Figure 7. Conformation of CHP3 is controlled by Ca²⁺ and target peptide (NHE1 CBD) binding.

Ca²⁺ (magenta) replaces Mg²⁺ (green) in EF-3 and induces the transition from the closed to the open conformation. Ca²⁺ binding affects the local flexibility in the N-lobe accelerating the tryptic cleavage and reduces the thermal stability of CHP3. In both, Ca²⁺- and Mg²⁺-bound states, CHP3 binds the target peptide CBD with nanomolar affinity (K_D values indicated, in brackets for myristoylated CHP3). Complex formation has no effect on the thermal stability of the Mg²⁺-bound state (closed conformation), whereas it strongly enhances thermal stability of the Ca²⁺-bound state (open conformation). Independent of the bound ion, both trypsin cleavage sites are protected from proteolysis in the CHP3:CBD complex.