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Sequence-specific Long Range networks in PSD-95/Discs Large/ZO-1 (PDZ) Domains Tune Their Binding Selectivity
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Per Jemth)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 31, 27167-27175 p.Article in journal (Refereed) Published
Abstract [en]

Protein-protein interactions mediated by modular protein domains are critical for cell scaffolding, differentiation, signaling, and ultimately, evolution. Given the vast number of ligands competing for binding to a limited number of domain families, it is often puzzling how specificity can be achieved. Selectivity may be modulated by intradomain allostery, whereby a remote residue is energetically connected to the functional binding site via side chain or backbone interactions. Whereas several energetic pathways, which could mediate intradomain allostery, have been predicted in modular protein domains, there is a paucity of experimental data to validate their existence and roles. Here, we have identified such functional energetic networks in one of the most common protein-protein interaction modules, the PDZ domain. We used double mutant cycles involving site-directed mutagenesis of both the PDZ domain and the peptide ligand, in conjunction with kinetics to capture the fine energetic details of the networks involved in peptide recognition. We performed the analysis on two homologous PDZ-ligand complexes and found that the energetically coupled residues differ for these two complexes. This result demonstrates that amino acid sequence rather than topology dictates the allosteric pathways. Furthermore, our data support a mechanism whereby the whole domain and not only the binding pocket is optimized for a specific ligand. Such cross-talk between binding sites and remote residues may be used to fine tune target selectivity.

Place, publisher, year, edition, pages
2011. Vol. 286, no 31, 27167-27175 p.
National Category
Biochemistry and Molecular Biology
Research subject
Anaesthesiology and Intensive Care
URN: urn:nbn:se:uu:diva-160821DOI: 10.1074/jbc.M111.239541ISI: 000293268700012PubMedID: 21653701OAI: oai:DiVA.org:uu-160821DiVA: diva2:453172
Available from: 2011-11-01 Created: 2011-11-01 Last updated: 2011-12-06Bibliographically approved
In thesis
1. Molecular Mechanisms of Folding and Binding in PDZ Domains
Open this publication in new window or tab >>Molecular Mechanisms of Folding and Binding in PDZ Domains
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

PDZ domains are one of the most abundant protein-protein interaction modules that mediate protein recognition by binding to short amino acid sequences in the cells. These globular protein domains usually consist of 80-100 amino acid residues that fold into six ß-strands and two a-helices. They are often found in tandem with other interacting modules such as SH3 and GK domains and even with other PDZ containing protein. These domains are involved in signal transduction, synapse formation and receptor translocation and have been implicated in certain diseases such as cervical cancer, Parkinson’s disease and cystic fibrosis.

In this thesis, I have characterized the folding and binding mechanism of three PDZ domains, i.e. SAP97 PDZ2, PSD95 PDZ3 and PTP-BL PDZ2.

I studied the folding mechanism of SAP97 PDZ2, which follows a triangular folding scheme. An intermediate was identified in the folding pathway of this domain, which was populated at equilibrium and may form from denatured or native state depending upon the experimental temperature. The results are suggestive of the plasticity of the folding landscape and reconcile the previous folding studies on PDZ domains (Paper I).

I have also identified allosteric networks in two PDZ domains i.e. PSD95 PDZ3 and PTP-BL PDZ2 by calculating the coupling free energy using the double mutant cycle analysis. Contrary to a previous study, the allosteric network was found to be sequence dependent rather than conserved among the PDZ domains. Moreover, the network identified for one ligand was found to be different from the network for another ligand for the same PDZ domain (Paper II).

Similarly, I have determined the allosteric networks in SAP97 PDZ2 that responds to the binding of peptides derived from the C-terminal end of HPV18 E6 protein. Importantly, the results corroborate the finding in Paper 2, i.e. the residues that are responsive to one peptide ligand are different from the residues that respond to the other peptide ligand (Paper II).

The results thus provide an insight about how the PDZ domain with simple topology and multiple binding partners may retain selectivity within the cell (Paper II and III).

I have also investigated the role of an auxiliary C-terminal helix (α-3) in the allosteric regulation of ligand binding in PSD95 PDZ3. The interaction of α-3 with the peptide Y-5 was found to be direct. Thus we proved in this study that the α-3 contribute towards the regulation of ligand binding in PSD95 PDZ3, and the decrease in affinity of ligand towards α-3 deleted mutant was due to the loss of direct interaction between the α-3 and the peptide ligand (Paper IV). A previous study has suggested that α-3 contributes towards regulation of ligand binding allosterically. But our results suggest that the interaction between the α-3 and Y-5 of the peptide is a direct one and therefore, this helix appears not to allosterically regulate the PDZ3 function. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 40 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 724
National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-160944 (URN)978-91-554-8221-3 (ISBN)
Public defence
2011-12-15, C10:301, BMC, Husargatan 3, Uppsala,, 10:00 (English)
Available from: 2011-11-23 Created: 2011-11-03 Last updated: 2011-11-24

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