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  • Research Article
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Mutagenesis and selection of PDZ domains that bind new protein targets

Abstract

PDZ domains are a recently characterized protein–recognition module. In most cases, PDZ domains bind to the C–terminal end of target proteins and are thought thereby to link these target proteins into functional signaling networks. We report the isolation of artificial PDZ domains selected via a mutagenesis screen in vivo, each recognizing a different C–terminal peptide. We demonstrate that the PDZ domains isolated can bind selectively to their target peptides in vitro and in vivo. Two of the target peptides chosen are the C–terminal ends of two cellular transmembrane proteins with which no known PDZ domains have been reported to interact. By targeting these artificial PDZ domains to the nucleus, interacting target peptides were efficiently transported to the same subcellular localization. One of the isolated PDZ domains was tested and shown to be efficiently directed to the plasma membrane when cotransfected with the full–length transmembrane protein in mammalian cells. Thus, artificial PDZ domains can be engineered and used to target intracellular proteins to different subcellular compartments.

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Figure 1: Relative CAT reporter gene activity of the artificial PDZ domains binding to different peptides in mammalian 293T cells.
Figure 2: In vitro binding analysis of different artificial PDZ domains to various target peptides.
Figure 3: Homology alignment of the PDZ domain variants isolated with the mutagenesis screen.
Figure 4: Subcellular localization of GFP–peptide and NLS–PDZ domain fusion proteins.
Figure 5: Membrane localization of the GFP–PDZ–ELK–L protein in a cell cotransfected with the full–length Elk–L protein.

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Acknowledgements

We are especially thankful to Ira Daar at the National Cancer Institute in Frederick, MD, for providing the Ephrin–B1 clone and antibody. We thank John Wade for mass spectroscopy and E.C. Nice for help with Biosensor analysis—both at the Howard Florey Institute, Melbourne, Australia. This work was supported by grants from the Swiss National Science Foundation and the Swiss Cancer League.

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Correspondence to Karin Moelling.

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Schneider, S., Buchert, M., Georgiev, O. et al. Mutagenesis and selection of PDZ domains that bind new protein targets . Nat Biotechnol 17, 170–175 (1999). https://doi.org/10.1038/6172

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