Dynamic Na+/H+ Exchanger 1 (NHE1):Calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation

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Abstract

Calmodulin (CaM) engages in Ca²⁺-dependent interactions with numerous proteins, including a still incompletely understood physical and functional interaction with the human Na⁺/H⁺-exchanger NHE1. Using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, and fibroblasts stably expressing wildtype and mutant NHE1, we discovered multiple accessible states of this functionally important complex existing in different NHE1:CaM stoichiometries and structures. We determined the NMR solution structure of a ternary complex in which CaM links two NHE1 cytosolic tails. In vitro, stoichiometries and affinities could be tuned by variations in NHE1:CaM ratio and calcium ([Ca²⁺]) and by phosphorylation of S648 in the first CaM-binding a-helix. In cells, Ca²⁺-CaM-induced NHE1 activity was reduced by mimicking S648 phosphorylation and by mutation of the first CaM-binding a-helix, whereas it was unaffected by inhibition of Akt, one of several kinases phosphorylating S648. Our results demonstrate a diversity of NHE1:CaM interaction modes and suggest that CaM may contribute to NHE1 dimerization and thereby augment NHE1 regulation. We propose that a similar structural diversity is of relevance to many other CaM complexes.

Data availability

Source data files are provided for Figure 1, 3, 5 and 6. Resonance assignments of the ternary complex of CaM and two H1 have been deposited in the Biological Magnetic Resonance Bank (BMRB) under ID code 34521. The atomic coordinates for the ternary complex of CaM and two H1 have been deposited in the Protein Data Bank under the ID code 6zbi.

The following data sets were generated

(2020) 1H, 13C and 15N chemical shift assignments of the 2:1 complex between NHE1 and calmodulin
Biological Magnetic Resonance Data Bank, 34521.

http://www.bmrb.wisc.edu/data_library/summary/index.php?bmrbId=34521
(2020) Ternary complex of Calmodulin bound to 2 molecules of NHE1
RCSB Protein Data Bank, 6ZBI.

http://www.rcsb.org/structure/6zbi

Article and author information

Author details

Lise M Sjøgaard-Frich

Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark

Competing interests
The authors declare that no competing interests exist.
Andreas Prestel

Structural Biology and NMR laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark

Competing interests
The authors declare that no competing interests exist.
Emilie S Pedersen

Structural Biology and NMR laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark

Competing interests
The authors declare that no competing interests exist.
Marc Severin

Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark

Competing interests
The authors declare that no competing interests exist.
Kristian Kølby Kristensen

Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark

Competing interests
The authors declare that no competing interests exist.
Johan G Olsen

Structural Biology and NMR laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark

Competing interests
The authors declare that no competing interests exist.
Birthe B Kragelund

Department of Biology, University of Copenhagen, Copenhagen, Denmark

For correspondence
bbk@bio.ku.dk

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-7454-1761
Stine Falsig Pedersen

Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark

For correspondence
sfpedersen@bio.ku.dk

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-3044-7714

Funding

Danish Research Councils (4181-00344)

Birthe B Kragelund

Novo Nordisk Fonden (NNF15OC0016670)

Birthe B Kragelund

Novo Nordisk Fonden (NNF18OC0034070)

Stine Falsig Pedersen

Novo Nordisk Fonden (NNF19OC0057241)

Stine Falsig Pedersen

Novo Nordisk Fonden (NNF18OC0032996)

Birthe B Kragelund

Villum Fonden

Birthe B Kragelund

Carlsbergfondet (CF20-0491)

Stine Falsig Pedersen

Novo Nordisk

Lise M Sjøgaard-Frich

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

Copyright

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.