title: Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus.
abstract: 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.
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
authors: O Andreas
authors: OA
authors: Karlsson
authors: Biotechnologie et Signalisation Cellulaire UMR 7242, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, BP 10413, F-67412 Illkirch, France.
authors: Juan
authors: J
authors: Ramirez
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
authors: Daniel
authors: D
authors: Öberg
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
authors: Tony
authors: T
authors: Malmqvist
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
authors: Åke
authors: Å
authors: Engström
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
authors: Maria
authors: M
authors: Friberg
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
authors: Celestine N
authors: CN
authors: Chi
authors: Department of Chemistry-BMC, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden.
authors: Mikael
authors: M
authors: Widersten
authors: Biotechnologie et Signalisation Cellulaire UMR 7242, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, BP 10413, F-67412 Illkirch, France.
authors: Gilles
authors: G
authors: Travé
authors: Department of Chemistry-BMC, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden.
authors: Mikael T I
authors: MT
authors: Nilsson
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
authors: Per
authors: P
authors: Jemth
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000074288:Discs Large Homolog 1 Protein; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D052162:Human papillomavirus 16 / Q000737:chemistry; D052161:Human papillomavirus 18 / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054731:PDZ Domains / Q000235:genetics*; D019151:Peptide Library; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C513314:DLG1 protein, human; D004268:DNA-Binding Proteins; D000074288:Discs Large Homolog 1 Protein; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; D008565:Membrane Proteins; D009856:Oncogene Proteins, Viral; D019151:Peptide Library; D011994:Recombinant Proteins; D012097:Repressor Proteins