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 α-helix. In cells, Ca2+-CaM-induced NHE1 activity was reduced by mimicking S648 phosphorylation and by mutation of the first CaM-binding α-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.
Highlights
Calmodulin (CaM) is a ubiquitously expressed EF-hand Ca2+-binding hub protein, which regulates a plethora 44 of Ca2+-dependent cellular processes such as ion transport, muscle contraction, proliferation and apoptosis [5, 13, 73, 74, 82]
We suggest that the high degree of diversity observed for NHE1s in a 2:1 (NHE1):CaM complexes may be relevant to other CaM complexes, in particular those involving membrane proteins
(h)NHE1622-657 (H1), hNHE1651-692 (H2) and hNHE1622-692 (H1H2); all based on previous studies [34], 115 sequence conservation and helix propensity calculations (Figure 1a-b)
Summary
Calmodulin (CaM) is a ubiquitously expressed EF-hand Ca2+-binding hub protein, which regulates a plethora 44 of Ca2+-dependent cellular processes such as ion transport, muscle contraction, proliferation and apoptosis [5, 13, 73, 74, 82]. CaM is a small, -helical protein consisting of two similar but not identical, N- and C-. Upon changes in the free cytosolic Ca2+ concentration, [Ca2+]i, CaM binds up to 48 four Ca2+ ions. This induces structural rearrangements exposing hydrophobic patches through which CaM interacts with numerous structurally and functionally different proteins [70, 73]. In most cases where this has been studied to date, CaM wraps both its lobes around its -helix-forming targets, exemplified by the interaction with myosin light chain kinase [30, 43]. Common features of CaM binding regions, such as high -helix propensity, net positive charge, and obligate hydrophobic docking residues, exist [73]. CaM engages with many membrane proteins and examples include the CaM substrates estrogen receptor alfa (ERα) [39, 84], the voltage-gated K+ channel Kv10.1 (eag1) [62, 80], and aquaporin 0 [20]
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