Dual Specificity PDZ- and 14-3-3-Binding Motifs: A Structural and Interactomics Study

Gógl, Gergő; Jané, Pau; Caillet‐Saguy, Célia; Kostmann, Camille; Bich, Goran; Cousido-Siah, A.; Nyitray, László; Vincentelli, Renaud; Wolff, Nicolas; Nominé, Yves; Sluchanko, Nikolai N.; Travé, Gilles

doi:10.1016/j.str.2020.03.010

Cited by 44 publications

(73 citation statements)

References 55 publications

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“…Protein concentrations were determined by far-UV absorption spectroscopy. A detailed protocol has been published previsouly [4].…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…Such affinity variations may result from differences of entropy of the free peptides, from altered interface contacts in the resulting PDZ-PBM complexes, or a combination of both. Accordingly, synthetic or recombinant PBMs employed for PDZ interactions generally include at least 9 to 11 residues [4], [5], [15], [17], [28], [50]. Indeed, the presence of distal sites altering PDZ-PBM binding has already been described [51], even at positions as far as at p-36 [52].…”

Section: Lessons From Distal Residues On the Pten Interactomementioning

confidence: 99%

“…a list of binding strengths in decreasing order exhibited by a given PBM towards the entire PDZome. The high accuracy and efficiency of the holdup assay has been validated previously [4], [15], [28], [30]. Very recently, a manual version of the holdup assay with purified samples and using widespread benchtop equipment has been implemented and has proven to be reliable [31].…”

Section: Introductionmentioning

confidence: 99%

“…PDZs recognize short linear motifs (called PDZ Binding Motif or PBMs) that follow particular sequence requirements and are mostly located at the extreme carboxy terminus of target proteins [2]. The human proteome contains 266 PDZ domains dispersed over 152 proteins [3] and thousands of presumably disordered C-termini matching a PBM consensus [4].…”

Section: Introductionmentioning

confidence: 99%

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Interactomic affinity profiling by holdup assay: acetylation and distal residues impact the PDZome-binding specificity of PTEN phosphatase

Jané

Gógl

Kostmann

et al. 2020

Preprint

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AbstractProtein domains often recognize short linear protein motifs composed of a core conserved consensus sequence surrounded by less critical, modulatory positions. Here we used an accurate experimental approach combining high-throughput holdup chromatographic assay and fluorescence polarization to measure quantitative binding affinity profiles of the PDZ domain-binding motif (PBM) of PTEN phosphatase towards the 266 known human PDZ domains. Inclusion of N-terminal flanking residues, acetylation or mutation of a lysine at a modulatory position significantly altered the PDZome-binding profile of the PTEN PBM. A specificity index is also introduced to quantify the specificity of a given PBM over the complete PDZome. Our results highlight the impact of modulatory residues and post-translational modifications on PBM interactomes and their specificity.

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“…Protein concentrations were determined by far-UV absorption spectroscopy. A detailed protocol has been published previsouly [4].…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Section: Lessons From Distal Residues On the Pten Interactomementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Interactomic affinity profiling by holdup assay: acetylation and distal residues impact the PDZome-binding specificity of PTEN phosphatase

Jané

Gógl

Kostmann

et al. 2020

Preprint

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“…Phosphorylated 16E6, 18E6, and 31E6 have been previously shown to bind to 14-3-3 proteins, albeit with different efficiencies 18,21,24 . In contrast, the binding of unphosphorylated or phosphomimetic E6 PBMs to 14-3-3 proteins was not detectable 21, 24 . This suggests that the phosphorylation-dependent interaction with 14-3-3 proteins is a general feature of various E6 PBMs.…”

Section: Introductionmentioning

confidence: 99%

Hierarchized phosphotarget binding by the seven human 14-3-3 isoforms

Gógl

Tugaeva

Eberling

et al. 2020

Preprint

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In tumors induced by high-risk mucosal human papillomaviruses (hrm-HPVs), HPV E6 oncoproteins inhibit apoptotic processes and sustain cell proliferation. E6 from all hrm-HPVs harbor a C-terminal short PDZ domain-binding motif (PBM), whose phosphorylation down-regulates PDZ binding but triggers E6 binding to 14-3-3 proteins. Here we classify PBMs of E6 proteins depending on their principle ability to be phosphorylated and subsequently acquire a 14-3-3-binding motif III consensus, (pS/pT)XX-COOH. Systematic competitive fluorescence polarization measurements show that the PBMs from four selected E6 oncoproteins bind all seven human 14-3-3 isoforms with distinct, wide-ranging affinities, obeying remarkable trends assigned to 14-3-3 isoform specificity and small E6 sequence variations. We crystallized the hrm-HPV18 E6 PBM bound to 14-3-3ζ, revealing a 14-3-3-motif III complex at 1.9 Å resolution. Using fluorescence polarization and crystallography, we also demonstrate that fusicoccin, a molecule that reinforces many known 14-3-3 complexes, destabilizes the 14-3-3-E6 interaction, indicating the druggability of that complex.

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Molecules interact. But how strong and how much?

2023

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Interactomics aims to characterize all interactions formed between molecules that comprise our body. Although it emerged from quantitative biophysics, it has devolved into a predominantly qualitative field of science over the past decades. Due to technical limitations at its onset, almost all tools in interactomics are qualitative, which persists in defining the discipline. Here, we argue that interactomics needs to return to a quantitative direction because the technical achievements of the last decade have overcome the original limitations that forced its current path. In contrast to qualitative interactomics which is constrained to charting lists of observed interactions, quantitative interactomics can also uncover answers to key questions such as the strength of interactions or how many of certain complexes can form in cells, thus providing researchers with more immediate proxies for understanding and predicting biological processes.

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Dual Specificity PDZ- and 14-3-3-Binding Motifs: A Structural and Interactomics Study

Abstract: Highlights d A large proportion of PDZ-binding motifs are phosphorylatable d Phosphorylated and phosphomimetic PBMs bind differently to PDZs and 14-3-3 proteins d These differences are demonstrated by X-ray analysis and affinity profiling

Cited by 44 publications

References 55 publications

Interactomic affinity profiling by holdup assay: acetylation and distal residues impact the PDZome-binding specificity of PTEN phosphatase

Interactomic affinity profiling by holdup assay: acetylation and distal residues impact the PDZome-binding specificity of PTEN phosphatase

Hierarchized phosphotarget binding by the seven human 14-3-3 isoforms

Molecules interact. But how strong and how much?

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