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The Transition State of Coupled Folding and Binding for a Flexible beta-Finger
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. ETH, Lab Phys Chem, Zurich, Switzerland. (Chi Celestine)
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.
2012 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 417, no 3, 253-261 p.Article 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.

Place, publisher, year, edition, pages
2012. Vol. 417, no 3, 253-261 p.
Keyword [en]
intrinsically disordered proteins, PDZ domain, binding kinetics, phi binding, flexible proteins
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-172818DOI: 10.1016/j.jmb.2012.01.042ISI: 000301805800010OAI: oai:DiVA.org:uu-172818DiVA: diva2:516223
Available from: 2012-04-17 Created: 2012-04-16 Last updated: 2017-10-16Bibliographically 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. AndreasChi, Celestine N.Engström, ÅkeJemth, Per

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