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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.

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1H-13C HSQC of selectively labeled-[13C]-methyl-Met CaM and its variants. The panels (A)–(C), (D)–(F), and (G)–(I) represent the spectra for CCM0, CCMΔ4, and CCMΔ5, respectively. Panels (A), (D), and (G) represent the protein spectra in the absence of Ca2+. (B), (E), and (H) are the protein spectra in the presence of Ca2+. (C), (F), and (I) are the spectra of proteins complexed with the MLCK peptide. The assignments shown in panels (D)–(I) are similar to those in panels (A)–(C). All NMR data were recorded at 1H frequency of 800 MHz.
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f9-7_35: 1H-13C HSQC of selectively labeled-[13C]-methyl-Met CaM and its variants. The panels (A)–(C), (D)–(F), and (G)–(I) represent the spectra for CCM0, CCMΔ4, and CCMΔ5, respectively. Panels (A), (D), and (G) represent the protein spectra in the absence of Ca2+. (B), (E), and (H) are the protein spectra in the presence of Ca2+. (C), (F), and (I) are the spectra of proteins complexed with the MLCK peptide. The assignments shown in panels (D)–(I) are similar to those in panels (A)–(C). All NMR data were recorded at 1H frequency of 800 MHz.

Mentions: The 1H-13C HSQC results for CCM0, CCMΔ4, and CCMΔ5 are shown in Figure 9. All the resonance assignments for variants displayed in panels D–I are according to a comparison with the spectra for CCM0. The results for the Ca2+-free forms are shown in panels A, D, and G. It is clear that the peaks that resonated from the residues in Ca2+-free N-lobe were not affected by the truncation of four or five residues at the C-terminus, indicating that this deletion does not affect the structure of the N-lobe in the absence of Ca2+. These results are consistent with those of the 1H-15N HSQC. Unlike the Met residues in the N-lobe, the peaks of M109, M124, and M144 in the C-lobe of CCMΔ4 and CCMΔ5 were shifted by the truncation. Thus, the conformation of the Ca2+-free C-lobe of the variants is different from that of CCM0. These findings are also consistent with the above-mentioned results for 1H-15N HSQC.


Roles of the C-terminal residues of calmodulin in structure and function
1H-13C HSQC of selectively labeled-[13C]-methyl-Met CaM and its variants. The panels (A)–(C), (D)–(F), and (G)–(I) represent the spectra for CCM0, CCMΔ4, and CCMΔ5, respectively. Panels (A), (D), and (G) represent the protein spectra in the absence of Ca2+. (B), (E), and (H) are the protein spectra in the presence of Ca2+. (C), (F), and (I) are the spectra of proteins complexed with the MLCK peptide. The assignments shown in panels (D)–(I) are similar to those in panels (A)–(C). All NMR data were recorded at 1H frequency of 800 MHz.
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Related In: Results  -  Collection

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

f9-7_35: 1H-13C HSQC of selectively labeled-[13C]-methyl-Met CaM and its variants. The panels (A)–(C), (D)–(F), and (G)–(I) represent the spectra for CCM0, CCMΔ4, and CCMΔ5, respectively. Panels (A), (D), and (G) represent the protein spectra in the absence of Ca2+. (B), (E), and (H) are the protein spectra in the presence of Ca2+. (C), (F), and (I) are the spectra of proteins complexed with the MLCK peptide. The assignments shown in panels (D)–(I) are similar to those in panels (A)–(C). All NMR data were recorded at 1H frequency of 800 MHz.
Mentions: The 1H-13C HSQC results for CCM0, CCMΔ4, and CCMΔ5 are shown in Figure 9. All the resonance assignments for variants displayed in panels D–I are according to a comparison with the spectra for CCM0. The results for the Ca2+-free forms are shown in panels A, D, and G. It is clear that the peaks that resonated from the residues in Ca2+-free N-lobe were not affected by the truncation of four or five residues at the C-terminus, indicating that this deletion does not affect the structure of the N-lobe in the absence of Ca2+. These results are consistent with those of the 1H-15N HSQC. Unlike the Met residues in the N-lobe, the peaks of M109, M124, and M144 in the C-lobe of CCMΔ4 and CCMΔ5 were shifted by the truncation. Thus, the conformation of the Ca2+-free C-lobe of the variants is different from that of CCM0. These findings are also consistent with the above-mentioned results for 1H-15N HSQC.

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.


Related in: MedlinePlus