Dynamic Na+/H+ Exchanger 1 (NHE1):Calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation
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.
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1H, 13C and 15N chemical shift assignments of the 2:1 complex between NHE1 and calmodulinBiological Magnetic Resonance Data Bank, 34521.
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Ternary complex of Calmodulin bound to 2 molecules of NHE1RCSB Protein Data Bank, 6ZBI.
Article and author information
Author details
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.
Reviewing Editor
- Lewis E Kay, University of Toronto, Canada
Version history
- Received: July 9, 2020
- Accepted: March 1, 2021
- Accepted Manuscript published: March 3, 2021 (version 1)
- Version of Record published: March 30, 2021 (version 2)
Copyright
© 2021, Sjøgaard-Frich et al.
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.
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