Conformational regulation and target-myristoyl switch of calcineurin B homologous protein 3

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Abstract

Calcineurin B homologous protein 3 (CHP3) is an EF-hand Ca²⁺-binding protein involved in regulation of cancerogenesis, cardiac hypertrophy and neuronal development through interactions with sodium/proton exchangers (NHEs) and signalling proteins. While the importance of Ca²⁺ binding and myristoylation for CHP3 function has been recognized, the underlying molecular mechanism remained elusive. In this study, we demonstrate that Ca²⁺ binding and myristoylation independently affect the conformation and functions of human CHP3. Ca²⁺ binding increased local flexibility and hydrophobicity of CHP3 indicative of an open conformation. The Ca²⁺-bound CHP3 exhibited a higher affinity for NHE1 and associated stronger with lipid membranes compared to the Mg²⁺-bound CHP3, which adopted a closed conformation. Myristoylation enhanced the local flexibility of CHP3 and decreased its affinity to NHE1 independently of the bound ion, but did not affect its binding to lipid membranes. The data exclude the proposed Ca²⁺-myristoyl switch for CHP3. Instead, a Ca²⁺-independent exposure of the myristoyl moiety is induced by binding of the target peptide to CHP3 enhancing its association to lipid membranes. We name this novel regulatory mechanism 'target-myristoyl switch'. Collectively, the interplay of Ca²⁺ binding, myristoylation, and target binding allows for a context-specific regulation of CHP3 functions.

Data availability

All data generated or analyzed during this study are included in the manuscript. Source files have been provided for Figures 1, 4, 6, and Figure 4-figure supplement 1. Data2Dynamics Software used for EC50 and KD determination is available at GitHub (https://github.com/Data2Dynamics/d2d).

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Florian Becker

Institute for Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-5889-8728
Simon Fuchs

Institute for Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany

Competing interests
No competing interests declared.
Lukas Refisch

Institute of Medical Biometry and Statistics, University of Freiburg, Freiburg, Germany

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-9049-1944
Friedel Drepper

Institute of Biology II, University of Freiburg, Freiburg, Germany

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-2043-5795
Wolfgang Bildl

Institute of Physiology, University of Freiburg, Freiburg, Germany

Competing interests
No competing interests declared.
Uwe Schulte

Institute of Physiology, University of Freiburg, Freiburg, Germany

Competing interests
Uwe Schulte, is an employee and shareholder of Logopharm GmbH that produces ComplexioLyte 47 used in this study..

"This ORCID iD identifies the author of this article:" 0000-0003-3557-0591
Shuo Liang

Institute for Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany

Competing interests
No competing interests declared.
Jonas Immanuel Heinicke

Institute for Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany

Competing interests
No competing interests declared.
Sierra C Hansen

Institute for Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany

Competing interests
No competing interests declared.
Clemens Kreutz

Institute of Medical Biometry and Statistics, University of Freiburg, Freiburg, Germany

Competing interests
No competing interests declared.
Bettina Warscheid

Institute of Biology II, University of Freiburg, Freiburg, Germany

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0001-5096-1975
Bernd Fakler

Institute of Physiology, University of Freiburg, Freiburg, Germany

Competing interests
Bernd Fakler, is shareholder of Logopharm GmbH. The company provides ComplexioLyte reagents to academic institutions on a non-profit basis..
Evgeny V Mymrikov

Institute of Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany

For correspondence
evgeny.mymrikov@biochemie.uni-freiburg.de

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0003-4817-6278
Carola Hunte

Institute for Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany

For correspondence
carola.hunte@biochemie.uni-freiburg.de

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-0826-3986

Funding

Deutsche Forschungsgemeinschaft (EXC-2189 - 390939984)

Clemens Kreutz
Bettina Warscheid
Bernd Fakler
Evgeny V Mymrikov
Carola Hunte

Deutsche Forschungsgemeinschaft (SFB 1381 - 403222702)

Uwe Schulte
Bettina Warscheid
Carola Hunte

Deutsche Forschungsgemeinschaft (SFB 1453 - 431984000)

Bernd Fakler
Carola Hunte

Deutsche Forschungsgemeinschaft (RTG 2202 - 278002225)

Carola Hunte

Bundesministerium für Bildung und Forschung (FKZ031L0080)

Clemens Kreutz

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.