig. 6a(B,D)). Therefore, we confirmed that the COO of Glu at the 12th position is bound to Ca2+ in the bidentate coordination mode15,16. Meanwhile, the spectral profiles for the peptide analogs corresponding to the site I and II of T-plastin were quite different from those obtained for the T-plastin headpiece. Bands at 1561 and 1565 cm−1 were detected for the peptide analogs corresponding to sites I and II of T-plastin, respectively (Fig. 6b(B,D)), while a band at 1555 cm−1 was observed for the T-plastin headpiece in the Ca2+-loaded state (Fig. 4a(C)). The bands at 1681 and 1625 cm−1 in the amide I’ region (Fig. 6b(B)) suggested that the peptide analogs aggregated in the Ca2+-loaded state and that this aggregation disturbed the affinity of them for Ca2+. The same profile was also observed for the peptide analog corresponding to site II of T-plastin in the apo state (Fig. 6b(C)). Bands at 1681 and 1625 cm−1 were not observed for the Ca2+-loaded state in Fig. 6b(D), which suggested that Ca2+ reduced the aggregation of the peptide. We attempted to reduce the aggregation of site II of T-plastin by substitution of amino acid residue. At the present stage, we were not able to obtain information regarding the Ca2+-coordination structure for site II of T-plastin. However, as for site I of plastin T, we found that the mutant 17-residue peptide (C9K), where the cysteine was substituted for lysine at the 9th position, did not aggregate. The FTIR spectra for the C9K peptide showed a band at 1560 cm−1 in the Ca2+-loaded state (data not shown), suggesting that the COO group of Glu at the 12th position is bound to Ca2+ in the mode of pseudo-bridging coordination rather than in the mode of bidentate coordination.

Figure 6
figure 6

Second-derivative spectra for synthetic peptide analogs of (A,B) the Ca2+-binding site I and (C,D) the Ca2+-binding II of (a) L- plastin and (b) T-plastin. (A) and (C) are in the apo state and (B) and (D) are in the Ca2+-loaded state.

Conclusions

The results obtained using the synthetic peptide analogs suggested that the lower sensitivity to Ca2+ in the T-plastin headpiece may be related to the susceptibility to aggregation for the two Ca2+-binding sites. ATR-FTIR spectroscopy in combination with the use of a synthetic peptide analog approach is promising for understanding the correlation of Ca2+-binding coordination and the aggregation of Ca2+ binding proteins.

Materials and Methods

Sample preparation for plastin headpieces

Hexahistidine (His6)-tagged L- and T-plastin headpieces (L-plastin Δ1‒100 and T-plastin Δ1‒103) were expressed using a modified pET28a vector harboring their DNA sequences and Escherichia coli BL21(DE3) according to a previous report9. The expressed proteins were purified using Ni-NTA agarose (Qiagen). After cleaving the His6-tag on the resin with AcTEV protease, the eluted plastin headpieces were treated with trichloroacetic acid to remove contaminating Ca2+ ions21. Further purification was performed by anion-exchange chromatography and size-exclusion chromatography with Resource-Q and Superdex 75 columns (GE Healthcare), respectively.

Sample preparation for the synthetic peptide analogs

We synthesized 17-residue peptide analogs corresponding to the two Ca2+-binding sites in the L- and T-plastins, as listed in Table 1, because the 17-residue peptide analogs for loop-helix F are the minimum number required for the Ca2+-binding property for site III of troponin C and site IV of akazara scallop troponin C22,23. The peptides were synthesized by the solid-phase method based on the Fmoc strategy17,22,23. A Fmoc-NH-SAL-PEG resin (Watanabe Chem.) solid-phase support containing the 4-[(2’,4’-dimethoxyphenyl) N-Fmoc-aminomethyl]phenoxyacetamido group, named Rink-amide linker24, was used to provide the peptides with C-terminal amide. The peptide chain was constructed in a stepwise manner for respective amino acid residues. The coupling reaction of a side-chain protected Fmoc-amino acid was carried out with an equimolar reagent system of HBTU-HOBt in DMF containing a double equivalence of N-methyl morpholine. In every synthetic cycle, Fmoc-protecting groups of the elongating peptide chains were deblocked with mixed reagents of piperidine-DBU-HOBt, with concentrations of 8%(v/v), 2%(v/v), and 2.5%(w/v) in DMF, respectively. The additive HOBt was used to suppress the side reaction of the aspartic residue caused by piperidine25. After completion of elongation, the peptides were harvested by cleavage reaction with trifluoroacetic acid (TFA) containing EDT (4%), TIPS (6%), and water (2%). The crude peptides were dissolved in a LiCl (4%) solution of DMF and purified by reverse-phase HPLC. The molecular weights of the peptides were confirmed by MALDI-TOFMS with AXIMA (Shimadzu). The TFA carried-over from HPLC purification was completely removed with a size-exclusion column, PD-10 (GE Healthcare), in 0.1 M ammonium bicarbonate buffer solution (pH 8.5) containing 0.1 M KCl, because TFA causes disruptive overlapping of the FTIR signals22,23. Finally, the collected fractions of peptides were desalted with PD-10 in pure water.

Table 1 The amino acid residue for the Ca2+-binding site for T- and L-plastins.

FTIR measurements

Most of the experiments described for FTIR measuremrnts were performed in the same manner as in our previous works17,26,27. ATR-FTIR measurements were carried out for the L- and T- plastin headpieces at room temperature using a Perkin-Elmer Spectrum-One Fourier transform infrared spectrometer equipped with an ATR unit and an MCT detector with a resolution of 2 cm−127. Interferograms from 200 scans were averaged for the series of measurements for L-plastin. On the other hand, interferograms from 500 scans were averaged for the series of measurements for the T-plastin headpiece since the sample concentration was lower (approximately half of the L-plastin headpiece concentration) due to partial aggregation17. Dry air gas was constantly pumped into the ATR unit of the spectrometer to suppress water vapor17. Approximately 10 μl of a sample solution was placed onto a Diamond/ZnSe 1-reflection top–plate (Perkin-Elmer). ATR-FTIR spectra for the solvents (buffer solutions) were measured in the same way. The treatment and analyses of the ATR-FTIR spectra have been described previously17,27. For the synthetic peptide analogs for the calcium binding sites of L- and T-plastins, ATR-FTIR measurements were also carried out for samples in a wet film under D2O vapor atmosphere to determine the absorbance intensity of the amide I’ and COO stretching modes17.

CD measurements

CD spectra were measured using a spectrometer J-720 (Jasco) at room temperature. The acquisition parameters were as follows: resolution, 0.2 nm; speed, 50 nm/min; response time, 2 s; bandwidth, 1 nm; and scan, 10. The 0.02 mM protein solution was prepared in 10 mM MOPS-KOH (pH 6.8), 100 mM KCl, and 0.05 mM EDTA for the M2+-free state and the same composition containing 2 mM MCl2, and for the M2+-loaded state. Each spectrum was subtracted with that from the buffer.