Proteome‐wide analysis of phospho‐regulated PDZ domain interactions
2018; Springer Nature; Volume: 14; Issue: 8 Linguagem: Inglês
10.15252/msb.20178129
ISSN1744-4292
AutoresGustav N. Sundell, Roland Arnold, Muhammad Ali, Piangfan Naksukpaiboon, Julien Orts, Peter Güntert, N. Celestine, Ylva Ivarsson,
Tópico(s)Cellular transport and secretion
ResumoMethod20 August 2018Open Access Source DataTransparent process Proteome-wide analysis of phospho-regulated PDZ domain interactions Gustav N Sundell Gustav N Sundell Department of Chemistry – BMC, Uppsala University, Uppsala, Sweden Search for more papers by this author Roland Arnold Corresponding Author Roland Arnold [email protected] Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Muhammad Ali Muhammad Ali orcid.org/0000-0002-8858-6776 Department of Chemistry – BMC, Uppsala University, Uppsala, Sweden Search for more papers by this author Piangfan Naksukpaiboon Piangfan Naksukpaiboon Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Julien Orts Julien Orts Laboratory of Physical Chemistry, ETH Zürich, Zürich, Switzerland Search for more papers by this author Peter Güntert Peter Güntert Laboratory of Physical Chemistry, ETH Zürich, Zürich, Switzerland Institute of Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Celestine N Chi Corresponding Author Celestine N Chi [email protected] Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden Search for more papers by this author Ylva Ivarsson Corresponding Author Ylva Ivarsson [email protected] orcid.org/0000-0002-7081-3846 Department of Chemistry – BMC, Uppsala University, Uppsala, Sweden Search for more papers by this author Gustav N Sundell Gustav N Sundell Department of Chemistry – BMC, Uppsala University, Uppsala, Sweden Search for more papers by this author Roland Arnold Corresponding Author Roland Arnold [email protected] Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Muhammad Ali Muhammad Ali orcid.org/0000-0002-8858-6776 Department of Chemistry – BMC, Uppsala University, Uppsala, Sweden Search for more papers by this author Piangfan Naksukpaiboon Piangfan Naksukpaiboon Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Julien Orts Julien Orts Laboratory of Physical Chemistry, ETH Zürich, Zürich, Switzerland Search for more papers by this author Peter Güntert Peter Güntert Laboratory of Physical Chemistry, ETH Zürich, Zürich, Switzerland Institute of Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Celestine N Chi Corresponding Author Celestine N Chi [email protected] Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden Search for more papers by this author Ylva Ivarsson Corresponding Author Ylva Ivarsson [email protected] orcid.org/0000-0002-7081-3846 Department of Chemistry – BMC, Uppsala University, Uppsala, Sweden Search for more papers by this author Author Information Gustav N Sundell1, Roland Arnold *,2, Muhammad Ali1, Piangfan Naksukpaiboon2, Julien Orts3, Peter Güntert3,4, Celestine N Chi *,5 and Ylva Ivarsson *,1 1Department of Chemistry – BMC, Uppsala University, Uppsala, Sweden 2Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK 3Laboratory of Physical Chemistry, ETH Zürich, Zürich, Switzerland 4Institute of Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany 5Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden *Corresponding author. Tel: +44 7936624996; E-mail: [email protected] *Corresponding author. Tel: +46 18 471 4557; E-mail: [email protected] *Corresponding author. Tel: +46 18 471 40 38; E-mail: [email protected] Molecular Systems Biology (2018)14:e8129https://doi.org/10.15252/msb.20178129 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract A key function of reversible protein phosphorylation is to regulate protein–protein interactions, many of which involve short linear motifs (3–12 amino acids). Motif-based interactions are difficult to capture because of their often low-to-moderate affinities. Here, we describe phosphomimetic proteomic peptide-phage display, a powerful method for simultaneously finding motif-based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C-terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR. We uncover site-specific phospho-regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho-regulation of motif-based interactions on a large scale. Synopsis The study presents phosphomimetic proteomic peptide phage display, a novel method for exploring phospho-regulated motif-based interactions. Application to PDZ domains reveals a site-specific phospho-regulation of PDZ-mediated interactions as a switching mechanism of interaction selectivity. Phosphomimetic proteomic peptide-phage display (ProP-PD) is a novel method for simultaneously finding motif-based interaction and identifying phosphorylation switches. Site-specific Ser/Thr phosphorylation events enable or disable PDZ domain interactions as revealed by phosphomimetic ProP-PD. The approach can be used to explore potential phospho-regulation of motif-based interactions on a large scale. Introduction Reversible protein phosphorylation is crucial for regulation of cellular processes and primarily occurs on Ser, Thr, and Tyr residues in eukaryotes (Seet et al, 2006). Phosphorylation may have different functional effects on the target protein, such as inducing conformational changes, altering cellular localization, or enabling or disabling interaction sites. Hundreds of thousands of such phosphosites have been identified in different cell lines and under different conditions (Olsen et al, 2006; Hornbeck et al, 2015). An unresolved question is which of these phosphosites are of functional relevance and not background noise caused by the off-target activity of kinases revealed by the high sensitivity in the mass spectrometry analysis. So far, only a minor fraction of identified phosphosites has been linked to functional effects, and only 35% of phosphosites are evolutionarily conserved, which would support that they play non-redundant functional roles (Landry et al, 2009). Sifting functional from non-functional phosphosites by experimental approaches is thus of fundamental importance in order to better understand the function of the phosphoproteome. Phosphosites are often located in the intrinsically disordered regions of proteins, which in eukaryotes are estimated to cover 30–40% of the populated protein sequence space (Ward et al, 2004). These regions are enriched in short linear motifs (SLiMs, 3–12 amino acids) that are recognized by modular domains (Van Roey et al, 2014). Discrete phosphorylation events may regulate and tune the strength of interactions. Phosphopeptide binding domains, such as the 14-3-3 proteins, serve as readers of this phosphorylation code (Yaffe et al, 1997; Reinhardt & Yaffe, 2013). Deciphering the phosphorylation code by linking the reader domains to their preferred binding phosphosites is a major challenge. Adding to the complexity, phosphorylation of a SLiM may have a switch-like effect, making the interaction stronger (enabling) with a given domain, while weakening interactions (disabling) with other domains (Van Roey et al, 2013), in other worlds regulating interactions. Such a switch-like mechanism was recently found to regulate interactions of the C-terminal region of PRTM5 with 14-3-3 proteins and PDZ (PSD-95/Discs-large/ZO-1) domains. Phosphorylation of the C-terminal tail of PRTM5 enables 14-3-3 interactions, while disabling PDZ domain interactions (Espejo et al, 2017). Although less common, phosphorylation switches also operate on the interaction interfaces of folded proteins, hundreds of which were suggested in recent analysis (Betts et al, 2017). There is a paucity of information linking phosphorylation enabled/disabled SLiMs to their binding partners, in part due to a lack of suitable experimental methods. Although affinity purification coupled to mass spectrometry (AP-MS) can be used to obtain information on phosphorylation states and interactions dynamically, the approach does not provide information with resolution at the level of the binding sites. In addition, interactions that rely on SLiMs are often elusive to AP-MS as they typically are of low-to-medium affinities, and exhibit rapid association/dissociation kinetics. Phosphopeptide libraries can be used to establish preferred binding motifs of phosphopeptide binding domains, but the number of sequences presented in these experiments is typically limited (Yaffe & Smerdon, 2004). In addition, Grossman and co-workers recently showed the use of yeast-two-hybrid (Y2H) for capturing phospho-Tyr-dependent interactions (Grossmann et al, 2015), but phospho-dependent interactions are otherwise typically not captured by Y2H. Thus, there is an urge for novel large-scale methods for charting phosphorylation-dependent SLiM-based interactions. Here, we describe a novel large-scale approach termed phosphomimetic proteomic peptide-phage display (phosphomimetic ProP-PD) developed to identify SLiM-based interactions that are regulated by Ser/Thr phosphorylation. Phosphomimetic ProP-PD is a further development of ProP-PD, which is a dedicated method for the large-scale identification of SLiM-based interactions (Ivarsson et al, 2014; Davey et al, 2017; Wu et al, 2017). We demonstrate that phosphomimetic ProP-PD is a straightforward approach for finding ligands of SLiM-binding domains and for simultaneously pinpointing Ser/Thr phosphorylation events with potential to enable or disable interactions. We showcase the approach by identifying phospho-regulated interactions of PDZ (PSD-95/Dlg/ZO-1) domains, which are among the most frequent interaction modules in eukaryote proteomes with about 267 instances in over human 150 proteins (Luck et al, 2011; Ivarsson, 2012). PDZ domains are well known for binding to C-terminal peptides with typical PDZ binding motifs (PDZbms; class I binding motif S/T-x-Φ-coo-, class II Φ-x-Φ-coo-, and class III D/E-x-Φ-coo-, where x is any amino acid and Φ is a hydrophobic amino acid), but also interact with internal PDZbms and phospholipids (Ivarsson, 2012). The last amino acid in the PDZbm is denoted p0, the upstream residue is denoted p-1, and so on. Although the PDZbms are the crucial determinants for binding, residues upstream contribute to the interactions (Tonikian et al, 2008). Based on a limited literature on phospho-regulation of PDZ domain interactions, phosphorylation of the Ser/Thr at p-2 of the class I PDZbm typically disables PDZ interactions (Cohen et al, 1996; Chetkovich et al, 2002; Choi et al, 2002; Hu et al, 2002; Tanemoto et al, 2002; Toto et al, 2017), while phosphorylation of other positions may either enable or disable interactions (Clairfeuille et al, 2016). In this study, we first create a phosphomimetic ProP-PD library displaying C-terminal regions of the human proteome that contain known or predicted phosphorylation sites, and the phosphomimetic mutants of these peptides. Second, we use this library in selections against the six PDZ domains of human Scribble and DLG1, well-characterized PDZ proteins with crucial roles in the postsynaptic density of excitatory neuronal synapses and in the establishment and maintenance of epithelial cell polarity (Humbert et al, 2008; Feng & Zhang, 2009). Third, we successfully identify known and novel interactions, with potential to be regulated by Ser/Thr phosphorylation, and uncover that Scribble PDZ interactions are enabled by p-3 phosphorylation. Finally, we provide the structural basis of such phosphopeptide binding, expand the analysis to an additional nine domains, and discuss the role of Ser/Thr phosphorylation as a switching mechanism of PDZ domain interactions. In this proof-of-concept study, we thus demonstrate that phosphomimetic ProP-PD is a powerful approach to identify SLiM-based interactions in the human proteome that are regulated by Ser/Thr phosphorylation. The approach is readily scalable and can be used to explore the potential phospho-regulation of human protein–protein interactions on a larger scale. Results Construction of a phosphomimetic ProP-PD library To design a specific phage display library for phosphomimetic ProP-PD, we scanned the human proteome for C-terminal regions containing known or predicted Ser/Thr phosphorylation sites based on phosphorylation databases and in silico predictions (see Materials and Methods). We identified 4,828 unique C-terminal peptides of human full-length proteins that contain one or multiple potential Ser/Thr phosphosites in their last nine amino acids. We designed the phosphomimetic ProP-PD library to comprehensively contain wild-type sequences and phosphomimetic mutants (Ser/Thr to Glu) thereof (7,626 phosphomimetic sequences, including combinations of multiple phosphorylation sites in one C-terminus), in order to allow the detection of interactions that are enabled or disabled by phosphomimetic mutations. The library design contains 24% of all non-redundant C-terminal peptides of human full-length proteins reported in the annotated section of SwissProt/UniProt (Table EV1). Of these sequences, 8.2% contain class I PDZ binding motifs, 6.2% contain class II binding motifs, and 3.4% match class III binding motifs. In case of the class I-containing peptides, 40.8% of the phosphorylation sites are at p-2 and are expected to disable PDZbm-dependent interactions. Oligonucleotides coding for the wild-type and phosphomimetic (Ser/Thr to Glu) peptides flanked by annealing sequences (Table EV2) were obtained as a custom oligonucleotide library and incorporated into a phagemid vector, thereby creating C-terminal fusion proteins of the M13 major coat protein p8, previously engineered for C-terminal display (Fuh et al, 2000; Fig 1). We confirmed 94.6% of the designed sequences by NGS and the vast majority of reads matched the library design. 62% of raw library sequences were of right length, and of those, non-synonymous mutations were present in 14.6% of the sequences. There were no major sequence biases in the constructed phage library (Fig EV1). The library coverage and quality were thus considered satisfactory. Figure 1. Schematics of the phosphomimetic ProP-PD approach and validationsWe designed a phosphomimetic ProP-PD library to display the C-terminal regions (nine amino acids) of human proteins containing known or putative Ser/Thr phosphorylation sites (4,827 peptides), and all phosphomimetic (Ser/Thr to Glu) variants thereof (7,626 peptides). Oligonucleotides encoding the sequences were incorporated into a phagemid designed for the display of peptides fused to the C-terminus of the major coat protein P8 of the M13 filamentous phage. The library was used in binding selections against immobilized PDZ domains. Binding-enriched phage pools were analyzed by NGS, followed by data analysis and validations. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Analysis of the phage selection results in comparison with the peptide representation in the naïve phage library A–D. Upper panels (A and B): the phosphomimetic ProP-PD experiment, lower panels (C and D): pre-phosphorylation of the wild-type library. Boxplots showing increased probability of a peptide being selected if being well represented in the initial library (y-axis: read count in the respective initial library sequencing, peptides with no read support not used). Scatter plots show the read-count after selection in the phage pools (x-axis) against the read-counts in the respective library sequencing (y-axis). Download figure Download PowerPoint Phosphomimetic ProP-PD selections against the PDZ domains of Scribble and DLG1 We tested the performance of the phosphomimetic ProP-PD library through selections against the immobilized PDZ domains of human Scribble (PDZ1, PDZ2, and PDZ3) and DLG1 (PDZ1, PDZ2, and PDZ3). Of these proteins, Scribble has previously been shown to interact with the p-1 phosphorylated PDZbm of MCC (colorectal mutant cancer protein; RPHTNETpSL-coo-; Pangon et al, 2012). In contrast, phosphorylation of the p-2 position has been shown to disable interaction with DLG1 PDZ2 (Adey et al, 2000). The experiment was thus designed to capture interactions that were enabled or disabled by phosphomimetic mutations. The selections were successful as judged by phage pool enzyme-linked immunosorbent assay (ELISA) and saturated after three rounds of selection. Binding-enriched phage pools were barcoded and analyzed by NGS, which resulted in a list of binding peptides ranked by their occurrence in the NGS results. Although we found increased sensitivity of a peptide being selected during the phage display if it is well represented in the naïve phage libraries (see boxplot figures of initial read support versus being selected or not selected; Fig EV1), there is no correlation of the selection strength with representation in the naïve library in any of the experiments reflecting the strong selection during phage display (Fig EV1). High-confidence sets of ligands (Fig 2A; Table EV3) were obtained by assigning cutoff values that filtered out non-specifically retained peptides lacking typical PDZbms. For each domain, we generated position weight matrices (PWMs) based on the ProP-PD (Fig 2B). All domains are class I binding PDZ domains, and the PWMs are in good agreement with previous studies (Tonikian et al, 2008; Ivarsson et al, 2014; Karlsson et al, 2016). Figure 2. Analysis of the phosphomimetic ProP-PD selection results Fractions of the sequencing counts for individual peptides (wild-type peptides indicated by red bars and phosphomimetic variants by blue bars) from NGS analysis of binding-enriched phage pools from selections against the PDZ domains of Scribble and DLG1. Peptides are sorted based on the site of the phosphomimetic mutations (corresponding to known or putative phosphosites). Standard deviations are indicated (n = 3 for all cases except for Scribble PDZ2 where n = 2). Peptide pairs for which there are significant differences between the fractions of NGS counts for wild-type and phosphomimetic peptides are indicated with * (multiple t-tests, significance level 0.05). Peptides with multiple phosphorylation sites have been omitted from this analysis for clarity. For further details, see Table EV3. Position weight matrices (PWMs, WebLogo3) representing the phosphomimetic ProP-PD selections data for the PDZ domains of Scribble and DLG1. The numbers of peptides used for the analysis are indicated. Scoring matrices of the effects of phosphomimetic mutations on the phosphomimetic ProP-PD results of the PDZ domains of Scribble and DLG1. Scores are calculated from the ratios between NGS counts of the phosphomimetic peptides and the sum of the NGS counts of the wild-type and phosphomimetic peptides. A score of 0 (red) indicates that the selection was dominated by wild-type peptides, and a score of 1 (blue) indicates that the selection was dominated by peptides with phosphomimetic mutations at the given position (n = 3 for all cases except for Scribble PDZ2 where n = 2). Positions marked with * have a score that is significantly different from the score 0.5 of a neutral effect as determined using multiple t-tests and correcting for multiple comparisons using a false discovery rate of 2.5%. The position for which only wild-type or phosphomimetic peptides are represented in the data set could not be subjected to this test. Download figure Download PowerPoint Analysis of the effects of phosphomimetic mutations We systematically evaluated the effects of phosphomimetic mutations at distinct positions of the PDZbms by analyzing the NGS counts received for each peptide pair (wild-type and phosphomimetic; Fig 2A). Essentially, we calculated the ratio between the NGS counts of a given phosphomimetic peptide and the sum of NGS counts of the phosphomimetic peptide and its wild type (Table EV3). For comparison between replicate experiments, we used the fraction of counts, rather than the raw counts, as the total amount of NGS reads differed between the sequencing rounds. We then calculated the average ratios for each position of the PDZbm. This resulted in a score in the range between 0 and 1 for each peptide position, where 0 indicates that the selection was dominated by wild-type peptides and suggests that phosphomimetic mutations at this position disable interactions. A score of 1 suggests that phosphomimetic mutations at the given position enable interactions. The scores for the different domains and peptide positions are summarized in matrices (Fig 2C). Inspection of the NGS frequency of the wild-type and mutant peptides and the matrices revealed common and distinct features. Among the shared features is that phosphomimetic mutations at p-2 disable interactions with all PDZ domains. Phosphorylation of this position is thus expected to serve as an "off-switch" for class I PDZ-mediated interactions. Interestingly, the analysis suggests that phosphomimetic mutations of the p-3 position enable interactions, in particular for Scribble PDZ1 and Scribble PDZ3, for which the differences are statistically significant (Fig 2C, Table EV4). Phosphomimetic mutations of the upstream positions −5 to −6 have instead negative effects on the interactions. Phosphorylation may thus enable or disable PDZ-mediated interactions in a site-specific manner. Microscale thermophoresis affinity measurements confirm phosphorylation switches To explore the extent to which the phosphomimetic ProP-PD results translate into affinity differences between unphosphorylated and phosphorylated peptides, we focused on Scribble PDZ1 ligands. We selected three distinct sets of peptides (unphosphorylated, phosphomimetic mutant, and phosphorylated) for affinity determination by microscale thermophoresis (MST; Fig 3A). The peptides were chosen in order to validate the effects of the putative enabling phosphorylation switches at the p-1 (MCC; HTNETSL-coo-) and p-3 (RPS6KA2, RLTSTRL-coo-) positions, and the disabling effect of the upstream p-6 position (TANC1, KRSFIESNV-coo-). Scribble PDZ1 was titrated against FITC-labeled wild-type, phosphomimetic, and phosphorylated peptides. All peptides bound to Scribble PDZ1 with micromolar affinities (0.6–101 μM KD values; Fig 3A). Consistent with the phosphomimetic ProP-PD results, Scribble PDZ1 has a higher affinity (3×) for the p-1 phosphorylated MCC peptide and the p-3 phosphorylated RPS6KA2 (5×), as compared to their unphosphorylated counterparts. The opposite is true for the TANC1 peptide (2× lower affinity for phosphopeptide; Fig 3A). Thus, there is a good qualitative agreement between the phosphomimetic ProP-PD data and the affinity differences between phosphorylated and unphosphorylated ligands as determined by MST for this domain. The effects of the phosphomimetic mutations are qualitatively, but not quantitatively, the same as the effects of ligand phosphorylation, which is expected due to the distinct properties of Glu in comparison with phospho-Ser/Thr. Figure 3. Scribble PDZ1 preferentially interacts with p-1 and p-3 phosphorylated ligands as shown by affinity determinations (MST and ITC), NMR structure, mutational analysis, and GST-pulldowns and co-immunoprecipitation experiments MST affinity measurements using FITC-labeled peptides (unphosphorylated, phosphorylated, and phosphomimetic variants) of MCC (p-1), RPS6KA2 (p-3), and TANC1 (p-6). A fixed peptide concentration (25–50 nM) was titrated with varying concentrations of Scribble PDZ1. KD values were determined using thermophoresis and T-Jump signal for data analysis (n = 3; error bars represent SD). Statistical assessment using ordinary one-way ANOVA for multiple comparisons confirmed significant differences (P ≤ 0.001) between the affinities for the phosphorylated peptides compared to the unphosphorylated peptides of MCC, RPS6KA2, and TANC1. For the comparison between the wild-type and phosphomimetic peptides, there were significant differences for the peptides of MCC (P ≤ 0.01) and RPS6KA2 (P ≤ 0.001), but not for TANC1. Insets show representative titrations Representative ITC titrations of Scribble PDZ1 with unphosphorylated and phosphorylated peptides of MCC, RPS6KA2, and MAPK12 (n = 3). Statistical assessment using multiple t-tests corrected using the Holm–Sidak method for multiple comparisons show a significant difference between the phosphorylated and unphosphorylated MCC (P = 0.00098) and RPS6KA2 (P = 0.0003) peptides. Differences between the thermodynamic parameters of Scribble PDZ1 when binding unphosphorylated or phosphorylated ligands (MCC and RPS6KA2) as determined in (B) (error bars represent SD, n = 3). Overlay of the previously published unliganded structure of Scribble PDZ1 (gray; PDB code 1X5Q) and the here determined NMR structure (blue; PDB code 6ESP) of the protein bound to MKRLTpSTRL-coo- (peptide not shown). The canonical PDZ binding grove is indicated by a dotted orange line. Structure of the phosphopeptide-bound Scribble PDZ1 showing three positively charged residues (K746, R762, and R801) surrounding the peptide binding pocket. Left panel: Equilibrium binding constants for the binding between Scribble PDZ1 wild type and mutants (K746A, R762A, and R801A) and RPS6KA2 (unphosphorylated and phosphorylated) as determined through ITC titrations (n = 3). Right panel: Changes in the thermodynamic parameters upon mutation (ΔΔHmutation and −TΔΔSmutation) as determined through ITC (error bars represent SD, n = 3). Section of the 15N-1H HSQC spectra of ligand-free wild-type Scribble PDZ1 (red) and the protein bound to the phospho-RPS6KA2 peptide (green), together with the spectra of the ligand-free R762A mutant (magenta) and the mutant bound to phospho-RPS6KA2 (cyan). Note that residue 762 experiences chemical shift perturbation only in the wild-type Scribble PDZ1. Slice of the 15N-1H HSQC spectra showing the R733 or R762A residue of the wild-type or mutant proteins. R733 is critical for mediating the carboxylate at the C-terminus, and its position does not change in the wild-type-bound or the R762A-bound form, showing that the carboxylate is still making the important interaction. GST-pulldown of full-length Flag-tagged RPS6KA2 wild type and mutants (S730A and S730E) and a truncated version (S730Δ) over-expressed in HEK293T cells with GST-tagged Scribble PDZ1. Detection was performed using an anti-Flag antibody. Co-IPs of GFP-tagged full-length Scribble with Flag-tagged full-length RPS6KA2 constructs as indicated. Detection was performed using an anti-Flag antibody and an anti-GFP antibody. GST-pulldown of full-length Flag-tagged RPS6KA1 wild type and a truncated version of the protein (S732Δ). Detection was performed using an anti-Flag antibody. Source data are available online for this figure. Source Data for Figure 3 [msb178129-sup-0011-SDataFig3.pdf] Download figure Download PowerPoint To evaluate the effects of ligand phosphorylation on DLG1 PDZ binding, we determined the affinities for the same set of peptides with DLG1 PDZ2 (Fig EV2; Table EV5), and found them to be in the μM range (2.7–95 μM). MST measurements with the p-1 modified MCC peptides revealed that phosphorylation of this site in this ligand has a disabling effect on DLG1 PDZ2 interactions (9×; KDMCC 6.4 μM; KDpMCC 57 μM). This seemingly contradicts the result reported in the position-specific matric, but importantly, the MCC ligand was not identified as a DLG1 PDZ2 in the phage selection, and it is possible that the results may not be generally extended to other ligands, as both domains and ligands are flexible and the results of phosphorylation may depend on the combination of amino acids at other ligand positions (e.g., the MCC peptide has a L at p0 instead of the V which is preferred by DLG1 PDZ2). For the p-3 modified RPS6KA2 peptide (Fig EV2), phosphomimetic mutation has a minor enabling effect on the interaction (1.3×). Both phosphorylation and phosphomimetic mutations of the p-6 position of the TANC1 peptide have minor disabling effects on DLG1 PDZ 2 binding (2.9× and 2.4×, respectively), consistent with the phosphomimetic ProP-PD results. Click here to expand this figure. Figure EV2. Microscale thermophoresis affinity measurements of FITC-labeled peptides and recombinant PDZ domainsA fixed peptide concentration (25–50 nM)
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