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Deciphering the kinetic binding mechanism of dimeric ligands, using a potent plasma-stable dimeric inhibitor of postsynaptic density protein-95 as an example
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. (Per jemth)
University of Copenhagen.
University of Copenhagen.
University of Copenhagen.
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2010 (Engelska)Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, nr 36, s. 28252-28260Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity towards their targets due to their ability to bind simultaneously to two binding sites and are therefore very attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide of PDZ1-2 of PSD-95 using protein engineering in combination with fluorescence polarisation, isothermal titration calorimetry and stopped-flow fluorimetry. Our experiments demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and we also find evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand, but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.

Ort, förlag, år, upplaga, sidor
2010. Vol. 285, nr 36, s. 28252-28260
Nyckelord [en]
protein-protein interactions, dimeric ligand, PDZ, conformational change, inhibitors
Nationell ämneskategori
Annan medicinsk grundvetenskap
Forskningsämne
Biokemi
Identifikatorer
URN: urn:nbn:se:uu:diva-126083DOI: 10.1074/jbc.M110.124040ISI: 000281404100068PubMedID: 20576616OAI: oai:DiVA.org:uu-126083DiVA, id: diva2:321843
Tillgänglig från: 2010-06-02 Skapad: 2010-06-02 Senast uppdaterad: 2018-01-12Bibliografiskt granskad
Ingår i avhandling
1. Post-synaptic Density Disc Large Zo-1 (PDZ) Domains: From Folding and Binding to Drug Targeting
Öppna denna publikation i ny flik eller fönster >>Post-synaptic Density Disc Large Zo-1 (PDZ) Domains: From Folding and Binding to Drug Targeting
2010 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2010. s. 42
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 573
Nyckelord
protein-protein interaction, protein folding, and drug design
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Forskningsämne
Biokemi
Identifikatorer
urn:nbn:se:uu:diva-126129 (URN)978-91-554-7836-0 (ISBN)
Disputation
2010-09-03, B22, BMC, Uppsala, 10:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2010-08-16 Skapad: 2010-06-03 Senast uppdaterad: 2010-08-25Bibliografiskt granskad

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