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Designing a Stapled Peptide Inhibitor of CHP‐NHE1 Protein‐Protein Interactions
Author(s) -
Bell Isaac,
Gufstason Jeffrey,
Provost Joseph
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.631.2
Subject(s) - chemistry , intracellular ph , extracellular , microbiology and biotechnology , gene isoform , biochemistry , biophysics , cytosol , biology , enzyme , gene
The Sodium Hydrogen Exchanger (NHE1) is a membrane transporter that exchanges an intracellular proton for an extracellular sodium ion. NHE1 plays a critical role interacting with, directing, and regulating proteins that drive cell motility. We have shown that NHE1 plays a pivotal role in pH homeostasis and migration of lung tumor cells. The Calcineurin homologous B protein isoforms 1 and 2 (CHP1/2) interact with NHE1 at the same 40aa domain (CHP binding domain (CBD)) both impacting NHE1 function. CHP1 is ubiquitously expressed and its interaction with NHE promotes basal activity. While CHP2 expression is limited to the gut in non‐diseased tissue, it is highly expressed in lung and other tumor cells. When CHP2 or NHE1 expression is diminished, tumor cell growth is nearly abrogated. This supports that CHP2‐NHE1 interactions have potential as an anticancer target. Models of both CHP proteins bound to NHE1 show each bind in the CBD of NHE but with subtle structural differences. To analyze the potential differences between CHP‐NHE binding, recombinant proteins were generated and used to determine the thermodynamics of CHP/NHE interaction through CD thermal melts. Recombinant CHP2 bound to NHE1 exhibited a lower free energy of binding (−1.08 kJ mol −1 ) compared to CHP1 (−0.48 kJ mol −1 ), a ΔΔG of 0.60 kJ mol −1 . Supporting this are in‐vitro ligand binding assays where we show a 2.23 fold greater affinity of CHP2‐NHE than CHP1‐NHE1. In order to create an effective peptide inhibitor, Large‐scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) computations were carried out using know crystal structures of each CHP isoform bound to NHE1. From these computations CHP1 was determined to have a two‐state binding interaction, whereas CHP2 had a one‐state binding interaction with NHE1. Unique initial binding contacts of NHE1 were determined for each CHP isoform. Stapled peptides mimicking the CHP‐NHE1 binding region were synthesized via a sulfenylation reaction with tryptophan amino acids. Peptides were then screened using a competitive fluorescent plate binding assay in which equal amounts of tagged CHP1/2 in the presence of NHE1. These peptide inhibitors will allow for increased CHP1‐NHE1 activity in targeted (cancer) cells while simultaneously reducing CHP2‐NHE1 activity. Support or Funding Information Supported in part by NSF Che 1664565 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .