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

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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.


Downfield-shifted regions in 1H-15N HSQC of uniformly 15N-labeled CaM and its variants in the presence of Ca2+. CCM0 (A), CCMΔ4 (B), and CCMΔ5 (C).
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f8-7_35: Downfield-shifted regions in 1H-15N HSQC of uniformly 15N-labeled CaM and its variants in the presence of Ca2+. CCM0 (A), CCMΔ4 (B), and CCMΔ5 (C).

Mentions: The downfield-shifted I27, I100, and N137 peaks of Ca2+-bound CCMΔ5 became weak, broad, and/or duplicated (Figure 8). The results are completely dissimilar to those of CCM0 and CCMΔ4. The spectrum strongly suggests that Ca2+-bound CCMΔ5 is in conformational equilibrium, whereas Ca2+-bound CCMΔ4 has a relatively stable conformation.


Roles of the C-terminal residues of calmodulin in structure and function
Downfield-shifted regions in 1H-15N HSQC of uniformly 15N-labeled CaM and its variants in the presence of Ca2+. CCM0 (A), CCMΔ4 (B), and CCMΔ5 (C).
© Copyright Policy
Related In: Results  -  Collection

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

f8-7_35: Downfield-shifted regions in 1H-15N HSQC of uniformly 15N-labeled CaM and its variants in the presence of Ca2+. CCM0 (A), CCMΔ4 (B), and CCMΔ5 (C).
Mentions: The downfield-shifted I27, I100, and N137 peaks of Ca2+-bound CCMΔ5 became weak, broad, and/or duplicated (Figure 8). The results are completely dissimilar to those of CCM0 and CCMΔ4. The spectrum strongly suggests that Ca2+-bound CCMΔ5 is in conformational equilibrium, whereas Ca2+-bound CCMΔ4 has a relatively stable conformation.

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.