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Roles of the C-terminal residues of calmodulin in structure and function

View Article: PubMed Central - PubMed

ABSTRACT

Electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, flow dialysis, and bioactivity measurements were employed to investigate the roles of the C-terminal residues of calmodulin (CaM). In the present study, we prepared a series of truncated mutants of chicken CaM that lack four (CCMΔ4) to eight (CCMΔ8) residues at the C-terminal end. It was found that CCMΔ4, lacking the last four residues (M145 to K148), binds four Ca2+ ions. Further deletion gradually decreased the ability to bind the fourth Ca2+ ion, and CCMΔ8 completely lost the ability. Interestingly, both lobes of Ca2+-sturated CCMΔ5 showed instability in the conformation, although limited part in the C-lobe of Ca2+-saturated CCMΔ4 was instable. Moreover, unlike CCMΔ4, structure of the C-lobe in CCMΔ5 bound to the target displayed dissimilarity to that of CaM, suggesting that deletion of M144 changes the binding manner. Deletion of the last five residues (M144 to K148) and further truncation of the C-terminal region decreased apparent capacity for target activation. Little contribution of the last four residues including M145 was observed for structural stability, Ca2+-binding, and target activation. Although both M144 and M145 have been recognized as key residues for the function, the present data suggest that M144 is a more important residue to attain Ca2+ induced conformational change and to form a proper Ca2+-saturated conformation.

No MeSH data available.


113Cd NMR of CaM and its variants. Panels (A) and (B), (C) and (D), and (E) and (F) represent the spectra for CCM0, CCMΔ4, and CCMΔ5, respectively. (A), (C), and (E) represent the protein spectra in the presence of Cd2+. (B), (D), and (F) represent the protein spectra in the presence of Cd2+ and skMLCKp. All NMR data were recorded at 113Cd resonance frequency of 110 MHz.
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f10-7_35: 113Cd NMR of CaM and its variants. Panels (A) and (B), (C) and (D), and (E) and (F) represent the spectra for CCM0, CCMΔ4, and CCMΔ5, respectively. (A), (C), and (E) represent the protein spectra in the presence of Cd2+. (B), (D), and (F) represent the protein spectra in the presence of Cd2+ and skMLCKp. All NMR data were recorded at 113Cd resonance frequency of 110 MHz.

Mentions: The results of the 113Cd-NMR experiments with CCM0, CCMΔ4, and CCMΔ5 are shown in Figure 10; data obtained in the absence of skMLCKp are shown on the left side. In the absence of the peptide, CCMΔ4 gave two peaks in the 113Cd-NMR spectrum, indicating that the C-lobe of CCMΔ4 can still bind two 113Cd ions; thus, CCMΔ4 binds four Ca2+ ions. This is consistent with the LC-MS results. However, the peak that appeared at higher field had some minor weak peaks at the bottom, suggesting the existence of conformational multiplicity in this EF-hand site. Unlike CCMΔ4, Cd2+-saturated CCMΔ5 gave only one peak in the 113Cd-NMR spectrum in the absence of the peptide (Figure 10E).


Roles of the C-terminal residues of calmodulin in structure and function
113Cd NMR of CaM and its variants. Panels (A) and (B), (C) and (D), and (E) and (F) represent the spectra for CCM0, CCMΔ4, and CCMΔ5, respectively. (A), (C), and (E) represent the protein spectra in the presence of Cd2+. (B), (D), and (F) represent the protein spectra in the presence of Cd2+ and skMLCKp. All NMR data were recorded at 113Cd resonance frequency of 110 MHz.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036782&req=5

f10-7_35: 113Cd NMR of CaM and its variants. Panels (A) and (B), (C) and (D), and (E) and (F) represent the spectra for CCM0, CCMΔ4, and CCMΔ5, respectively. (A), (C), and (E) represent the protein spectra in the presence of Cd2+. (B), (D), and (F) represent the protein spectra in the presence of Cd2+ and skMLCKp. All NMR data were recorded at 113Cd resonance frequency of 110 MHz.
Mentions: The results of the 113Cd-NMR experiments with CCM0, CCMΔ4, and CCMΔ5 are shown in Figure 10; data obtained in the absence of skMLCKp are shown on the left side. In the absence of the peptide, CCMΔ4 gave two peaks in the 113Cd-NMR spectrum, indicating that the C-lobe of CCMΔ4 can still bind two 113Cd ions; thus, CCMΔ4 binds four Ca2+ ions. This is consistent with the LC-MS results. However, the peak that appeared at higher field had some minor weak peaks at the bottom, suggesting the existence of conformational multiplicity in this EF-hand site. Unlike CCMΔ4, Cd2+-saturated CCMΔ5 gave only one peak in the 113Cd-NMR spectrum in the absence of the peptide (Figure 10E).

View Article: PubMed Central - PubMed

ABSTRACT

Electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, flow dialysis, and bioactivity measurements were employed to investigate the roles of the C-terminal residues of calmodulin (CaM). In the present study, we prepared a series of truncated mutants of chicken CaM that lack four (CCMΔ4) to eight (CCMΔ8) residues at the C-terminal end. It was found that CCMΔ4, lacking the last four residues (M145 to K148), binds four Ca2+ ions. Further deletion gradually decreased the ability to bind the fourth Ca2+ ion, and CCMΔ8 completely lost the ability. Interestingly, both lobes of Ca2+-sturated CCMΔ5 showed instability in the conformation, although limited part in the C-lobe of Ca2+-saturated CCMΔ4 was instable. Moreover, unlike CCMΔ4, structure of the C-lobe in CCMΔ5 bound to the target displayed dissimilarity to that of CaM, suggesting that deletion of M144 changes the binding manner. Deletion of the last five residues (M144 to K148) and further truncation of the C-terminal region decreased apparent capacity for target activation. Little contribution of the last four residues including M145 was observed for structural stability, Ca2+-binding, and target activation. Although both M144 and M145 have been recognized as key residues for the function, the present data suggest that M144 is a more important residue to attain Ca2+ induced conformational change and to form a proper Ca2+-saturated conformation.

No MeSH data available.