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Ligand binding to the PDZ domains of postsynaptic density protein 95
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Univ Rome, Fdn Cenci Bolognetti, Ist Pasteur, Dipartimento Sci Biochim A Rossi Fanelli Sapienza, I-00185 Rome, Italy.;Univ Rome, CNR, Ist Biol & Patol Mol, I-00185 Rome, Italy..
Univ Copenhagen, Ctr Biopharmaceut, Dept Drug Design & Pharmacol, Univ Pk 2, DK-2100 Copenhagen, Denmark..
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Univ Copenhagen, Ctr Biopharmaceut, Dept Drug Design & Pharmacol, Univ Pk 2, DK-2100 Copenhagen, Denmark..
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2016 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 29, no 5, 169-175 p.Article in journal (Refereed) Published
Resource type
Text
Abstract [en]

Cellular scaffolding and signalling is generally governed by multidomain proteins, where each domain has a particular function. Postsynaptic density protein 95 (PSD-95) is involved in synapse formation and is a typical example of such a multidomain protein. Protein-protein interactions of PSD-95 are well studied and include the following three protein ligands: (i) N-methyl-d-aspartate-type ionotropic glutamate receptor subunit GluN2B, (ii) neuronal nitric oxide synthase and (iii) cysteine-rich protein (CRIPT), all of which bind to one or more of the three PDZ domains in PSD-95. While interactions for individual PDZ domains of PSD-95 have been well studied, less is known about the influence of neighbouring domains on the function of the respective individual domain. We therefore performed a systematic study on the ligand-binding kinetics of PSD-95 using constructs of different size for PSD-95 and its ligands. Regarding the canonical peptide-binding pocket and relatively short peptides (up to 15-mer), the PDZ domains in PSD-95 by and large work as individual binding modules. However, in agreement with previous studies, residues outside of the canonical binding pocket modulate the affinity of the ligands. In particular, the dissociation of the 101 amino acid CRIPT from PSD-95 is slowed down at least 10-fold for full-length PSD-95 when compared with the individual PDZ3 domain.

Place, publisher, year, edition, pages
2016. Vol. 29, no 5, 169-175 p.
Keyword [en]
CRIPT, GluN2B, Kinetics, PDZ domain, PSD-95
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-298245DOI: 10.1093/protein/gzw004ISI: 000376351600002PubMedID: 26941280OAI: oai:DiVA.org:uu-298245DiVA: diva2:945549
Funder
Swedish Research Council, 2012-5096
Available from: 2016-07-01 Created: 2016-07-01 Last updated: 2017-01-13Bibliographically 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)
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Available from: 2017-02-07 Created: 2017-01-13 Last updated: 2017-02-15

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Karlsson, O. AndreasAndersson, EvaChi, Celestine N.Jemth, Per
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