Computational Analysis of CHP‐NHE Protein‐Protein Interaction

The Sodium Hydrogen Exchanger (NHE1) is an ATP independent membrane transporter that exchanges an intracellular proton for an extracellular sodium ion. In addition to maintaining intracellular pH homeostasis, NHE1 plays a critical role interacting with proteins on the leading edge of migrating proteins controlling directed cell motility. Regulation of NHE1 is complex. Phosphorylation, lipid binding and protein‐protein interactions all take place at the 300 amino acid carboxyl terminal regulating NHE1 protein exchange and protein interactions. Two NHE1 binding proteins whose roles are not fully clear are the calcineurin homologous protein isoforms 1 and 2 (CHP1 and CHP2). While both CHP1 and CHP2 bind to nearly the same domain of NHE1 (CHP Binding Domain amino acids 503–545; NHE1‐CBD), each has a unique effect on NHE1 function. Because CHP2 is primarily expressed in gut and transformed tumor cells, the interaction between CHP and NHE is a potential anticancer target. In order to quantify the interaction between the CHP1‐NHE and the CHP2‐NHE we created GST fusion protein of NHE1‐CBD with a thrombin cleavage site between fusion proteins as well as 6XHis‐CHP1 and 6XHis‐CHP2. Each protein was expressed and purified from bacterial culture. GST‐NHE1‐CBD was cleaved from its fusion partner on the column. After purification each protein was dialyzed in 10mM K‐ phosphate, 100mM NaCl, and 0.5mM Ca 2+ , pH of 7.4 Circular Dichroism (CD) wavelength scan of each protein was determined to identify the helical nature of the protein; CHP1 with 51.60% helical structure, CHP2 with a 49.74% helical structure, and NHE1 with a 40.91% helical structure in the CBD. Thermal folding of each protein alone or in combination was determined following CD as a function of temperature. Melting point (Tm) for CHP1 (56.33+/−0.72 °C), and NHE1‐CBD (59.43+/−0.62 °C) alone increased to 59.73 +/−0.21 °C when the two proteins were combined. We determine the van't hoff enthalpy and entropy of unfolding to estimate the binding constant of protein interaction for CHP1‐NHE1 and CHP2‐NHE1. From the experimental data collected, the theoretical temperature at which each construct denatures; for both the CHP2‐NHE construct and CHP1‐NHE construct were inputed into a Large‐scale Atomic/Molecular Massively Parallel Simulator (LAMMPS). Results from the LAMMPS analysis were then inputted into a Weighted Histogram Analysis Method (WHAM) in order to generate specific base‐pair interactions in the binding and unbinding in each of the two CHP isoforms and NHE. WHAM analysis also provides a theoretical depiction of the intermediate steps of binding, helping to determine if the process is one‐stage or a step‐wise process. This data along with pdb files created at set intervals during the LAMMPS simulation will be used to create a visual representation of the binding of the different constructs. This work will help predict the binding mechanism of CHP‐NHE1 interaction to identify potential therapeutic targets blocking CHP2 but not CHP1 from NHE1.