Peptide Binding Properties of the Three PDZ Domains of Bazooka (Drosophila Par-3)

PLOS ONE, Dec 2019

The Par complex is a conserved cell polarity regulator. Bazooka/Par-3 is scaffold for the complex and contains three PDZ domains in tandem. PDZ domains can act singly or synergistically to bind the C-termini of interacting proteins. Sequence comparisons among Drosophila Baz and its human and C. elegans Par-3 counterparts indicate a divergence of the peptide binding pocket of PDZ1 and greater conservation for the pockets of PDZ2 and PDZ3. However, it is unclear whether the domains from different species share peptide binding preferences, or if their tandem organization affects their peptide binding properties. To investigate these questions, we first used phage display screens to identify unique peptide binding profiles for each single PDZ domain of Baz. Comparisons with published phage display screens indicate that Baz and C. elegans PDZ2 bind to similar peptides, and that the peptide binding preferences of Baz PDZ3 are more similar to C. elegans versus human PDZ3. Next we quantified the peptide binding preferences of each Baz PDZ domain using single identified peptides in surface plasmon resonance assays. In these direct binding studies, each peptide had a binding preference for a single PDZ domain (although the peptide binding of PDZ2 was weakest and the least specific). PDZ1 and PDZ3 bound their peptides with dissociation constants in the nM range, whereas PDZ2-peptide binding was in the µM range. To test whether tandem PDZ domain organization affects peptide binding, we examined a fusion protein containing all three PDZ domains and their normal linker regions. The binding strengths of the PDZ-specific peptides to single PDZ domains and to the PDZ domain tandem were indistinguishable. Thus, the peptide binding pockets of each PDZ domain in Baz are not obviously affected by the presence of neighbouring PDZ domains, but act as isolated modules with specific in vitro peptide binding preferences.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0086412&type=printable

Peptide Binding Properties of the Three PDZ Domains of Bazooka (Drosophila Par-3)

et al. (2014) Peptide Binding Properties of the Three PDZ Domains of Bazooka (Drosophila Par-3). PLoS ONE 9(1): e86412. doi:10.1371/journal.pone.0086412 Peptide Binding Properties of the Three PDZ Domains of Bazooka (Drosophila Par-3 ) Cao Guo Yu 0 Raffi Tonikian 0 Corinna Felsensteiner 0 Jacquelyn R. Jhingree 0 Darrell Desveaux 0 Sachdev S. Sidhu 0 Tony J. C. Harris 0 Francois Schweisguth, Institut Pasteur, France 0 1 Department of Cell & Systems Biology, University of Toronto , Toronto, Ontario , Canada , 2 Terrence Donnelly Centre for Cellular and Biomolecular Research, and Banting and Best Department of Medical Research, and Department of Molecular Genetics, University of Toronto , Toronto, Ontario , Canada , 3 Centre for the Analysis of Genome Evolution & Function, University of Toronto , Toronto, Ontario , Canada The Par complex is a conserved cell polarity regulator. Bazooka/Par-3 is scaffold for the complex and contains three PDZ domains in tandem. PDZ domains can act singly or synergistically to bind the C-termini of interacting proteins. Sequence comparisons among Drosophila Baz and its human and C. elegans Par-3 counterparts indicate a divergence of the peptide binding pocket of PDZ1 and greater conservation for the pockets of PDZ2 and PDZ3. However, it is unclear whether the domains from different species share peptide binding preferences, or if their tandem organization affects their peptide binding properties. To investigate these questions, we first used phage display screens to identify unique peptide binding profiles for each single PDZ domain of Baz. Comparisons with published phage display screens indicate that Baz and C. elegans PDZ2 bind to similar peptides, and that the peptide binding preferences of Baz PDZ3 are more similar to C. elegans versus human PDZ3. Next we quantified the peptide binding preferences of each Baz PDZ domain using single identified peptides in surface plasmon resonance assays. In these direct binding studies, each peptide had a binding preference for a single PDZ domain (although the peptide binding of PDZ2 was weakest and the least specific). PDZ1 and PDZ3 bound their peptides with dissociation constants in the nM range, whereas PDZ2-peptide binding was in the mM range. To test whether tandem PDZ domain organization affects peptide binding, we examined a fusion protein containing all three PDZ domains and their normal linker regions. The binding strengths of the PDZ-specific peptides to single PDZ domains and to the PDZ domain tandem were indistinguishable. Thus, the peptide binding pockets of each PDZ domain in Baz are not obviously affected by the presence of neighbouring PDZ domains, but act as isolated modules with specific in vitro peptide binding preferences. - Funding: TJCH and DD hold Tier 2 Canada Research Chairs. Work supported by a Canadian Institute of Health Research (CIHR) operating grant (MOP 82829) to TH, a CIHR operating grant (MOP 93684) to SSS, and a Natural Sciences and Engineering Research Council of Canada Discovery Grant to DD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Cell polarity is fundamental to cell biology. For a cell to migrate directionally, constrict apically, transport material vectorally, or divide asymmetrically, distinguishing one end of the cell from the other is essential [1,2,3,4,5]. The Par complex is a core polarity regulator [6,7,8,9]. It is made up of the adaptor protein Par-6, atypical protein kinase C (aPKC), and the scaffold protein Bazooka (Baz)/Par-3. The complex is conserved across animals, and functions in epithelial cell polarity, epithelial morphogenesis, asymmetric cell division, axon outgrowth, and cancer progression. Similar to its counterparts in other species, Drosophila Baz contains an N-terminal oligomerization domain, three PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains, a C-terminal aPKC binding region, and a C-terminal lipid binding region (Fig. 1A). In vivo structure-function analyses have revealed that multiple domains in Baz can act redundantly to localize the protein, and that specific domains are important for Baz activity as a polarity protein [10,11,12]. PDZ domains are configured to bind the C-termini of protein interaction partners [13,14,15]. Specifically, the PDZ domain is formed from six b-strands (bAF) and two a-helices (aAB). The peptide binding pocket is formed between strand bB and helix aB. Natural protein interactions involving this site occur with KD values the low mM range. Within the C-termini of targets, binding specificity is strongly influenced by the C-terminal amino acid residue (position P0) and the second internal amino acid residue (position P2). Specific interaction partners have been found to bind to the PDZ domains of Baz and its counterparts in other species. For Baz, wh (...truncated)


This is a preview of a remote PDF: https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0086412&type=printable

Cao Guo Yu, Raffi Tonikian, Corinna Felsensteiner, Jacquelyn R. Jhingree, Darrell Desveaux, Sachdev S. Sidhu, Tony J. C. Harris. Peptide Binding Properties of the Three PDZ Domains of Bazooka (Drosophila Par-3), PLOS ONE, 2014, 1, DOI: 10.1371/journal.pone.0086412