Comparative analysis of PDZ-binding motifs in the diacylglycerol kinase family.

Diacylglycerol kinases (DGKs) control local and temporal amounts of diacylglycerol (DAG) and phosphatidic acid (PA) by converting DAG to PA through phosphorylation in cells. Certain DGK enzymes possess C-terminal sequences that encode potential PDZ-binding motifs (PBMs), which could be involved in their recruitment into supramolecular signaling complexes. In this study, we used two different interactomic approaches, quantitative native holdup (nHU) and qualitative affinity purification (AP), both coupled to mass spectrometry (MS) to investigate the PDZ partners associated with the potential PBMs of DGKs. Complementing these results with site-specific affinity interactomic data measured on isolated PDZ domain fragments and PBM motifs, as well as evolutionary conservation analysis of the PBMs of DGKs, we explored functional differences within different DGK groups. All our results indicate that putative PBM sequences of type II enzymes, namely DGKδ, DGKη, and DGKκ, are likely to be nonfunctional. In contrast, type IV enzymes, namely DGKζ and DGKι, possess highly promiscuous PBMs that interact with a set of PDZ proteins with very similar affinity interactomes. The combination of various interactomic assays and evolutionary analyses provides a useful strategy for identifying functional domains and motifs within diverse enzyme families.

PDZome-wide and structural characterization of the PDZ-binding motif of VANGL2.

VANGL2 is a core component of the non-canonical Wnt/Planar Cell Polarity signaling pathway that uses its highly conserved carboxy-terminal type 1 PDZ-binding motif (PBM) to bind a variety of PDZ proteins. In this study, we characterize and quantitatively assess the largest VANGL2 PDZome-binding profile documented so far, using orthogonal methods. The results of our holdup approach support VANGL2 interactions with a large panel of both long-recognized and unprecedented PDZ domains. Truncation and point mutation analyses of the VANGL2 PBM establish that, beyond the strict requirement of the P-0 / V521 and P-2 / T519 amino acids, upstream residues, including E518, Q516 and R514 at, respectively, P-3, P-5 and P-7 further contribute to the robustness of VANGL2 interactions with two distinct PDZ domains, SNX27 and SCRIBBLE-PDZ3. In agreement with these data, incremental amino-terminal deletions of the VANGL2 PBM causes its overall affinity to progressively decline. Moreover, the holdup data establish that the PDZome binding repertoire of VANGL2 starts to diverge significantly with the truncation of E518. A structural analysis of the SYNJ2BP-PDZ/VANGL2 interaction with truncated PBMs identifies a major conformational change in the binding direction of the PBM peptide after the P-2 position. Finally, we report that the PDZome binding profile of VANGL2 is dramatically rearranged upon phosphorylation of S517, T519 and S520. Our crystallographic approach illustrates how SYNJ2BP accommodates a S520-phosphorylated PBM peptide through the ideal positioning of two basic residues, K48 and R86. Altogether our data provides a comprehensive view of the VANGL2 PDZ network and how this network specifically responds to the post-translation modification of distinct PBM residues. These findings should prove useful in guiding future functional and molecular studies of the key PCP component VANGL2.

The Human PDZome 2.0: Characterization of a New Resource to Test for PDZ Interactions by Yeast Two-Hybrid.

PSD95-disc large-zonula occludens (PDZ) domains are globular modules of 80-90 amino acids that co-evolved with multicellularity. They commonly bind to carboxy-terminal sequences of a plethora of membrane-associated proteins and influence their trafficking and signaling. We previously built a PDZ resource (PDZome) allowing us to unveil human PDZ interactions by Yeast two-hybrid. Yet, this resource is incomplete according to the current knowledge on the human PDZ proteome. Here we built the PDZome 2.0 library for Yeast two-hybrid, based on a PDZ library manually curated from online resources. The PDZome2.0 contains 305 individual clones (266 PDZ domains in isolation and 39 tandems), for which all boundaries were designed based on available PDZ structures. Using as bait the E6 oncoprotein from HPV16, a known promiscuous PDZ interactor, we show that PDZome 2.0 outperforms the previous resource.

Evidence for direct interaction between the oncogenic proteins E6 and E7 of high-risk human papillomavirus (HPV).

Human papillomaviruses (HPVs) are DNA tumor viruses that infect mucosal and cutaneous epithelial cells of more than 20 vertebrates. High-risk HPV causes about 5% of human cancers worldwide, and the viral proteins E6 and E7 promote carcinogenesis by interacting with tumor suppressors and interfering with many cellular pathways. As a consequence, they immortalize cells more efficiently in concert than individually. So far, the networks of E6 and E7 with their respective cellular targets have been studied extensively but independently. However, we hypothesized that E6 and E7 might also interact directly with each other in a novel interaction affecting HPV-related carcinogenesis. Here, we report a direct interaction between E6 and E7 proteins from carcinogenic HPV types 16 and 31. We demonstrated this interaction via cellular assays using two orthogonal methods: coimmunoprecipitation and flow cytometry-based FRET assays. Analytical ultracentrifugation of the recombinant proteins revealed that the stoichiometry of the E6/E7 complex involves two E7 molecules and two E6 molecules. In addition, fluorescence polarization showed that (I) E6 binds to E7 with a similar affinity for HPV16 and HPV31 (in the same micromolar range) and (II) that the binding interface involves the unstructured N-terminal region of E7. The direct interaction of these highly conserved papillomaviral oncoproteins may provide a new perspective for studying HPV-associated carcinogenesis and the overall viral life cycle.

ProFeatMap: a highly customizable tool for 2D feature representation of protein sets.

Motivation: Studies of sets of proteins are a central point in biology. In particular, the application of omics in the last decades has generated lists of several hundreds or thousands of proteins or genes. However, these lists are often not inspected globally, possibly due to the lack of tools capable of simultaneously visualizing the feature architectures of a large number of proteins. Results: Here, we present ProFeatMap, an intuitive Python-based website. For a given set of proteins, it allows to display features such as domains, repeats, disorder or post-translational modifications and their organization along the sequences, into a highly customizable 2D map. Starting from a user-defined protein list of UniProt accession codes, ProFeatMap extracts the most important annotated features available for each protein from one of the well-established databases such as Uniprot or InterPro, allocates shapes and colors, potentially depending on quantitative or qualitative data and sorts the protein list based on homologous feature content. The resulting publication-quality map allows even large protein families to be explored, and to classify them based on shared features. It can help to gain insights, for example, feature redundancy or feature pattern, that were previously overlooked. ProFeatMap is freely available on the web at: https://profeatmap.pythonanywhere.com/. Availability and implementation: Source code is freely accessible at https://github.com/profeatmap/ProFeatMap under the GPL license. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Native holdup (nHU) to measure binding affinities from cell extracts.

Characterizing macromolecular interactions is essential for understanding cellular processes, yet most methods currently used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to estimate equilibrium binding constants of protein interactions directly from cell extracts. Compared to other pull-down-based assays, nHU requires less sample preparation and can be coupled to any analytical methods as readouts, such as Western blotting or mass spectrometry. We use nHU to explore interactions of SNX27, a cargo adaptor of the retromer complex and find good agreement between in vitro affinities and those measured directly from cell extracts using nHU. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.

The cooperative folding of annexin A2 relies on a transient nonnative intermediate.

Annexins (Anxs) are a family of highly homologous proteins that bind and aggregate lipid vesicles in the presence of calcium. All members of the family contain a variable N-terminus determining specific functions, followed by a conserved core region responsible for the general calcium-dependent lipid-binding property. The core structure consists of four homologous domains (DI-DIV), each consisting of a right-handed super-helix of five α-helices. We present data from a combination of site-directed mutagenesis, NMR, and circular dichroism showing that the G25-D34 region of the N-terminus as well as the contacts between residues D38A, R63A, and Q67A of AnxA2-DI are crucial for the autonomous folding and stability of DI of AnxA2. However, we also show that the folding of the full-length protein is very robust in that mutations and truncations that disrupted the folding of AnxA2-DI did not abolish the folding of full-length AnxA2, only lowering its thermal stability. This robustness of the folding of full-length AnxA2 is likely to be mediated by the existence of at least one transient nonnative intermediate as suggested by our kinetic data using stopped-flow fluorescence experiments. We also show that hydrophobic amino acids in AnxA2-DI involved in interfacial contacts with AnxA2-DIV are important for the cooperative folding and stability of the full-length protein. Mutating all of the V57E-V98R-G101Y residues in AnxA2-DI did not affect the folding of the domain, only its stability, but prevented the cooperative folding of the full-length protein. Our collective results favor a highly cooperative and robust folding process mediated by alternative intermediate steps. Since AnxA2 is a multifunctional protein involved in several steps of the progression of cell transformation, these data on structure and folding pathways are therefore crucial to designing anticancer drugs targeting AnxA2.

Quantitative fragmentomics allow affinity mapping of interactomes.

Human protein networks have been widely explored but most binding affinities remain unknown, hindering quantitative interactome-function studies. Yet interactomes rely on minimal interacting fragments displaying quantifiable affinities. Here, we measure the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer. We calculate interactomic distances, identify hot spots for viral interference, generate binding profiles and specificity logos, and explain selected cases by crystallographic studies. Mass spectrometry experiments on cell extracts and literature surveys show that quantitative fragmentomics effectively complements protein interactomics by providing affinities and completeness of coverage, putting a full human interactome affinity survey within reach. Finally, we show that interactome hijacking by the viral PBM of human papillomavirus E6 oncoprotein substantially impacts the host cell proteome beyond immediate E6 binders, illustrating the complex system-wide relationship between interactome and function.

A scalable strategy to solve structures of PDZ domains and their complexes.

The human PDZome represents one of the largest globular domain families in the human proteome, with 266 instances. These globular domains typically interact with C-terminal peptide motifs found in thousands of human proteins. Despite previous efforts, not all PDZ domains have experimentally solved structures and most of their complexes remain to be solved. Here, a simple and cost-effective strategy is proposed for the crystallization of PDZ domains and their complexes. A human annexin A2 fusion tag was used as a crystallization chaperone and the structures of nine PDZ domains were solved, including five domains that had not yet been solved. Finally, these novel experimental structures were compared with AlphaFold predictions and it is speculated how predictions and experimental methods could cooperate in order to investigate the structural landscapes of entire domain families and interactomes.

Promiscuity mapping of the S100 protein family using a high-throughput holdup assay.

S100 proteins are small, typically homodimeric, vertebrate-specific EF-hand proteins that establish Ca2+-dependent protein-protein interactions in the intra- and extracellular environment and are overexpressed in various pathologies. There are about 20 distinct human S100 proteins with numerous potential partner proteins. Here, we used a quantitative holdup assay to measure affinity profiles of most members of the S100 protein family against a library of chemically synthetized foldamers. The profiles allowed us to quantitatively map the binding promiscuity of each member towards the foldamer library. Since the library was designed to systematically contain most binary natural amino acid side chain combinations, the data also provide insight into the promiscuity of each S100 protein towards all potential naturally occurring S100 partners in the human proteome. Such information will be precious for future drug design to interfere with S100 related pathologies.

High-Risk Mucosal Human Papillomavirus 16 (HPV16) E6 Protein and Cutaneous HPV5 and HPV8 E6 Proteins Employ Distinct Strategies To Interfere with Interferon Regulatory Factor 3-Mediated Beta Interferon Expression.

Persistent infection with some mucosal α-genus human papillomaviruses (HPVs; the most prevalent one being HPV16) can induce cervical carcinoma, anogenital cancers, and a subset of head and neck squamous cell carcinoma (HNSCC). Cutaneous ß-genus HPVs (such as HPV5 and HPV8) associate with skin lesions that can progress into squamous cell carcinoma with sun exposure in Epidermodysplasia verruciformis patients and immunosuppressed patients. Here, we analyzed mechanisms used by E6 proteins from the α- and ß-genus to inhibit the interferon-ß (IFNB1) response. HPV16 E6 mediates this effect by a strong direct interaction with interferon regulatory factor 3 (IRF3). The binding site of E6 was localized within a flexible linker between the DNA-binding domain and the IRF-activation domain of IRF3 containing an LxxLL motif. The crystallographic structure of the complex between HPV16 E6 and the LxxLL motif of IRF3 was solved and compared with the structure of HPV16 E6 interacting with the LxxLL motif of the ubiquitin ligase E6AP. In contrast, cutaneous HPV5 and HPV8 E6 proteins bind to the IRF3-binding domain (IBiD) of the CREB-binding protein (CBP), a key transcriptional coactivator in IRF3-mediated IFN-ß expression. IMPORTANCE Persistent HPV infections can be associated with the development of several cancers. The ability to persist depends on the ability of the virus to escape the host immune system. The type I interferon (IFN) system is the first-line antiviral defense strategy. HPVs carry early proteins that can block the activation of IFN-I. Among mucosal α-genus HPV types, the HPV16 E6 protein has a remarkable property to strongly interact with the transcription factor IRF3. Instead, cutaneous HPV5 and HPV8 E6 proteins bind to the IRF3 cofactor CBP. These results highlight the versatility of E6 proteins to interact with different cellular targets. The interaction between the HPV16 E6 protein and IRF3 might contribute to the higher prevalence of HPV16 than that of other high-risk mucosal HPV types in HPV-associated cancers.

A Computational Protocol to Analyze PDZ/PBM Affinity Data Obtained by High-Throughput Holdup Assay.

The holdup assay is an automated high-throughput comparative chromatographic retention approach that allows to measure quantitative binding intensities (BI) for a large number of domain-motif pairs and deduce equilibrium binding affinity constants. We routinely apply this approach to obtain quantitative binding specificity profiles of particular PDZ-binding motifs (PBMs) toward the full library of known human PDZ domains (the PDZome). The quality of the electropherograms extracted from the capillary electrophoresis instrument at the final step of the holdup assay may vary, influencing the accuracy and reproducibility of the measurement. By using bioinformatic tools, we can solve these issues to extract more reliable BIs by means of a better superimposition of the electropherograms. The protocol presented in this chapter describes the main principles and strategies of our curated method to process holdup data and new ways to plot and compare the BIs for the PBM-PDZ interactions. For this particular protocol, all the necessary computing commands are freely available in open Python packages.

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

The seven 14-3-3 isoforms are highly abundant human proteins encoded by similar yet distinct genes. 14-3-3 proteins recognize phosphorylated motifs within numerous human and viral proteins. Here, we analyze by X-ray crystallography, fluorescence polarization, mutagenesis and fusicoccin-mediated modulation the structural basis and druggability of 14-3-3 binding to four E6 oncoproteins of tumorigenic human papillomaviruses. 14-3-3 isoforms bind variant and mutated phospho-motifs of E6 and unrelated protein RSK1 with different affinities, albeit following an ordered affinity ranking with conserved relative KD ratios. Remarkably, 14-3-3 isoforms obey the same hierarchy when binding to most of their established targets, as supported by literature and a recent human complexome map. This knowledge allows predicting proportions of 14-3-3 isoforms engaged with phosphoproteins in various tissues. Notwithstanding their individual functions, cellular concentrations of 14-3-3 may be collectively adjusted to buffer the strongest phosphorylation outbursts, explaining their expression variations in different tissues and tumors.

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

Protein domains often recognize short linear protein motifs composed of a core conserved consensus sequence surrounded by less critical, modulatory positions. PTEN, a lipid phosphatase involved in phosphatidylinositol 3-kinase (PI3K) pathway, contains such a short motif located at the extreme C-terminus capable to recognize PDZ domains. It has been shown that the acetylation of this motif could modulate the interaction with several PDZ domains. Here we used an accurate experimental approach combining high-throughput holdup chromatographic assay and competitive fluorescence polarization technique to measure quantitative binding affinity profiles of the PDZ domain-binding motif (PBM) of PTEN. We substantially extended the previous knowledge towards the 266 known human PDZ domains, generating the full PDZome-binding profile of the PTEN PBM. We confirmed that inclusion of N-terminal flanking residues, acetylation or mutation of a lysine at a modulatory position significantly altered the PDZome-binding profile. A numerical specificity index is also introduced as an attempt 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.

Structure of High-Risk Papillomavirus 31 E6 Oncogenic Protein and Characterization of E6/E6AP/p53 Complex Formation.

The degradation of p53 is a hallmark of high-risk human papillomaviruses (HPVs) of the alpha genus and HPV-related carcinogenicity. The oncoprotein E6 forms a ternary complex with the E3 ubiquitin ligase E6-associated protein (E6AP) and tumor suppressor protein p53 targeting p53 for ubiquitination. The extent of p53 degradation by different E6 proteins varies greatly, even for the closely related HPV16 and HPV31. HPV16 E6 and HPV31 E6 display high sequence identity (∼67%). We report here, for the first time, the structure of HPV31 E6 bound to the LxxLL motif of E6AP. HPV16 E6 and HPV31 E6 are structurally very similar, in agreement with the high sequence conservation. Both E6 proteins bind E6AP and degrade p53. However, the binding affinities of 31 E6 to the LxxLL motif of E6AP and p53, respectively, are reduced 2-fold and 5.4-fold compared to 16 E6. The affinity of E6-E6AP-p53 ternary complex formation parallels the efficacy of the subsequent reaction, namely, degradation of p53. Therefore, closely related E6 proteins addressing the same cellular targets may still diverge in their binding efficiencies, possibly explaining their different phenotypic or pathological impacts.IMPORTANCE Variations of carcinogenicity of human papillomaviruses are related to variations of the E6 and E7 interactome. While different HPV species and genera are known to target distinct host proteins, the fine differences between E6 and E7 of closely related HPVs, supposed to target the same cellular protein pools, remain to be addressed. We compare the oncogenic E6 proteins of the closely related high-risk HPV31 and HPV16 with regard to their structure and their efficiency of ternary complex formation with their cellular targets p53 and E6AP, which results in p53 degradation. We solved the crystal structure of 31 E6 bound to the E6AP LxxLL motif. HPV16 E6 and 31 E6 structures are highly similar, but a few sequence variations lead to different protein contacts within the ternary complex and, as quantified here, an overall lower binding affinity of 31 E6 than 16 E6. These results align with the observed lower p53 degradation potential of 31 E6.

Benchtop holdup assay for quantitative affinity-based analysis of sequence determinants of protein-motif interactions.

Many protein-protein interactions are mediated by short linear peptide motifs binding to cognate proteins or protein domains. Such interactions often display affinities in the mid-micromolar range that are challenging to quantify accurately, especially when the motifs harbor single-point mutations. Here, we present a manual benchtop assay for determining affinities of weak interactions between a purified protein and a peptide array representing mutants of a target motif. The assay is based on the "holdup" principle, a chromatographic approach allowing sensitive detection of weak interactions at equilibrium and accurate estimation of their binding free energy. We tested two alternative setups using, as a readout, either capillary electrophoresis or fluorescence. Using this approach, we studied the amino acid sequence determinants of the interactions between HPV16 E6 viral oncoprotein and single-point mutants of its prototypical target LXXLL motif from the E3 ubiquitin ligase E6AP. Comparing SPOT peptide array and holdup approaches revealed a good agreement for most interactions except the weakest ones, which were only detected by holdup assay. In addition, the strongest interactions were validated by Surface-Plasmon Resonance. The manual holdup procedure proposed here can be readily adapted for accurate evaluation of a wide variety of protein-motif interactions displaying low to medium affinities.

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

Protein-protein interaction motifs are often alterable by post-translational modifications. For example, 19% of predicted human PDZ domain-binding motifs (PBMs) have been experimentally proven to be phosphorylated, and up to 82% are theoretically phosphorylatable. Phosphorylation of PBMs may drastically rewire their interactomes, by altering their affinities for PDZ domains and 14-3-3 proteins. The effect of phosphorylation is often analyzed by performing "phosphomimetic" mutations. Here, we focused on the PBMs of HPV16-E6 viral oncoprotein and human RSK1 kinase. We measured the binding affinities of native, phosphorylated, and phosphomimetic variants of both PBMs toward the 266 human PDZ domains. We co-crystallized all the motif variants with a selected PDZ domain to characterize the structural consequence of the different modifications. Finally, we elucidated the structural basis of PBM capture by 14-3-3 proteins. This study provides novel atomic and interactomic insights into phosphorylatable dual specificity motifs and the differential effects of phosphorylation and phosphomimetic approaches.

Conformational editing of intrinsically disordered protein by α-methylation.

Intrinsically disordered proteins (IDPs) constitute a large portion of "Dark Proteome" - difficult to characterize or yet to be discovered protein structures. Here we used conformationally constrained α-methylated amino acids to bias the conformational ensemble in the free unstructured activation domain of transcriptional coactivator ACTR. Different sites and patterns of substitutions were enabled by chemical protein synthesis and led to distinct populations of α-helices. A specific substitution pattern resulted in a substantially higher binding affinity to nuclear coactivator binding domain (NCBD) of CREB-binding protein, a natural binding partner of ACTR. The first X-ray structure of the modified ACTR domain - NCBD complex visualized a unique conformation of ACTR and confirmed that the key α-methylated amino acids are localized within α-helices in the bound state. This study demonstrates a strategy for characterization of individual conformational states of IDPs.

A novel small-molecule inhibitor of the human papillomavirus E6-p53 interaction that reactivates p53 function and blocks cancer cells growth.

Despite prophylactic vaccination campaigns, human papillomavirus (HPV)-induced cancers still represent a major medical issue for global population, thus specific anti-HPV drugs are needed. Since the ability of HPV E6 oncoprotein to promote p53 degradation is linked to tumor progression, E6 has been proposed as an ideal target for cancer treatment. Using the crystal structure of the E6/E6AP/p53 complex, we performed an in silico screening of small-molecule libraries against a highly conserved alpha-helix in the N-terminal domain of E6 involved in the E6-p53 interaction. We discovered a compound able to inhibit the E6-mediated degradation of p53 through disruption of E6-p53 binding both in vitro and in cells. This compound could restore p53 intracellular levels and transcriptional activity, reduce the viability and proliferation of HPV-positive cancer cells, and block 3D cervospheres formation. Mechanistic studies revealed that the compound anti-tumor activity mainly relies on induction of cell cycle arrest and senescence. Our data demonstrate that the disruption of the direct E6-p53 interaction can be obtained with a small-molecule compound leading to specific antitumoral activity in HPV-positive cancer cells and thus represents a new approach for anti-HPV drug development.