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

Abstract

Calcineurin B homologous proteins (CHPs) belong to the EF-hand Ca²⁺ -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 Ca²⁺ with high affinity. Biochemical data indicated that Ca²⁺ can regulate their functions. Experimental evidence for Ca²⁺ -modulated structural changes was lacking. With a newly established fluorescent probe hydrophobicity (FPH) assay, we detected Ca²⁺ -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 Ca²⁺ in CHP2, it is still accessible for the dye in CHP1. Both CHPs interacted with CBD in the presence and absence of Ca²⁺ . 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 Ca²⁺ , whereas Ca²⁺ -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.

Keywords: Ca2+ signaling; EF-hand Ca2+-binding proteins; NHE1; calcineurin B homologous proteins; protein-protein interaction.