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Computer-aided design of a PDZ domain to recognize new target sequences

Abstract

PDZ domains are small globular domains that recognize the last 4–7 amino acids at the C-terminus of target proteins. The specificity of the PDZ–ligand recognition is due to side chain–side chain interactions, as well as the positioning of an α-helix involved in ligand binding. We have used computer-aided protein design to produce mutant versions of a Class I PDZ domain that bind to novel Class I and Class II target sequences both in vitro and in vivo, thus providing an alternative to primary antibodies in western blotting, affinity chromatography and pull-down experiments. Our results suggest that by combining different backbone templates with computer-aided protein design, PDZ domains could be engineered to specifically recognize a large number of proteins.

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Figure 1
Figure 2: Ribbon diagrams of the PDZ–ligand complexes.
Figure 3: Fluorescence polarization binding assays.
Figure 4: Interactions between the PDZ domains and their target peptides in vitro.
Figure 5: Interactions between the PDZ domains and their target peptides in vitro and in vivo.

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Acknowledgements

We thank M. Kress who kindly provided us with the plasmid containing the cDNA of the mouse Eg5 kinesin, the members of our laboratories for helpful discussions and the EMBL genomics and protein expression core facilities for their support. This work was financed by a grant from the 'Fundación Ramón Areces' and grants from the European Union.

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Correspondence to Cayetano Gonzalez.

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The techonology described in this article is being patented and has been licensed by a company. S.D.H., V.R. and M.S.S. are employees of this company, of which L.S. and C.G. are scientific founders

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Reina, J., Lacroix, E., Hobson, S. et al. Computer-aided design of a PDZ domain to recognize new target sequences. Nat Struct Mol Biol 9, 621–627 (2002). https://doi.org/10.1038/nsb815

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