In vivo Crystals Reveal Critical Features of the Interaction Between Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and the PDZ2 Domain of Na +/H + Exchange Cofactor NHERF1

Eleanor Martin, Alessandro Barbieri, Robert Ford, Robert C Robinson

Research output: Contribution to journalArticlepeer-review

Abstract

Crystallization of recombinant proteins has been fundamental to our understanding of protein function, dysfunction and molecular recognition. However, this information has often been gleaned under extremely non-physiological protein, salt, and H+ concentrations. Here, we describe the development of a robust Inka1-Box (iBox)–PAK4cat system that spontaneously crystallizes in several mammalian cell types. The semi-quantitative assay described here allows the measurement of in-vivo protein­–protein interactions using a novel GFP-linked reporter system which produces fluorescent readouts from protein crystals. We combined this assay with in-vitro X-ray crystallography and molecular dynamics studies to characterize the molecular determinants of the interaction between PDZ2 domain of Na+/H+ exchange regulatory cofactor NHE-RF1 (NHERF1) and cystic fibrosis transmembrane conductance regulator (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis. These experiments revealed the crystal structure of the extended PDZ domain of NHERF1 and indicated, contrary to what has been previously reported, that residue selection at positions −1 and −3 of the PDZ-binding motif influences the affinity and specificity of the NHERF1 PDZ2–CFTR interaction. Our results suggest that this system could be utilized to screen additional protein–protein interactions, provided they can be accommodated within the spacious iBox-PAK4cat lattice.
Original languageEnglish
JournalJ. Biol. Chem.
Early online date2 Feb 2020
DOIs
Publication statusPublished - 2020

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