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Characterization and Engineering of Protein-Protein Interactions Involving PDZ Domains
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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. , p. 39
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1292
Keywords [en]
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: urn:nbn:se:uu:diva-312872ISBN: 978-91-554-9798-9 (print)OAI: oai:DiVA.org:uu-312872DiVA, id: diva2:1065196
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
List of papers
1. The Transition State of Coupled Folding and Binding for a Flexible beta-Finger
Open this publication in new window or tab >>The Transition State of Coupled Folding and Binding for a Flexible beta-Finger
2012 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 417, no 3, p. 253-261Article in journal (Refereed) Published
Abstract [en]

Flexible and fully disordered protein regions that fold upon binding mediate numerous protein protein interactions. However, little is known about their mechanism of interaction. One such coupled folding and binding occurs when a flexible region of neuronal nitric oxide synthase adopts a beta-finger structure upon binding to its protein ligand, a PDZ [PSD-95 (postsynaptic density protein-95)/Discs large/ZO-1] domain from PSD-95. We have analyzed this binding reaction by protein engineering combined with kinetic experiments. Mutational destabilization of the beta-finger changed mainly the dissociation rate constant of the proteins and, to a lesser extent, the association rate constant. Thus, mutation affected late events in the coupled folding and binding reaction. Our results therefore suggest that the native binding interactions of the beta-finger are not present in the rate-limiting transition state for binding but form on the downhill side in a cooperative manner. However, by mutation, we could destabilize the beta-finger further and change the rate-limiting step such that an initial conformational change becomes rate limiting. This switch in rate-limiting step shows that multistep binding mechanisms are likely to be found among flexible and intrinsically disordered regions of proteins.

Keywords
intrinsically disordered proteins, PDZ domain, binding kinetics, phi binding, flexible proteins
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-172818 (URN)10.1016/j.jmb.2012.01.042 (DOI)000301805800010 ()
Available from: 2012-04-17 Created: 2012-04-16 Last updated: 2017-10-16Bibliographically approved
2. Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus
Open this publication in new window or tab >>Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 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.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-258849 (URN)10.1038/srep09382 (DOI)000351373500005 ()25797137 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2015-07-23 Created: 2015-07-20 Last updated: 2017-12-04Bibliographically approved
3. Improved affinity at the cost of decreased specificity: a recurring theme in PDZ-peptide interactions.
Open this publication in new window or tab >>Improved affinity at the cost of decreased specificity: a recurring theme in PDZ-peptide interactions.
Show others...
2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 34269Article in journal (Refereed) Published
Abstract [en]

The E6 protein from human papillomavirus (HPV) plays an important role during productive infection and is a potential drug target. We have previously designed a high affinity bivalent protein binder for the E6 protein, a fusion between a helix from the E6 associated protein and PDZØ9, an engineered variant (L391F/K392M) of the second PDZ domain from synapse associated protein 97 (SAP97 PDZ2). How the substitutions improve the affinity of SAP97 PDZ2 for HPV E6 is not clear and it is not known to what extent they affect the specificity for cellular targets. Here, we explore the specificity of wild type SAP97 PDZ2 and PDZØ9 through proteomic peptide phage display. In addition, we employ a double mutant cycle of SAP97 PDZ2 in which the binding kinetics for nine identified potential cellular peptide ligands are measured and compared with those for the C-terminal E6 peptide. The results demonstrate that PDZØ9 has an increased affinity for all peptides, but at the cost of specificity. Furthermore, there is a peptide dependent coupling free energy between the side chains at positions 391 and 392. This corroborates our previous allosteric model for PDZ domains, involving sampling of intramolecular energetic pathways.

National Category
Natural Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-306579 (URN)10.1038/srep34269 (DOI)000384758300001 ()27694853 (PubMedID)
Funder
Swedish Research Council
Available from: 2016-10-29 Created: 2016-10-29 Last updated: 2018-09-02Bibliographically approved
4. Ligand binding to the PDZ domains of postsynaptic density protein 95
Open this publication in new window or tab >>Ligand binding to the PDZ domains of postsynaptic density protein 95
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2016 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 29, no 5, p. 169-175Article in journal (Refereed) Published
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.

Keywords
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:nbn:se:uu:diva-298245 (URN)10.1093/protein/gzw004 (DOI)000376351600002 ()26941280 (PubMedID)
Funder
Swedish Research Council, 2012-5096
Available from: 2016-07-01 Created: 2016-07-01 Last updated: 2017-11-28Bibliographically approved

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