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A sequential binding mechanism in a PDZ domain
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH. (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.
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2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 30, 7089-7097 p.Article in journal (Refereed) Published
Abstract [en]

Conformational selection and induced fit are two well-known mechanisms of allosteric protein-ligand interaction. Some proteins, like ubiquitin, have recently been found to exist in multiple conformations at equilibrium, suggesting that the conformational selection may be a general mechanism of interaction, in particular for single-domain proteins. Here, we found that the PDZ2 domain of SAP97 binds its ligand via a sequential (induced fit) mechanism. We performed binding experiments using SAP97 PDZ2 and peptide ligands and observed biphasic kinetics with the stopped-flow technique, indicating that ligand binding involves at least a two-step process. By using an ultrarapid continuous-flow mixer, we then detected a hyperbolic dependence of binding rate constants on peptide concentration, corroborating the two-step binding mechanism. Furthermore, we found a similar dependence of the rate constants on both PDZ and peptide concentration, demonstrating that the PDZ2-peptide interaction involves a precomplex, which then undergoes a conformational change, and thereby follows an induced fit mechanism.

Place, publisher, year, edition, pages
2009. Vol. 48, no 30, 7089-7097 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-119523DOI: 10.1021/bi900559kISI: 000268231800002PubMedID: 19496620OAI: oai:DiVA.org:uu-119523DiVA: diva2:300369
Available from: 2010-02-26 Created: 2010-02-26 Last updated: 2017-10-16Bibliographically approved
In thesis
1. Post-synaptic Density Disc Large Zo-1 (PDZ) Domains: From Folding and Binding to Drug Targeting
Open this publication in new window or tab >>Post-synaptic Density Disc Large Zo-1 (PDZ) Domains: From Folding and Binding to Drug Targeting
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Understanding how proteins fold and bind is interesting since these processes are central to most biological activity. Protein folding and protein-protein interaction are by themselves very complex but using a good and robust system to study them could ease some of the hurdles.

In this thesis I have tried to answer some of the fundamental questions of protein folding and binding. I chose to work with PDZ domains, which are protein domains consisting of 90-100 amino acids. They are found in more than 400 human proteins and function mostly as protein-protein interaction units. These proteins are very stable, easy to express and purify and their folding reaction is reversible under most laboratory conditions.

I have characterized the interaction of PSD-95 PDZ3 domain with its putative ligand under different experimental conditions and found out that its binding kinetics is sensitive to salt and pH.  I also demonstrated that the two conserved residues R318 and H372 in PDZ3 are responsible for the salt and pH effect, respectively, on the binding reaction. Moreover, I determined that for PSD 95 PDZ3 coupling of distal residues to peptide binding was better described by a distance relationship and there was a very weak evidence of an allosteric network. Further, I showed that another PDZ domain, SAP97 PDZ2 undergoes conformational change upon ligand binding.

Also, I characterized the binding mechanism of a dimeirc ligand/PDZ1-2 tandem interaction and showed that despite its apparent complexity the binding reaction is best described by a square scheme. Additionally, I determined that for the SAP 97 PDZ/HPV E6 interaction that all three PDZ domains each bind one molecule of the E6 protein and that a set of residues in the PDZ2 of SAP 97 could operate in an unexpected long-range manner during E6 interaction.

Finally, I showed that perhaps all members in the PDZ family could fold via a three state folding mechanism. I characterized the folding mechanism of five different PDZ domains having similar overall fold but different primary structure and the results indicate that all five fold via an intermediate with two transition states. Transition state one is rate limiting at low denaturant concentration and vice versa for transition state two. Comparing and characterizing the structures of the transition states of two PDZ domains using phi value analysis indicated that their early transition states are less similar as compared to their late transition states.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 42 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 573
Keyword
protein-protein interaction, protein folding, and drug design
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-126129 (URN)978-91-554-7836-0 (ISBN)
Public defence
2010-09-03, B22, BMC, Uppsala, 10:15 (English)
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Available from: 2010-08-16 Created: 2010-06-03 Last updated: 2010-08-25Bibliographically approved

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Engström, ÅkeJemth, Per

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