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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-15N HSQC of uniformly 15N-labeled CaM and its variants in the absence of Ca2+. The NMR spectra for CCM0 (A), CCMΔ4 (B), and CCMΔ5 (C) were recorded at a 1H frequency of 750 MHz. For clarity, only selected residues are labeled with their residue name and number. The positions of the peaks that are absent in panel (B) have been labeled using boxes.
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f6-7_35: 1H-15N HSQC of uniformly 15N-labeled CaM and its variants in the absence of Ca2+. The NMR spectra for CCM0 (A), CCMΔ4 (B), and CCMΔ5 (C) were recorded at a 1H frequency of 750 MHz. For clarity, only selected residues are labeled with their residue name and number. The positions of the peaks that are absent in panel (B) have been labeled using boxes.

Mentions: For the next NMR study, we performed heteronuclear two-dimensional 1H-15N HSQC experiments to obtain detailed information on the global structures. The 1H-15N HSQC spectra of CaM and its variants in the absence of Ca2+ are shown in Figure 6. Several peaks observed in the NMR spectrum of apo-CCM0 disappeared within that of apo-CCMΔ4. The missing peaks included G98, A102, T117, D131, V136, and N137, all of which are located in the C-lobe. Although the deletion was performed at EF4, the fourresidue truncation induced a global conformational change in the whole C-lobe, that is, both EF3 and EF4 were affected. These results seem to be reasonable because EF3 and EF4 form a globular domain. In contrast, the peaks that resonated from the residues in the N-lobe of apo-CCMΔ4 appeared at chemical shift positions identical to those observed in the spectrum of apo-CCM0. The four-residue deletion at the C-terminus induces structural changes at the C-lobe, but not at the N-lobe. The NMR spectra of apo-CCMΔ4 and apo-CCMΔ5 were very similar, suggesting that their conformation is very similar.


Roles of the C-terminal residues of calmodulin in structure and function
1H-15N HSQC of uniformly 15N-labeled CaM and its variants in the absence of Ca2+. The NMR spectra for CCM0 (A), CCMΔ4 (B), and CCMΔ5 (C) were recorded at a 1H frequency of 750 MHz. For clarity, only selected residues are labeled with their residue name and number. The positions of the peaks that are absent in panel (B) have been labeled using boxes.
© Copyright Policy
Related In: Results  -  Collection

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

f6-7_35: 1H-15N HSQC of uniformly 15N-labeled CaM and its variants in the absence of Ca2+. The NMR spectra for CCM0 (A), CCMΔ4 (B), and CCMΔ5 (C) were recorded at a 1H frequency of 750 MHz. For clarity, only selected residues are labeled with their residue name and number. The positions of the peaks that are absent in panel (B) have been labeled using boxes.
Mentions: For the next NMR study, we performed heteronuclear two-dimensional 1H-15N HSQC experiments to obtain detailed information on the global structures. The 1H-15N HSQC spectra of CaM and its variants in the absence of Ca2+ are shown in Figure 6. Several peaks observed in the NMR spectrum of apo-CCM0 disappeared within that of apo-CCMΔ4. The missing peaks included G98, A102, T117, D131, V136, and N137, all of which are located in the C-lobe. Although the deletion was performed at EF4, the fourresidue truncation induced a global conformational change in the whole C-lobe, that is, both EF3 and EF4 were affected. These results seem to be reasonable because EF3 and EF4 form a globular domain. In contrast, the peaks that resonated from the residues in the N-lobe of apo-CCMΔ4 appeared at chemical shift positions identical to those observed in the spectrum of apo-CCM0. The four-residue deletion at the C-terminus induces structural changes at the C-lobe, but not at the N-lobe. The NMR spectra of apo-CCMΔ4 and apo-CCMΔ5 were very similar, suggesting that their conformation is very similar.

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