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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.


Amino acid sequences of chicken CaM and its variants. The amino acids are represented by a one-letter code. Only EF4 is aligned in this figure. The red letters indicate the residues in the F-helix, and the blue regions represent Ca2+-binding loops.
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f1-7_35: Amino acid sequences of chicken CaM and its variants. The amino acids are represented by a one-letter code. Only EF4 is aligned in this figure. The red letters indicate the residues in the F-helix, and the blue regions represent Ca2+-binding loops.

Mentions: CaM is widely distributed in eukaryotes and its amino acid sequences are highly conserved; the only exception identified to date is Saccharomyces cerevisiae CaM (yCaM). The sequence homology between yCaM and the CaMs of other species is approximately 60%. The differences are concentrated in the EF4 site of yCaM: two residues are missing and 15 residues are substituted compared to EF4 of chicken CaM12–14. Because of such dissimilarity, the EF4 of yCaM does not bind Ca2+. However, it has been reported that the EF4 F-helix of yCaM is essential for target activation13– 16. Thus, it remains unclear how the C-terminal residues of CaM contribute to its structure and function. In this study, we investigated the roles of the C-terminal residues of CaM by using a series of deletion mutants of intact chicken CaM (CCM0); for example, CCMΔ4 is lacking the four C-terminal residues of CCM0 (Fig. 1). We measured their capacity to bind Ca2+ by mass spectrometry (MS) and flow-dialysis. Circular dichroism (CD) was employed to monitor the Ca2+-induced conformational change. In addition, we obtained various NMR data to discuss their structural characteristics in the absence and presence of Ca2+ and/or target. Their biological activities for calcineurin (CN) were also examined.


Roles of the C-terminal residues of calmodulin in structure and function
Amino acid sequences of chicken CaM and its variants. The amino acids are represented by a one-letter code. Only EF4 is aligned in this figure. The red letters indicate the residues in the F-helix, and the blue regions represent Ca2+-binding loops.
© Copyright Policy
Related In: Results  -  Collection

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

f1-7_35: Amino acid sequences of chicken CaM and its variants. The amino acids are represented by a one-letter code. Only EF4 is aligned in this figure. The red letters indicate the residues in the F-helix, and the blue regions represent Ca2+-binding loops.
Mentions: CaM is widely distributed in eukaryotes and its amino acid sequences are highly conserved; the only exception identified to date is Saccharomyces cerevisiae CaM (yCaM). The sequence homology between yCaM and the CaMs of other species is approximately 60%. The differences are concentrated in the EF4 site of yCaM: two residues are missing and 15 residues are substituted compared to EF4 of chicken CaM12–14. Because of such dissimilarity, the EF4 of yCaM does not bind Ca2+. However, it has been reported that the EF4 F-helix of yCaM is essential for target activation13– 16. Thus, it remains unclear how the C-terminal residues of CaM contribute to its structure and function. In this study, we investigated the roles of the C-terminal residues of CaM by using a series of deletion mutants of intact chicken CaM (CCM0); for example, CCMΔ4 is lacking the four C-terminal residues of CCM0 (Fig. 1). We measured their capacity to bind Ca2+ by mass spectrometry (MS) and flow-dialysis. Circular dichroism (CD) was employed to monitor the Ca2+-induced conformational change. In addition, we obtained various NMR data to discuss their structural characteristics in the absence and presence of Ca2+ and/or target. Their biological activities for calcineurin (CN) were also examined.

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.