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Unique Structural Changes in Calcium-Bound Calmodulin Upon Interaction with Protein 4.1R FERM Domain: Novel Insights into the Calcium-dependent Regulation of 4.1R FERM Domain Binding to Membrane Proteins by Calmodulin

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Abstract

Calmodulin (CaM) binds to the FERM domain of 80 kDa erythrocyte protein 4.1R (R30) independently of Ca2+ but, paradoxically, regulates R30 binding to transmembrane proteins in a Ca2+-dependent manner. We have previously mapped a Ca2+-independent CaM-binding site, pep11 (A264KKLWKVCVEHHTFFR), in 4.1R FERM domain and demonstrated that CaM, when saturated by Ca2+ (Ca2+/CaM), interacts simultaneously with pep11 and with Ser185 in A181KKLSMYGVDLHKAKD (pep9), the binding affinity of Ca2+/CaM for pep9 increasing dramatically in the presence of pep11. Based on these findings, we hypothesized that pep11 induced key conformational changes in the Ca2+/CaM complex. By differential scanning calorimetry analysis, we established that the C-lobe of CaM was more stable when bound to pep11 either in the presence or absence of Ca2+. Using nuclear magnetic resonance spectroscopy, we identified 8 residues in the N-lobe and 14 residues in the C-lobe of pep11 involved in interaction with CaM in both of presence and absence of Ca2+. Lastly, Kratky plots, generated by small-angle X-ray scattering analysis, indicated that the pep11/Ca2+/CaM complex adopted a relaxed globular shape. We propose that these unique properties may account in part for the previously described Ca2+/CaM-dependent regulation of R30 binding to membrane proteins.

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Abbreviations

4.1R:

Protein 4.1R

CaM:

Calmodulin

CD:

Circular dichroism spectroscopy

DSC:

Differential scanning calorimetry

FERM:

Four.one–ezrin–radixin–moesin

NMR:

Nuclear magnetic resonance

SAXS:

Small-angle X-ray scattering

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Acknowledgments

The authors thank Dr. Philippe Gascard, Department of Pathology, University of California, San Francisco, USA, for critical reading and editing of the manuscript. The authors thank also Professors Norio Matsushima, Division of Physics, Center for Medical Education, Sapporo Medical University (Hokkaido, Japan), and Yoshinobu Izumi, Yamagata University (Yamagata, Japan), for their useful discussion of the SAXS data. Lastly, the authors thank Dr. Yasushi Sakaguchi, DKSH Management Ltd./DKSH Holding Ltd. (Tokyo, Japan) for his technical assistance with DSC measurements. This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education Culture, Sport, Science and Technology of Japan (KAKENHI) 15570123 to WN.

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Authors and Affiliations

  1. Center for Geo-Environmental Science, Graduate School of Engineering and Resource Science, Akita University, Tegata-Gakuên 1-1, Akita, 010-8502, Japan

    Wataru Nunomura

  2. Department of Life Science, Graduate School of Engineering and Resource Science, Akita University, Tegata-Gakuên 1-1, Akita, 010-8502, Japan

    Wataru Nunomura & Hideki Wakui

  3. Center for Nano Materials and Technology, Japan Advanced Institute of Science and Technology, Asahidai 1-1, Nōmi, Ishikawa, 923-1292, Japan

    Noriyoshi Isozumi & Shinya Ohki

  4. Department of Bioengineering, Nagaôka University of Technology, Kamitomiôka 1603-1, Nagaôka, Niigata, 940-2188, Japan

    Shigeyoshi Nakamura & Shun-ichi Kidokoro

  5. Graduate School of Science and Engineering, Yamagata University, Jyōnan 4-3-16, Yonezawa, Yamagata, 992-8510, Japan

    Yuji Jinbo

  6. Department of Biochemistry, Tokyo Women’s Medical University, Kawada 8-1, Shinjuku, Tokyo, 162-8666, Japan

    Yuichi Takakuwa

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  1. Wataru Nunomura
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  2. Noriyoshi Isozumi
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Correspondence to Wataru Nunomura.

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Nunomura, W., Isozumi, N., Nakamura, S. et al. Unique Structural Changes in Calcium-Bound Calmodulin Upon Interaction with Protein 4.1R FERM Domain: Novel Insights into the Calcium-dependent Regulation of 4.1R FERM Domain Binding to Membrane Proteins by Calmodulin. Cell Biochem Biophys 69, 7–19 (2014). https://doi.org/10.1007/s12013-013-9758-6

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