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

Abstract

Abbreviations: CaM, calmodulin; CBD, CHP-binding region of sodium/proton exchanger 1 (residues 503 to 545); CHP, calcineurin B homologous protein; CV, column volumes; EFCaBPs, EF-hand Ca 2+ -binding proteins; FPH assay, fluorescent probe hydrophobicity assay; ITC, isothermal titration calorimetry; MBP, maltose-binding protein; MST, microscale thermophoresis; NHE1, sodium proton exchanger 1; PAGE, polyacrylamide gel electrophoresis. AbstractCalcineurin B homologous proteins (CHPs) belong to the EF-hand Ca 2+ -binding protein (EFCaBP) family. They have multiple important functions including the regulation of the Na + /H + exchanger 1 (NHE1). The human isoforms CHP1 and CHP2 share high sequence similarity, but have distinct expression profiles with CHP2 levels for instance increased in malignant cells. These CHPs bind Ca 2+ with high affinity.Biochemical data indicated that Ca 2+ can regulate their functions. Experimental evidence for Ca 2+ -modulated structural changes was lacking. With a newly established fluorescent probe hydrophobicity (FPH) assay, we detected Ca 2+ -induced conformational changes in both CHPs. These changes are in line with an opening of their hydrophobic pocket that binds the CHP-binding region (CBD) of NHE1. Whereas the pocket is closed in the absence of Ca 2+ in CHP2, it is still accessible for the dye in CHP1. Both CHPs interacted with CBD in the presence and absence of Ca 2+ .Isothermal titration calorimetry (ITC) analysis revealed high binding affinity for both CHPs to CBD with equilibrium dissociation constants (K D s) in the nanomolar range.The K D for CHP1:CBD was not affected by Ca 2+ , whereas Ca 2+ -depletion increased the K D 7-fold for CHP2:CBD showing a decreased affinity. The data indicate an isoform specific regulatory interaction of CHP1 and CHP2 with NHE1. K E Y W O R D Scalcineurin B homologous proteins, Ca 2+ signaling, EF-hand Ca 2+ -binding proteins, NHE1, proteinprotein interaction 3254 | LIANG et AL.

Calcineurin homologous protein isoform 2 supports tumor survival via the sodium hydrogen exchanger isoform 1 in non-small cell lung cancer

“…Yet unknown is the potential for other factors (outside of simple expression) to drive CHP2-NHE1 binding and activity. At physiological calcium concentrations, both CHP isoforms bind to NHE1 with nearly the same nanomolar affinities 45 similar to our work with recombinant proteins (data not shown). While quantitative comparisons CHP1 and CHP2 levels are sparse, a review of the protein abundance database (PAX-db.org) identifies that for a handful of non-diseased tissues the ratio of CHP1:CHP2 expression ranges from 6 to over 30 in the same tissue.…”

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

“…To analyse how these conformational dynamics affect CHP3 function, we measured affinities of CHP3 and mCHP3 for the target peptide CBD with microscale thermophoresis (MST) in Ca 2+ - and in Mg 2+ -bound states. We used the system established earlier for quantification of binding affinities of CHP isoforms (CHP1, CHP2, CHP3) to NHE1, in which the target peptide CBD was fused to maltose-binding protein (MBP-CBD) for its stabilization (15, 33). We conducted the MST experiments with three biological replicates per sample and five individual titration series (technical replicates) per biological replicate (Figure 5).…”

“…The conformational changes of the protein in response to ion binding were probed with the fluorescence probe hydrophobicity (FPH) assay (15) and by monitoring the intrinsic tryptophan fluorescence. The buffer of the protein solution was exchanged to the assay buffer (20 mM HEPES, 150 mM NaCl, 1 mM TCEP, 2 mM MgCl 2 , pH 7.2) containing 1 mM EGTA using Zeba TM spin desalting columns (Thermo Fisher Scientific Inc., Waltham, MA, USA).…”

“…Next, we analysed the effect of Ca 2+ binding in the presence of Mg 2+ and of the N-terminal myristoylation on CHP3 conformation. For this purpose, we optimized the FPH (fluorescence probe hydrophobicity) assay, previously developed to monitor Ca 2+ -induced conformational changes of CHPs (15), by using the dye ProteOrange (Lumiprobe) at defined micromolar concentration (see Materials and Methods). In this assay, the fluorescence of the dye strongly increases upon its binding to hydrophobic protein surfaces (39).…”

“…All three CHPs were shown to undergo conformational changes upon Ca 2+ binding (4, 15). Notably, CHP3 has a lower affinity for Ca 2+ (0.8 µM) (4) in comparison to CHP1 and CHP2 (K D values below 100 nM) (16, 17).…”