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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.


Target activation by CaM and its variants. Results of the CN pNPPase assay (n=3 and 2 for CCM0 and variants, respectively). CCM0, CCMΔ4, CCMΔ5, CCMΔ6, CCMΔ7, and CCMΔ8. Detailed experimental procedures are described in Materials and Methods.
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f11-7_35: Target activation by CaM and its variants. Results of the CN pNPPase assay (n=3 and 2 for CCM0 and variants, respectively). CCM0, CCMΔ4, CCMΔ5, CCMΔ6, CCMΔ7, and CCMΔ8. Detailed experimental procedures are described in Materials and Methods.

Mentions: Finally, we measured the biological activity of intact CaM and its variants (Figure 11). In the biochemical experiments, we employed CN as a CaM target. CCMΔ4 and CCMΔ5 seemed to activate CN to the same extent as intact CaM. The apparent affinity for CN decreased with an increase in the deletion at the C-terminal. Interestingly, CCMΔ4 showed slightly lower activation compared with intact CaM, suggesting that M144 may have a specific role in CN activation. Even CCMΔ6 retained ability to activate CN. This is also an interesting finding, because it lacks the two Met residues, M144 and M145, contributing to the target binding7,8. In contrast, CCMΔ7 slightly activated CN, and CCMΔ8 lost the ability of target activation.


Roles of the C-terminal residues of calmodulin in structure and function
Target activation by CaM and its variants. Results of the CN pNPPase assay (n=3 and 2 for CCM0 and variants, respectively). CCM0, CCMΔ4, CCMΔ5, CCMΔ6, CCMΔ7, and CCMΔ8. Detailed experimental procedures are described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

f11-7_35: Target activation by CaM and its variants. Results of the CN pNPPase assay (n=3 and 2 for CCM0 and variants, respectively). CCM0, CCMΔ4, CCMΔ5, CCMΔ6, CCMΔ7, and CCMΔ8. Detailed experimental procedures are described in Materials and Methods.
Mentions: Finally, we measured the biological activity of intact CaM and its variants (Figure 11). In the biochemical experiments, we employed CN as a CaM target. CCMΔ4 and CCMΔ5 seemed to activate CN to the same extent as intact CaM. The apparent affinity for CN decreased with an increase in the deletion at the C-terminal. Interestingly, CCMΔ4 showed slightly lower activation compared with intact CaM, suggesting that M144 may have a specific role in CN activation. Even CCMΔ6 retained ability to activate CN. This is also an interesting finding, because it lacks the two Met residues, M144 and M145, contributing to the target binding7,8. In contrast, CCMΔ7 slightly activated CN, and CCMΔ8 lost the ability of target activation.

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.