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Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, 9382Article in journal (Refereed) Published
Abstract [en]

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 (K-d = 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.

Place, publisher, year, edition, pages
2015. Vol. 5, 9382
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-258849DOI: 10.1038/srep09382ISI: 000351373500005PubMedID: 25797137OAI: oai:DiVA.org:uu-258849DiVA: diva2:842895
Funder
Swedish Cancer Society
Available from: 2015-07-23 Created: 2015-07-20 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Characterization and Engineering of Protein-Protein Interactions Involving PDZ Domains
Open this publication in new window or tab >>Characterization and Engineering of Protein-Protein Interactions Involving PDZ Domains
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The work presented in this thesis has contributed with knowledge to several aspects of protein-protein interaction involving PDZ domains. A substantial amount of our proteome contains regions that are intrinsically disordered but fold upon ligand interaction. The mechanism by which disordered regions bind to their ligands is one important piece of the puzzle to understand why disorder is beneficial. A region in the PDZ domain of nNOS undergoes such a disorder-to-order transition to form a b-sheet in the binding pocket of its partner. By studying the kinetics of interaction, in combination with mutations that modulate the stability of the aforementioned region, we demonstrate that the binding mechanism consists of multiple steps in which the native binding interactions of the b-sheet are formed cooperatively after the rate-limiting transition state. These mechanistic aspects may be general for the binding reactions of intrinsically disordered protein regions, at least upon formation of β-sheets.  

            The second part of this thesis deals with the engineering of proteins for increasing affinity in protein-protein interaction. Infection by high-risk human papillomavirus (hrHPV) can lead to cancer, and the viral E6 protein is an attractive drug target. E6 from hrHPV natively interacts with the well-characterized PDZ2 domain in SAP97, which we used as a scaffold to develop a high affinity bivalent binder of hrHPV E6. We initially increased PDZ2's affinity for E6 6-fold, but at the cost of decreased specificity. Attaching a helix that binds E6 at a distant site, increasing the affinity another14-fold, completed the design.

            The final work of this thesis investigates if binding studies conducted with isolated PDZ domains is representative of the full-length proteins they belong to. It has been suggested that ligand binding in PDZ domains can be influenced by factors such as adjacent domains and interactions outside of the binding pocket. We studied these aspects for the three PDZ domains of PSD-95 and found that they on the whole function in an independent manner with short peptides as ligands, but that interactions outside of the PDZ binding-pocket may be present. The representative length of the PDZ interaction partner should therefore be considered.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 39 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1292
Keyword
intrinsically disordered protein regions, PDZ domain, binding kinetics, protein engineering, interaction mechanism, specificity, PDZbody
National Category
Biochemistry and Molecular Biology Biophysics
Research subject
Chemistry with specialization in Biophysics
Identifiers
urn:nbn:se:uu:diva-312872 (URN)978-91-554-9798-9 (ISBN)
Public defence
2017-03-03, B42, Biomedicinskt Centrum, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2017-02-07 Created: 2017-01-13 Last updated: 2017-02-15

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Karlsson, O. AndreasÖberg, DanielEngström, ÅkeChi, Celestine N.Widersten, MikaelJemth, Per

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