Calcineurin B homologous protein 3 binds with high affinity to the CHP binding domain of the human sodium/proton exchanger NHE1

Abstract

The Na+/H+ exchanger NHE1 is critical for cell vitality as it controls intracellular pH and cell volume. Its functionality is influenced by calcineurin B homologous proteins (CHPs). The human isoform CHP3 is important for transport of NHE1 to the plasma membrane and for its activity. Here, we characterized the binding interaction of human CHP3 with the regulatory domain of NHE1. The exact binding site of CHP3 was previously debated. CHP3 as well as both regions of NHE1 in question were produced and purified. CHP3 specifically formed stable complexes with the CHP-binding region (CBD) of NHE1 (residues 503–545) in size-exclusion chromatography (SEC), but not with the C-terminal region (CTD, residues 633–815). CTD was functional as shown by Ca2+-dependent binding of calmodulin in SEC analysis. CHP3 bound with high affinity to CBD with an equilibrium dissociation constant (KD) of 56 nM determined by microscale thermophoresis. The high affinity was substantiated by isothermal calorimetry analysis (KD = 3 nM), which also revealed that the interaction with CBD is strongly exothermic (ΔG° = −48.6 kJ/mol, ΔH = −75.3 kJ/mol, −TΔS° = 26.7 kJ/mol). The data provide insights in the molecular mechanisms that underlie the regulatory interaction of CHP3 and NHE1 and more general of calcineurin homologous proteins with their target proteins.

Calcium affects CHP1 and CHP2 conformation and their interaction with sodium/proton exchanger 1

“…The ITC measurements were performed in the presence and absence of Ca 2+ with three independently produced and purified biological samples per protein (Figure ). The triple‐shift procedure was applied to correct for the concentration of active CHP1, CHP2, and CBD (Supplemental Tables S3‐S6) as described in the previous study . Subsequently, global nonlinear regression of the data points using an one set of sites model was performed and the confidence of the fit was calculated with error support plane analysis (Supplemental Figures S6‐S9 and Supplemental Tables S3‐S6).…”

“…Production and purification of human CaM and maltose-binding protein (MBP)-CBD was performed essentially as described earlier. 24 The production and purification of CHP3 was performed with HIC and gel filtration as described for other EFCaBPs. 25 The protein concentration was determined spectroscopically with NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA) or with the Cary 4000 UV-vis spectrophotometer for the microscale thermophoresis (MST) and ITC measurements (Agilent technologies, Mulgrave, Australia) using calculated extinction coefficients…”

“…The optimized experimental conditions were then used for the thermodynamic characterization by ITC as recently described. 24 Three biological replicates with data points averaged for five technical replicates were measured for Ca 2+ -free CHP1 and Ca 2+ -bound CHP2 (Figure 4). The T-jump analysis was chosen for the calculation of the binding isotherms.…”

“…Protein concentration for CHP1, CHP2, and MBP-CBD and efficiency of labeling were determined as described previously. 24 The extinction coefficient of ɛ 650 = 250 000 M −1 cm −1 was used for the dye NT647. A correction factor of 0.028 was used to correct the additional dye absorption at 280 nm.…”

“…22,23 All CHPs bind to the same amphipathic helix in the regulatory C-terminal domain of NHE1 (residues 503-545, designated as CBD or CHP-binding region). 13,24 Conceptually, CHPs could compete for binding to CBD when simultaneously expressed in the same cell. CHP1 and CHP3 were both shown to be present in mouse Purkinje cells.…”