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In-vivo crystals reveal protein interactions

Martin, E. R.; Barbieri, A.; Ford, R. C.; Robinson, R. C.

2019-09-26 biochemistry
10.1101/778779 bioRxiv
Show abstract

Crystallisation of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under non-physiological extremes of protein, salt, and H+ concentrations. Here, we describe the development of the robust iBox-PAK4cat system that spontaneously crystallises in several mammalian cell types. The developments described here allow the quantitation of in-vivo protein-protein interactions using a novel GFP-linked reporter system. Here, we have combined this assay with in-vitro X-ray crystallography and molecular dynamics studies characterise the molecular determinants of the interaction between NHERF1 PDZ2 and CFTR, a protein complex pertinent to the genetic disease cystic fibrosis. These studies have revealed the crystal structure of the extended PDZ domain of NHERF1, and indicated, contrary to previous reports, that residue selection at -1 and -3 PDZ-binding motif positions influence the affinity and specificity of the interaction. The results presented here demonstrate that the iBox-PAK4cat assay could easily be utilised to screen other protein-protein interactions.

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