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Martin, Eleanor R. School of Biological Sciences, Faculty of Biology Medicine and Health, Michael Smith Building, The University of Manchester
Barbieri, Alessandro School of Biological Sciences, Faculty of Biology Medicine and Health, Michael Smith Building, The University of Manchester
Ford, Robert C. School of Biological Sciences, Faculty of Biology Medicine and Health, Michael Smith Building, The University of Manchester
Robinson, Robert C. Research Institute for Interdisciplinary Science, Okayama University ORCID Kaken ID researchmap
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 nonphysiological 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 that 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 the 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.
Keywords
PDZ domain
X-ray crystallography
molecular modeling
protein complex
protein crystallization
crystal structure
cystic fibrosis transmembrane conductance regulator (CFTR)
cystic fibrosis
ion channel
protein-protein interaction
SLC9A3 regulator 1 (SLC9A3R1)
Published Date
2020-04-03
Publication Title
Journal of Biological Chemistry (JBC)
Volume
volume295
Issue
issue14
Publisher
American Society for Biochemistry and Molecular Biology
Start Page
4464
End Page
4476
ISSN
0021-9258
NCID
AA00251083
Content Type
Journal Article
language
英語
OAI-PMH Set
岡山大学
Copyright Holders
© 2020 Martin et al.
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isVersionOf https://doi.org/10.1074/jbc.RA119.012015
License
https://creativecommons.org/licenses/by-nc-nd/4.0/