Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus.

Publisher: Nature Publishing Group Country of Publication: England NLM ID: 101563288 Publication Model: Electronic Cited Medium: Internet ISSN: 2045-2322 (Electronic) Linking ISSN: 20452322 NLM ISO Abbreviation: Sci Rep Subsets: MEDLINE

Original Publication: London : Nature Publishing Group, copyright 2011-

Peptide Library*; Adaptor Proteins, Signal Transducing/*chemistry ; DNA-Binding Proteins/*chemistry ; Membrane Proteins/*chemistry ; Oncogene Proteins, Viral/*chemistry ; PDZ Domains/*genetics ; Repressor Proteins/*chemistry; Adaptor Proteins, Signal Transducing/genetics ; Adaptor Proteins, Signal Transducing/metabolism ; Amino Acid Sequence ; Binding Sites ; DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism ; Discs Large Homolog 1 Protein ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Gene Expression ; HeLa Cells ; Human papillomavirus 16/chemistry ; Human papillomavirus 18/chemistry ; Humans ; Immunoprecipitation ; Membrane Proteins/genetics ; Membrane Proteins/metabolism ; Models, Molecular ; Molecular Sequence Data ; Oncogene Proteins, Viral/genetics ; Oncogene Proteins, Viral/metabolism ; Protein Binding ; Protein Structure, Secondary ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Repressor Proteins/genetics ; Repressor Proteins/metabolism

Chronic infection by high risk human papillomavirus (HPV) strains may lead to cancer. Expression of the two viral oncoproteins E6 and E7 is largely responsible for immortalization of infected cells. The HPV E6 is a small (approximately 150 residues) two domain protein that interacts with a number of cellular proteins including the ubiquitin ligase E6-associated protein (E6AP) and several PDZ-domain containing proteins. Our aim was to design a high-affinity binder for HPV E6 by linking two of its cellular targets. First, we improved the affinity of the second PDZ domain from SAP97 for the C-terminus of HPV E6 from the high-risk strain HPV18 using phage display. Second, we added a helix from E6AP to the N-terminus of the optimized PDZ variant, creating a chimeric bivalent binder, denoted PDZbody. Full-length HPV E6 proteins are difficult to express and purify. Nevertheless, we could measure the affinity of the PDZbody for E6 from another high-risk strain, HPV16 (Kd = 65 nM). Finally, the PDZbody was used to co-immunoprecipitate E6 protein from HPV18-immortalized HeLa cells, confirming the interaction between PDZbody and HPV18 E6 in a cellular context.

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0 (Adaptor Proteins, Signal Transducing)
0 (DLG1 protein, human)
0 (DNA-Binding Proteins)
0 (Discs Large Homolog 1 Protein)
0 (E6 protein, Human papillomavirus type 16)
0 (E6 protein, Human papillomavirus type 18)
0 (Membrane Proteins)
0 (Oncogene Proteins, Viral)
0 (Peptide Library)
0 (Recombinant Proteins)
0 (Repressor Proteins)

Date Created: 20150324 Date Completed: 20160104 Latest Revision: 20201209

20221213

PMC4369733

10.1038/srep09382

25797137